SUSPECTED PRECURSOR-TARGETED IMMUNE-MEDIATED ANEMIA IN DOGS By Cynthia de Assumpção Lucidi A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Pathobiology – Doctor of Philosophy 2015 ABSTRACT SUSPECTED PRECURSOR-TARGETED IMMUNE-MEDIATED ANEMIA IN DOGS By Cynthia de Assumpção Lucidi Immune-mediated hemolytic anemia (IMHA) results in regenerative anemia and is the most well studied immune-mediated anemia in dogs. However, the underlying cause of IMHA remains unknown, and mortality remains a problem. In a different condition, dogs with suspected precursor-targeted immune-mediated anemia (PIMA) present with nonregenerative anemia and ineffective erythropoiesis, which have occasionally been associated with phagocytosis of erythroid precursors (rubriphagocytosis) or myelofibrosis. The pathogenesis of PIMA has not yet been determined, but roles for immunoglobulin and/or complement have been suspected. Additional involvement of apoptosis-like mechanisms with phosphatidylserine (PS) exposure is possible in both canine IMHA and PIMA based on previous studies in people and dogs. Our central hypothesis is that PIMA is part of a spectrum of immune-mediated anemias including IMHA, in which IgG, with or without the contribution of phosphatidylserine (PS), target and promote phagocytosis of different stages of erythroid cells, simultaneously or independently. The rationale was that better characterizing the pathogenesis of PIMA and its association with IMHA will ultimately help establish diagnostic criteria, identify appropriate therapeutic strategies based on knowledge of the pathogenesis of the diseases, and raise veterinary awareness of PIMA. This dissertation first describes a retrospective study of dogs with PIMA with the main goal of helping characterize canine PIMA and facilitate its recognition and diagnosis. Then it describes the development of flow cytometric assays for RBC and erythroid precursor IgG, including a Percoll gradient separation for erythroid populations. These methods were used for isolation of erythroid populations and their assessment for IgG and PS in IMHA and PIMA dogs, healthy dogs, and sick dogs with no evidence of IMHA or PIMA. We found that IMHA dogs had significantly higher IgG and PS when compared to healthy and non-IMHA dogs. Additionally, we showed that a subset of PIMA dogs had increased IgG-positive erythroid precursors when compared to healthy and non-IMHA dogs, and erythroid precursors from most tested PIMA dogs had more PS-positive erythroid precursors than healthy dogs; however, no sick dogs without PIMA were tested for comparison. These findings suggest a role for IgG in canine PIMA and for PS in canine IMHA and PIMA. Finally, we demonstrated the expression of DEA1.1 on canine erythroid precursors from rubriblasts through metarubricytes. Copyright by CYNTHIA DE ASSUMPÇÃO LUCIDI 2015 This dissertation is dedicated to all dogs, for their example of loyalty, cheerfulness, forgiveness, and unconditional love, and for making the world a better place. v ACKNOWLEDGEMENTS My eternal gratitude goes first to my mom and dad for their support and unconditional love. I cannot imagine the sacrifice it was for them to have their youngest child move to a different and far away country to pursue her dreams, while staying supportive and keeping a smile on their faces regardless of all the worries, longing, and fears they had. I would not exist without you, I would not be where I am without you, and I would not be who I am without you. I will be forever thankful for all you represent to me, and all you have done for me. I love you deeply, and I hope I can always honor you and the values you have given me. I would also like to thank my mentor, colleague, and friend, Dr. Michael Scott. We have been working together for 9 years (since 2006), when he opened the doors of his lab for me to fly from Brazil and develop my master’s project with him. He completely changed my life when he asked me to consider doing a PhD at MSU under his supervision. That was an offer I could not decline, especially because I had deep appreciation for Dr. Scott’s  work. He offered me the opportunity to pursue my PhD working on canine bone marrow and hematology, my passion, and I will always be thankful to him for this amazing opportunity! I have so many things to thank Dr. Scott for, including his constant support and availability, him sharing with me his brilliant mind and so much knowledge, his patience in teaching me how to improve my writing and presentation skills, him teaching me his approach and thoughts on science and research ethics, him helping me become more professional, and above all him pushing me to do and be my best, always. Thank you so much, Dr. Scott! v My deep thanks also go to my co-advisor, Dr. Gerlach, who embraced me into the BLD department and HLA lab, provided scientific and financial support, offered professional coaching, and especially gave me wonderful co-workers/friends as part of his BLD/HLA family. My PhD would not have been possible without you and I will never be able to thank you enough for that! I also want to thank Dr. Thomas and Dr. Meek, for being part of my research committee and for their guidance, support, and valuable input and ideas. I am grateful for all the support I received during my PhD from so many people, including: the Clinical Pathology Lab and the Small Animal Clinics faculty and staff for their help identifying cases and samples; Drs. Erickson, Gravelyn and Hollinger for their help identifying cases and collecting data on selected cases; Rose Wahl, Dr. Aulbach, MPI Research, Dr. Komaromy, and Kristen Koehl for their assistance in obtaining samples from healthy dogs; the staff at Medical Records, especially LuAnn Longstreth, for retrieving uncountable patients records for me in a timely manner and with a smile on their faces; Sue McQuiston and Dr. King for their help and expertise with flow cytometry; Dr. Jackson for his assistance with statistical analyses; Dr. Yuzbasiyan-Gurkan and her graduate students, and Dr. Mahdukar for their help with cell culture; John Davis for his input with research designs, troubleshooting, and chemistry questions; to the HLA team for their help, assistance, and friendship, and especially to Sue Forney for her guidance with flow cytometry and finding things in the lab throughout my master’s and PhD degree programs; Drs. Garratty, Sluyter, Judd, Lachmann, and all scientists who gracefully donated their time to clarify my questions through email and hugely helped me advance with my project; and to the BLD department, the MSU Graduate School, the vi department of Pathobiology and Diagnostic Investigation, and the College of Veterinary Medicine for their financial support. I thank my friends and family, especially my husband, Mark Farrugia, for his love, support, understanding, and cheering during the second half of my PhD. It was quite an adventure to have both of us write and defend our PhD dissertations at the same time, and I am so glad I had you by my side through this process. I also thank my sister for always believing in me, for her friendship, and for being an inspiration throughout my life. Finally, I thank God for opening so many doors for me, for planting the vocation for veterinary medicine (in especial clinical pathology) in my heart, for giving me strength to follow my dreams, and for putting so many wonderful people on my life. I hope I can always be strong enough to keep following my call and pursuing my dreams. I strongly encourage everyone who crosses my path to follow their dreams and seek selfrealization too. vii TABLE OF CONTENTS LIST OF TABLES...................................................................................................... xi LIST  OF  FIGURES………………………………………………..………………………. xii KEY  TO  ABBREVIATIONS…….………………………………………………………… xiv CHAPTER 1. Introduction and literature review…………………………………….… BACKGROUND………………………………………………………………….. Overview of erythropoiesis…………………………………………....…. Canine IMHA…………………………………………………….………… Canine PIMA…………………………………………….………………… Human PIMA counterpart……………………….……………………..… Phosphatidylserine and apoptosis in immune-mediated anemias...… SUMMARY……………………………………………………………………...… HYPOTHESIS AND SPECIFIC  AIMS………………………………………….. Aim  1…...…………………………………………………………………… Aim  2…...…………………………………………………………………… Aim 3…...…………………………………………………………………… Aim  4…...…………………………………………………………………… OUTLINE OF STUDIES……………………………………………………….… Chapter  2…………………………………………………………………… Chapter  3…………………………………………………………………… Chapter  4…………………………………………………………………… Appendix  A……………………………………………………………….… Appendix  B……………………………………………………………….… REFERENCES.............................................................................................. 1 2 2 5 7 11 12 16 18 18 18 19 19 20 20 20 21 22 22 23 CHAPTER 2. Histologic and Cytologic Bone Marrow Findings in Dogs with Ineffective Erythropoiesis and Phagocytosis of Erythroid Precursors – Suspected PrecursorTargeted Immune-Mediated  Anemia………………………........................................ 32 ABSTRACT…………………………………………………………………...…… 33 INTRODUCTION……………………………………………………………..…… 35 MATERIAL AND  METHODS…………………………………………………..… 38 Samples…………………………………………………………………...… 38 Clinical and laboratory  data……………………………………………..… 42 Data  analysis………………...…………………………………………...… 43 RESULTS………………………………………………………………………...… 44 CBC  findings………………………………………………………………… 45 Bone  marrow  findings……………………………………………………… 47 Other  findings………………..……………………………………………… 53 Clinical  outcomes…………………………………………………………… 54 Group  differences………………………………………………………...… 55 DISCUSSION…………………………………………………………………….… 57 REFERENCES............................................................................................... 65 viii CHAPTER 3. Flow Cytometric Assays for Canine Immune-mediated Anemias; Detection of DEA1.1 on Canine Erythroid Precursors.............................................................. 69 ABSTRACT…………………………………………………………..…………… 70 INTRODUCTION……………………………………………………..………...… 71 MATERIAL  AND  METHODS……………………………………….…………… 73 Dogs……..…………………………………………………...…………..… 73 Bone  marrow  collection…...………………………………...……….…… 73 RBC sample  preparation…..………………………………...…………… 74 Flow cytometric  assay  for  RBC  IgG………………………...…………… 74 Clinical  assessment  of  the  RBC  IgG  assay………………...…...……… 77 Fractionation  of  bone  marrow  cells………………………...……...…..… 78 Preparation of bone marrow fractions  for  flow  cytometry…...……...…. 78 Flow cytometric identification  of  bone  marrow  nRBCs……...……….… 79 IgG  detection  on  nRBCs………………………………………...………… 80 Statistical  analysis……………………………………………...………..… 81 RESULTS……………………………………………………………...………...… 82 RBC IgG assay characterization….………………………...………….… 82 Clinical  assessment  of  the  RBC  IgG  assay..……………...………..…… 83 Bone marrow fractions  and  detection  of  nRBCs………...……………… 84 IgG detection  on  nRBCs…………………………………...……………… 88 DISCUSSION……………………………………………………...…………….… 90 REFERENCES............................................................................................... 97 CHAPTER 4. Exploring the Roles of IgG and Phosphatidylserine in Canine Regenerative and Nonregenerative Immune-Mediated Anemias............................. ABSTRACT…………………………………………………………………...…… INTRODUCTION………………………………………………………………..… MATERIAL AND  METHODS………………………………………………..…… Animals….………………………………………………………………..… RBC  samples  and  isolation….………………………………………….… Bone  marrow  samples  and  erythroid  precursor  isolation…..………..… RBC  testing  for  IgG………………………….……………………………... RBC testing  for  PS……………………………………………………….… Erythroid  precursor  testing  for  IgG  and  PS…………..………………..… Statistical analysis………………………………………………………..… RESULTS………………………………………………………………………...… RBC IgG and  PS  in  IMHA……….….…………………………………...… Erythroid precursor IgG and PS in PIMA………..……………………..… Clinical and diagnostic findings in PIMA dogs...………………………… DISCUSSION………………………...………………………………………….… REFERENCES............................................................................................... 101 102 103 106 106 107 108 109 110 113 116 118 118 121 126 128 134 CHAPTER 5. Summary and Conclusions..………………………………………….… SUMMARY OF FINDINGS..………..………………………………………….… LIMITATIONS  AND  UNANSWERED  QUESTIONS….…………….……….… FUTURE DIRECTIONS..……………………………………………………....… 138 139 144 146 ix APPENDICES……………………………..…...……................................................... APPENDIX A. Assessment of Canine  C3….................................................. Complement involvement in immune-mediated  anemias………….… Detection  of  complement  fragment  C3  in  dogs…………………...…… C3-coated  positive  control  cells……………………………………….… Reactivity of anti-[dog C3] antibodies  to  canine  C3b  and/or  C3d…… Conclusions  and  future  directions…………………………………….… Aim 1………….…………………………………………………..… Aim 2………….…………………………………………………..… REFERENCES.................................................................................... APPENDIX B. Development and Characterization of a Phagocytic Assay for Canine Erythroid Cells……………...…………………..................................... Introduction……………………………………………………………...… Mediators  of  phagocytosis…………………………………………….… Erythroid precursor phagocytosis  in  PIMA…………………………..… Erythrocyte phagocytosis  in  IMHA……………………………………… Erythroid cell phagocytosis in apoptotic  conditions…………………… Rationale………………………………………………………………...… Experimental design and  methods……………………………………… Macrophages……………………………………………………..… Phagocytic assay…………………………………………………… Dogs and samples..………………………………………………… Expected outcomes.................................................................... Summary of experiment results and pitfalls encountered.................... Outcome and future directions............................................................. REFERENCES..................................................................................... x 149 150 151 151 155 156 159 160 162 163 167 168 168 170 171 171 172 172 172 173 176 176 176 181 183 LIST OF TABLES Table 1: Features and categories for cytologic and histologic assessment of bone marrow aspirate and core specimens...………………………………………………… 39 Table 2: Hematologic findings in the 25 dogs fulfilling diagnostic criteria for PIMA.. 46 Table 3: Features of the four bone marrow patterns apparent in the 25 dogs with PIMA……………………………………………………………………………………...… 48 Table 4: Selected findings in late-stage PIMA dogs grouped by degree of collagen myelofibrosis…………………………………………………………………………….… 52 Table 5: Number of IMHA and unhealthy non-IMHA dogs with negative and positive RBC IgG results………………………………………………………………………...… 84 Table 6: Relationship between the stage of rubriphagocytosis (RP) reported on bone marrow examination and the Percoll fractions of bone marrow with IgG and/or phosphatidylserine (PS) positivity in PIMA dogs…………………………………….… 126 Table 7: Methods of complement fixation to erythrocytes, and complement fragments expected to be fixed as a result……………………………………………………….… 156 Table 8: C3 detection on canine and human erythrocytes after various treatments for C3b and C3d fixation…………………………………………………………………...… 158 Table 9: C3 detection on canine erythrocytes using the Nordic anti-[dog C3c] antibody.................................................................................................................... 159 Table 10: Percentage of J774 macrophages engulfing DEA1.1-positive erythrocytes after incubation with different dilutions of anti-DEA1.1 typing serum to yield decreasing IgG positivity documented by flow cytometry………………………………………..… 175 Table 11: Phagocytosis of bone marrow-derived nucleated cells after incubation with J774 macrophages for different time periods………………………………………..… 177 Table 12: Percentage of J774 macrophages engulfing IgG-coated erythrocytes after incubation with mixtures of erythrocytes and white blood cells of varying WBC:RBC ratio but constant total number………………………………………………………………… 180 Table 13: Percentage of J774 macrophages engulfing IgG-coated erythrocytes after incubation with mixtures of erythrocytes and white blood cells (WBC) of varying WBC:RBC ratio and fixed RBC number………………………………………………… 181 xi LIST OF FIGURES Figure 1: Cytophagia in aspirates of canine bone marrow……...…………………… 41 Figure 2: Macrophage cytoplasmic fragments containing erythroid precursors..… 42 Figure 3: Four histologic patterns identified in the 25 dogs with PIMA.................... 49 Figure 4: Mild (A), moderate (B), and severe (C) collagen myelofibrosis in dogs with late-stage PIMA........................................................................................................ 50 Figure 5: Effect of RBC agglutinates on IgG detection............................................ 76 Figure 6: Dose-response curve for RBC IgG.......................................................... 83 Figure 7: Canine bone marrow fractions after separation using a Percoll gradient………………………………………………………......................................... 85 Figure 8: Flow cytometric identification of potentially contaminating cell populations based on cell sorting of non-erythroid regions…...................................................... 86 Figure 9: Cytologic findings in sorted samples from erythroid regions of LDS751 versus CD18 dot plots.......................................................................................................... 87 Figure 10: nRBC IgG detection using DEA1.1-positive cells coated with anti-DEA1.1 IgG………..…........................................................................................................... 89 Figure 11: Median fluorescence intensity (MdFI) versus % positive events for assessing IgG positivity…......................................................................................................... 92 Figure 12: Representative dot plots of flow cytometric IgG fluorescence (vertical axis) versus forward scatter (horizontal axis) of red blood cells (RBCs)........................... 110 Figure 13: Representative frequency histograms of flow cytometric phosphatidylserine (PS) fluorescence (horizontal axis) on red blood cells (RBCs)................................. 112 Figure 14: Representative dot plots of flow cytometric IgG fluorescence (vertical axis) versus forward scatter (horizontal axis) of erythroid precursors (nRBCs)................. 114 Figure 15: Representative dot plots of flow cytometric phosphatidylserine (PS) fluorescence (vertical axis) versus forward scatter (horizontal axis) of erythroid precursors (nRBCs)………………………………………………………………………. 116 Figure 16: IgG positivity on RBCs from healthy dogs (n = 20) and dogs with IMHA (n = 11) or without IMHA (non-IMHA, n = 9)..................................................................... 119 xii Figure 17: Phosphatidylserine (PS) positivity on RBCs from healthy dogs (n = 20) and dogs with IMHA (n = 11) or without IMHA (non-IMHA, n = 9)................................... 120 Figure 18: IgG positivity on erythroid precursors from the mid (A) and bottom (B) Percoll fractions of bone marrow from healthy dogs (n = 10) and dogs with PIMA (n = 17 for mid, n = 16 for bottom), without PIMA (non-PIMA, n = 7), or with unclear diagnoses (n = 11)………..……........................................................................................................ 123 Figure 19: PS positivity on erythroid precursors from the mid (A) and bottom (B) Percoll fractions of bone marrow from healthy dogs (n = 5) and dogs with PIMA (n = 5 for mid and bottom), without PIMA (non-PIMA, n = 2 for mid, n = 1 for bottom), or with unclear diagnoses (n = 2)...................................................................................................... 124 Figure 20: IgG (A) and PS (B) positivity on RBCs from healthy dogs (n = 20) and dogs with PIMA (n = 14 for IgG, n = 7 for PS), without PIMA (non-PIMA, n = 6 for IgG, n = 2 for PS), or with unclear diagnoses (n = 9 for IgG, n = 4 for PS)……......................... 125 Figure 21: Representative images of phagocytic assay using murine J774 macrophages adherent to Millicell slides......................................................................................... 174 Figure 22: Number of phagocytized bone marrow-derived nucleated cells from the mid and bottom Percoll fractions of a dog after IgG coating and incubation with J774 macrophages for different time intervals................................................................... 178 xiii KEY TO ABBREVIATIONS AIHA: Autoimmune hemolytic anemia AIMF: Autoimmune myelofibrosis BFU-E: Burst-forming units-erythroid BSA: Bovine serum albumin BzATP:  2′(3′)-O-(4-Benzoylbenzoyl)adenosine  5′-triphosphate triethylammonium C3: Third component of complement C4: Fourth component of complement Ca2+: Calcium CBC: Complete blood count CD: Cluster of differentiation CFU-E: Colony-forming units-erythroid CR1: Complement receptor 1 CR2: Complement receptor 2 CV: Coefficient of variation DAT: Direct antiglobulin test DCPAH: Diagnostic Center for Population and Animal Health DEA: Dog erythrocyte antigen EDTA: Ethylenediaminetetraacetic acid EGTA: Ethylene glycol tetraacetic acid ELISA: Enzyme-linked immunosorbent assay EPO: Erythropoietin EPOR: EPO receptor xiv FDP: Fibrin degradation products FITC: Fluorescein isothiocyanate FSC: Forward scatter GSH: Reduced glutathione Hct: Hematocrit IFN: Interferon IMHA: Immune-mediated hemolytic anemia IMN: Immune-mediated neutropenia IMT: Immune-mediated thrombocytopenia M:E: Myeloid to erythroid ratio MAC: Membrane attack complex MCV: Median corpuscular volume MDA: Malondialdehyde MdFI: Median fluorescence intensity MF: Myelofibrosis MFI: Mean fluorescence intensity mRNA: Messenger ribonucleic acid MSU VMC: Michigan State University Veterinary Medical Center NIMA: Nonregenerative immune-mediated anemia nRBCs: Nucleated red blood cells NRIMHA: Nonregenerative IMHA PBS: Phosphate-buffered saline PE: Phycoerythrin PIMA: Precursor-targeted immune-mediated anemia xv PRCA: Pure red cell aplasia PS: Phosphatidylserine RBCs: Red blood cells RI: Reference interval ROS: Reactive oxygen species RP: Rubriphagocytosis SCF: Stem cell factor SD: Standard deviation SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SSC: Side scatter TGF-β: Tumor growth factor β TNF-R: TNF receptor TNF: Tumor necrosis factor WBC: White blood cells WRI: Within reference interval xvi CHAPTER 1 Introduction and Literature Review 1 BACKGROUND Overview of erythropoiesis In normal erythropoiesis, stem cells commit to the erythroid lineage, developing into erythroid progenitors that are represented by burst-forming units-erythroid (BFU-E) and colony-forming units-erythroid (CFU-E), and subsequently into morphologically recognizable erythroid precursors from rubriblasts through prorubricytes, basophilic rubricytes, polychromatophilic rubricytes, and metarubricytes. After this stage, erythroid cells extrude their nucleus, leave the bone marrow, and are called reticulocytes, which then mature into red blood cells (RBCs) in the circulation. During this process, erythroid maturation is pyramidal, represented by fewer of the more immature forms, and progressively higher numbers of the more mature stages. Several cytokines and growth factors are involved in erythropoiesis, with the primary proerythropoietic stimulus coming from erythropoietin (EPO), a hormone produced mainly in the kidneys. EPO interacts with the EPO receptor (EPOR) on the surface of erythroid precursors, stimulating their survival and differentiation. EPOR is expressed in stem cells and progenitor cells, and decreases gradually as erythroid maturation progresses (Olver 2010). Hypoxia can up-regulate EPO up to 1,000 fold, stimulating increased erythropoiesis (Olver 2010). Other proerythropoietic factors include: 1) IL-3, which acts on stem cells and progenitors to induce proliferation and selfrenewal, and primes progenitor cells to more vigorously respond to EPO, and 2) stem cell factor (SCF, or c-kit ligand), which enhances the activity of EPO towards proliferation and differentiation of erythroid progenitors and precursors. During erythroid differentiation, several molecules including transcription factors, growth factor receptors, and proteins that are important for erythroid cell structure and/or 2 function are expressed on the cell membrane (Testa 2004; Chen et al., 2009). Some of these markers are expressed in early but not late stage erythroid precursors, or vice versa, while others are expressed in all stages throughout development and including mature erythrocytes (Chen et al., 2009; Okumura et al., 1992; Spivak 2005). Some of these membrane proteins are used to characterize canine blood groups, and have been described on mature erythrocytes from dogs. Eight major dog erythrocyte antigen (DEA) types have been recognized, and typing sera have been produced and applied in blood typing in order to minimize transfusion reactions following blood transfusions (Hale 1995). DEA1.1 is the most clinically relevant alloantigen because of its potential to cause acute hemolytic reactions after incompatible blood transfusions (24, 25), and a frequency of approximately 50% in the canine population (Swisher et al. 1961; Hale 1995; Kessler et al. 2010; Lucidi et al. 2011). Macrophages play a critical role in erythropoiesis and are part of the erythropoietic niche. They form erythroid islands throughout the marrow, in which macrophages are located in the center and are surrounded by erythroid precursors. The roles of the macrophage involve development of erythroid cells, transfer of iron to developing erythroid precursors to synthesize hemoglobin (Chasis et al., 2008), and removal of apoptotic hematopoietic cells or cell nuclei (Testa 2004; Angelucci et al., 2002; Yoshida et al., 2005; Chasis et al., 2008). They also have a role in clearance of senescent RBCs from the circulation. Erythropoiesis and apoptotic signals are closely related. Phosphatidylserine (PS), a phospholipid present in cell membranes, is normally restricted to the inner leaflet of the plasma membrane (Balasubramanian et al., 2003). This asymmetric assembly is promoted by two enzymes: aminophospholipid translocase, which specifically transports 3 PS from the outer to the inner leaflet of the plasma membrane in an ATP-dependent manner, and scramblase, which randomly translocates lipids across the lipid bilayer in a Ca2+-dependent but ATP-independent manner. Extruded nuclei from metarubricytes rapidly expose phosphatidylserine on their surface, promoting phagocytosis by adjacent macrophages (Yoshida et al., 2005; Chasis et al., 2008). Apoptosis is involved in the maintenance of normal homeostasis of the erythropoietic system. In vitro studies suggest that in health, a small number of erythroid precursors die prematurely in the bone marrow before reaching full development (Samson et al., 1981). Death is due to apoptotic changes, and involves ligands and cell death receptors of the tumor necrosis factor (TNF) and TNF receptor (TNF-R) superfamilies, respectively (de Maria et al., 1999; Testa 2004). These molecules are down-regulated in response to EPO, allowing for increased erythropoiesis. On the other hand, these molecules are increased and provide the main mechanism for control of erythropoiesis when EPO production is low. One other proposed mechanism for regulation of erythropoiesis is that more mature precursors apply a Fas-mediated cytotoxic effect on more immature precursors within erythroid islands, in a negativefeedback fashion (de Maria et al., 1999). Apoptotic erythroid precursors are rapidly engulfed by adjacent macrophages (Angelucci et al., 2002). The average life span for canine erythrocytes is approximately 100 days (Garon et al., 2010), and through erythropoiesis, millions of erythrocytes are produced daily in order to maintain enough mature erythrocytes to provide adequate oxygenation of body tissues. Processes leading to anemia may involve increased erythrocyte loss, increased erythrocyte destruction, or decreased production in the bone marrow. These studies will 4 focus on two types of anemia in dogs, involving primarily increased erythrocyte destruction and decreased bone marrow production. Canine IMHA In immune-mediated hemolytic anemia (IMHA), a common and well characterized hematologic disorder in many breeds of dogs, immunoglobulins or complement become associated with the surface of RBCs and mediate erythrocyte destruction through a type II hypersensitivity immune reaction. The most common types of immunoglobulins involved are IgG and IgM, but IgA can also be involved in a minority of cases (Barker et al., 1992; Slappendel 1979; McCullough 2003). IgG molecules are monomeric (i.e., they possess only two antigen binding sites) and mediate RBC destruction mostly through phagocytosis (extravascular hemolysis) resulting from Fc receptors on macrophages binding to the Fc portion of the RBC-associated IgG molecules. Some immunoglobulins that bind to RBCs promote fixation of C1, with subsequent generation of C3 on the cell membrane. This can lead to activation of other complement components (C5, C6, C7, C8, C9) and generation of a membrane attack complex that forms transmembrane channels in the RBC membrane, allowing entry of water and electrolytes, and ultimately promoting intravascular hemolysis. Complement can also mediate RBC phagocytosis through complement receptors on macrophages, which bind C3. Because of their pentameric structure, IgM molecules are more efficient than IgG in activating and fixing complement, as well as in promoting RBC agglutination. Although rarely documented, if antibodies are directed against RBC autoantigens and there is no underlying cause for a secondary immune response, the condition is considered idiopathic and known as autoimmune hemolytic anemia (AIHA). The 5 mechanisms that cause primary IMHA (i.e., AIHA) are unknown, but are likely to involve a failure in natural regulation of autoreactive lymphocytes (Day 2010). When IMHA occurs in association with neoplasia, infections, or drug administration, it is generally regarded as secondary. In these cases, antibodies may attach to RBC membranes not through normal RBC epitopes but via surface organisms, altered RBC membrane proteins, or foreign proteins. Immunoglobulins may also bind to RBCs by recognition of alloantigens, such as in blood transfusion reactions or neonatal isoerythrolysis. And in some species, C3-bearing immune complexes can bind to complement receptor on the RBC membrane and cause removal of the RBC (and therefore of the immunecomplexes) by a bystander effect. This is not documented in the dog. Canine IMHA most frequently occurs as a single hematologic disease; however, it may also occur concurrently with other immune-mediated disorders such as immunemediated thrombocytopenia (IMT), or be part of a systemic immune-mediated condition such as systemic lupus erythematosus (Grindem et al., 1983; Jackson et al., 1985; Klag et al., 1993). In IMHA, a regenerative bone marrow response and reticulocytosis are expected within 3-5 days of the onset of anemia, restoring the hematocrit by increased erythropoiesis if hemolysis is controlled by treatment. A regenerative anemia is typically considered to be IMHA when present with one or more of the following: 1) positivity for RBC surface-associated immunoglobulin or C3, 2) RBC agglutination, and 3) spherocytosis (Klag et al., 1993; Kellerman et al., 1995; Wilkerson et al., 2000; Quigley et al., 2001; McCullough 2003; Kucinskiene et al., 2005; Wardrop 2005). In addition, plasma may be hemolyzed and hemoglobinuria may be present with overwhelming intravascular hemolysis, and hyperbilirubinemia and ghost cells may occur. 6 Several immunoassays have been used for detection of canine RBC-associated immunoglobulins  and  C3,  including  Coombs’  (Overman  et al., 2007; Piek et al., 2012; Warman et al., 2008), gel column (Caviezel et al., 2014; Piek et al., 2012), and flow cytometry tests (Wilkerson et al., 2000; Quigley et al., 2001; Kucinskiene et al., 2005). Of these, flow cytometry has been shown to be the most sensitive method for detection of RBC bound antibodies in both canine (Wilkerson et al., 2000; Quigley et al., 2001; Kucinskiene et al., 2005) and human samples (Wang et al., 2001; Chaudhary et al., 2006; Lin et al., 2009). Despite progress in understanding the disease (McCullough 2003; Day 1999; Tan et al., 2012), relapses (Piek et al., 2008), complications, and mortality are still problematic (Swan et al., 2013), and often associated with steroid therapy or thromboembolic events (Carr et al., 2002; Klein et al., 1989; Reimer et al., 1999; Swan et al., 2013; Piek et al., 2008). Although there is a clear association between IMHA and a prothrombotic state (Fenty et al., 2011), the cause of thromboembolism in dogs with IMHA has not yet been clearly determined (Kidd et al., 2013). Canine PIMA In contrast to the classical regenerative presentation of IMHA, as many as 33-58% of dogs reported to have evidence of immune-mediated RBC destruction lack an appropriate regenerative bone marrow response at the time of presentation, in a condition frequently called nonregenerative IMHA (NRIMHA) (Klag et al., 1993; Kellerman et al., 1995; Burgess et al., 1997; Jonas et al., 1987). Although many of these dogs may have been assessed before stimulated erythropoiesis led to reticulocytosis, i.e., in a preregenerative state (Klag et al., 1993; Kellerman et al., 1995; Burgess et al., 7 1997), few have had persistent anemia accompanied by impaired erythropoiesis (Klag et al., 1993; Jonas et al., 1987). Bone marrow assessment of these dogs has usually revealed erythroid hypercellularity, indicating ineffective erythropoiesis, or less commonly erythroid hypocellularity, interpreted as pure red cell aplasia (PRCA) (Jonas et al., 1987; Stockham et al., 1980; Weiss et al 1982; Holloway et al., 1990; Scott-Moncrieff et al., 1995; McManus 2000; Stokol et al., 2000; Weiss 2002; Weiss 2008). An additional group of dogs with persistent hyporegenerative anemia and similar bone marrow findings have no evidence of hemolysis but have a similar clinical course, and have also been considered to have NRIMHA or nonregenerative immune-mediated anemia (NIMA) (Weiss et al 1982; Scott-Moncrieff et al., 1995; McManus 2000; Stokol et al., 2000; Weiss 2002). Because these dogs are suspected to have immune-mediated targeting of their erythroid precursors, the condition will be referred to henceforth as precursortargeted immune-mediated anemia (PIMA). Phagocytosis of erythroid precursors, which we refer to as rubriphagocytosis, has been reported sporadically in dogs with suspected PIMA (Jonas et al., 1987; Stockham et al., 1980; Holloway et al., 1990; McManus 2000), but one investigator (McManus 2000) found that many of 44 dogs with NRIMHA had increased marrow macrophages, rubriphagocytosis, and macrophages containing basophilic granules consistent with degraded cellular material. These findings were rare in marrow from dogs with traditional IMHA or hemorrhagic anemia. There is no study to date showing how rubriphagocytosis and the stage at which it occurs during erythroid development may be associated with bone marrow abnormalities and expected clinical outcome in PIMA. 8 An additional bone marrow abnormality seen in some dogs with suspected PIMA is myelofibrosis. Reticulin myelofibrosis was reported in 16 of 16 tested dogs with PIMA (Stokol et al., 2000), and has been associated with autoimmune disorders in humans in a condition recently characterized as autoimmune myelofibrosis (Vergara-Lluri et al., 2014). A relationship between PIMA and myelofibrosis could also be related to an immune-mediated mechanism, and supports the concept of autoimmune-associated myelofibrosis that has been suggested in dogs (Villiers et al., 1999; Reagan et al., 1992). Alternatively, myelofibrosis could be secondary to a state of chronic inflammation in the bone marrow of PIMA dogs, possibly accompanied by an increase in fibrotic signaling and tumor growth factor β (TGF-β) (Ueha et al., 2012). Other mechanisms for myelofibrosis in anemia that have been considered include: 1) persistent increases in EPO from high pharmacologic doses of recombinant human erythropoietin (Bader et al., 1992) or in dogs with inherited pyruvate kinase deficiency (Prasse et al. 1975; Searcy et al., 1979), 2) hemosiderin accumulation (Hoff et al., 1991), and 3) increased numbers of dysplastic bone marrow megakaryocytes as in people with primary myelofibrosis (Thiele et al., 2009), but there is no strong evidence to indicate these mechanisms could be the cause of myelofibrosis in PIMA. Current lack of systematic assessment of core biopsies of dogs with PIMA limits understanding of how myelofibrosis may relate to this condition. Over 200 dogs with some form of suspected PIMA have been described (Klag et al., 1993; Kellerman et al., 1995; Burgess et al., 1997; Jonas et al., 1987; Stockham et al., 1980; Weiss et al., 1982; Holloway et al., 1990; Scott-Moncrieff et al., 1995; McManus 2000; Stokol et al., 2000; Weiss 2002; Weiss 2008; Weiss 1986). Suspicion of an immune-mediated pathogenesis (Klag et al., 1993; Kellerman et al., 1995; Burgess et al., 1997; Jonas et al., 1987; Stockham et al., 1980; Holloway et al., 1990; Stokol et al., 9 2000; Weiss 2008) is reasonable but unproven, and is based on concurrent evidence of IMHA in many dogs, some favorable responses to immunosuppressive therapy, apparent relapses after withdrawal of steroids, rubriphagocytosis, which may reflect immune-targeting of erythroid precursors, and evidence that some dogs with PRCA have a circulating inhibitor to erythropoiesis that is in the IgG-containing fraction of plasma (Weiss 1986). In the most complete clinical study published to date, 43 dogs with PRCA or NRIMA were described, including a wide range of breeds and ages with a median age of 6.5 years and over-representation by spayed females and Labrador retrievers (Stokol et al., 2000). Evidence of concurrent IMHA was frequent: 20 of 35 tested animals had Coombs’  test  positivity,  23  of  43  had  many  spherocytes,  and  2  of  43  dogs  had  RBC agglutination. Treatment with a variety of immunosuppressive protocols was associated with complete, partial, and poor response in 55%, 18%, and 27% of the dogs, respectively. Time to initial response was 1-10 weeks, and six dogs (21%) relapsed after tapering or stopping treatment. The mortality rate was 28%. Others have reported variable and slow treatment responses with relapses (Jonas et al., 1987; Stockham et al., 1980; Scott-Moncrieff et al., 1995) and mortality rates up to 44% (Weiss 2008). Therapeutic protocols commonly used for regenerative IMHA seem to be less successful in dogs with PIMA. Despite suspicion and evidence for erythroid lineage immune targeting in PIMA, lack of response in many dogs, slow responses for animals that improve with treatment, and absence of evidence of concurrent IMHA in many PIMA dogs suggest that there is more to learn about the pathogenesis and treatment of this condition. 10 Human PIMA counterpart Some human cases of PRCA and reticulocytopenic AIHA resemble cases of canine PIMA, with or without evidence of mature RBC targeting (Meyer et al., 1978; Hedge et al., 1977; Conley et al., 1982; van de Loosdrecht et al., 2000; Jacobs et al., 1985; Crosby et al., 1956; Wranne et al., 1970; Liesveld et al., 1987; Cazzola et al., 1983; Keefer et al., 1988). Although rarely reported, plasma and RBC eluates of some patients with reticulocytopenic AIHA contain IgG reactive with erythrocytes and erythroid precursors (Mangan et al., 1984; Meyer et al., 1978), sometimes via distinctly different antibodies (Mangan et al., 1984). Such patient antibodies have been associated with inhibition of in vitro erythropoiesis (Meyer et al., 1978; Mangan et al., 1984), and either bone marrow erythroid hyperplasia (ineffective erythropoiesis) (Conley et al., 1982; Cazzola et al., 1983) or erythroid hypoplasia in vivo (Mangan et al., 1984). Similarly, many patients with acquired idiopathic PRCA have plasma with demonstrable stageselective inhibition of erythropoiesis prior to the BFU-E, between the BFU- E and CFUE, or after the CFU-E (Charles et al., 1996). In one isolated case of reticulocytopenic AIHA associated with thymoma, anemia was attributed not only to an antibody-mediated mechanism, but also to T-cell-mediated in vitro suppression of erythropoiesis (Taniguchi et al., 1988). Direct detection of IgG on the surface of erythroid precursors has been shown in one person with reticulocytopenic AIHA (van de Loosdrecht et al., 2000), and phagocytosis of erythroid precursors has been rarely reported in people with reticulocytopenic AIHA (Keefer et al., 1988). Although infrequently reported, human reticulocytopenic AIHA seems to resemble PIMA clinically, since multiple drug protocols are often required, splenectomy is sometimes necessary, responses are typically slow, relapses are not uncommon, and there is substantial morbidity associated with 11 prolonged therapy (Charles et al., 1996; Dessypris et al., 2003; Kwong et al., 1996; Beard et al., 1978). Phosphatidylserine and apoptosis in immune-mediated anemias Apoptosis of nucleated cells is typically characterized by cell shrinkage, chromatin condensation, nuclear fragmentation, mitochondrial membrane permeabilization, cell membrane blebbing, and breakdown of PS asymmetry of the plasma membrane (Ziegler et al., 2004). Cells exposing PS at the cell surface are recognized by macrophages, which are equipped with receptors specific for PS (Fadok et al., 2000) and rapidly engulf and degrade the affected cells. It has been shown that, despite not having a nucleus, erythrocytes can undergo a similar process, eryptosis, which is characterized by cell shrinkage, cell membrane scrambling, exposure of PS, and consequently triggering of phagocytosis by macrophages (Lang et al., 2015). This process is stimulated by increased Ca2+ entry into the cell, and seems to be one of the mechanisms involved in programmed RBC death, therefore participating in erythrocyte turnover (Lang et al., 2005; Lang et al., 2012; Kuypers et al., 2004; Bosman et al., 2005; Garratty 2008). Examples of conditions that may promote eryptosis are hyperosmolarity, oxidative stress, energy depletion, and hyperthermia (Lang et al., 2005; Lang et al., 2012; Kuypers et al., 2004). Oxidative stress of a severity much less than that required to cause morphologic changes indicative of oxidative damage to RBCs (eccentrocytes, Heinz bodies) has been associated with decreased RBC survival, with or without involvement of antibodies. In people with thalassemia, oxidative damage was shown to cause increased PS exposure on RBCs, contributing to increased erythrophagocytosis and development of 12 anemia (Kuypers et al., 1998; Amer et al., 2008). In vitro treatment of thalassemic RBCs with an antioxidant compound reduced their sensitivity to hemolysis and phagocytosis by macrophages (Amer et al., 2008). In mice, drug-induced oxidative stress has triggered erythrophagocytosis and anemia. Interestingly, in this study, anemia was accompanied by spherocytosis (Noh et al., 2010), suggesting that increased oxidative stress may contribute to spherocyte formation. Also in mice, decreased antioxidant capacity has been related to the appearance of RBCs with increased membrane IgG and increased sensitivity to lysis (Lee et al., 2004). The investigators proposed that a chronic increase in oxidative stress sensitized RBCs with IgG and promoted IMHA (Lee et al., 2004). In contrast, the binding of anti-A IgG to human A antigen (of the ABO group) on human RBCs in vitro induced Ca2+ entry and subsequent PS exposure, suggesting that eryptosis may contribute to RBC removal following an immune reaction against the A antigen (Attanasio et al., 2007), and creating controversy as to which event (immunoglobulin binding or increased oxidative stress/PS exposure) happens first. To date, the relationship between increased oxidative stress, PS exposure, and IgG binding to RBCs is not altogether clear. Oxidative stress and apoptotic-like mechanisms have been suggested to play a role in canine IMHA because of the demonstration of plasma antibodies against two distinct RBC proteins in IMHA dogs: Prx2, which is involved with the antioxidative metabolism, and calpain, which plays a role in apoptotic pathways (Tan et al., 2012). It has been proposed that oxidative stress or binding of autoreactive antibodies could lead to eryptosis in IMHA (Tan et al., 2012). In support of this idea is the finding of increased plasma malondialdehyde (MDA) concentrations, a marker of oxidative stress, in IMHA dogs when compared to healthy dogs (Pesillo et al., 2002). Currently, there is no clearly 13 established relationship between oxidative stress, PS exposure, IgG binding, and increased RBC destruction in immune-mediated anemias in humans or dogs. Phosphatidylserine exposure may be involved not only with accelerated destruction of RBCs, but also with destruction of erythroid precursors in hyporegenerative immunemediated anemias. This has been evidenced by a person with reticulocytopenic AIHA and positivity for IgG and PS on bone marrow erythroid nucleated cells (van de Loosdrecht et al., 2000). Despite PS positivity, erythroid cells with morphologic abnormalities characteristic of apoptosis were not observed in bone marrow cytologic evaluations of this patient. Apoptosis ultimately results in prominent cellular morphologic changes, but early apoptotic events can trigger PS exposure and may mediate macrophage phagocytosis before the cell appearance is affected (Martin et al., 1995). Therefore, apoptosis cannot be excluded as a potential contributor to ineffective erythropoiesis in dogs with PIMA. Increased PS exposure in eryptotic RBCs has been reported to cause excessive erythrocyte adhesion to the vascular endothelium, potentially promoting microvascular occlusion and contributing to thrombosis (Borst et al., 2012). The presence of PS in the outer leaflet of the plasma membrane of RBCs from beta-thalassemia patients has been suggested to account for the chronic hypercoagulable state, frequent thrombotic events, and platelet activation that are seen in these patients (Borenstain-Ben Yashar et al., 1993; Musallam et al. 2012). A prothrombotic state and development of thromboembolic disease are frequent complications in dogs with IMHA, and are associated with poor prognosis and increased mortality (Carr et al., 2002; Klein et al., 1989; Fenty et al., 2011; Swan et al., 2013; Piek et al., 2008). The pathogenesis of this prothrombotic state in dogs with IMHA has not yet been determined. Thrombosis has been reported in at 14 least two PIMA dogs (Jonas et al., 1987; Stokol et al., 2000) and deserves further evaluation. There are no studies to date showing involvement of increased PS exposure on canine RBCs or erythroid precursors in IMHA or PIMA. However, there is enough information to suggest that cell membrane scrambling and eryptotic mechanisms may contribute to canine immune-mediated anemias in an immunologic (secondary to immunoglobulin binding) or non-immunologic (independent of immunoglobulin binding) fashion. Phosphatidylserine exposure may contribute to anemia and/or ineffective erythropoiesis and rubriphagocytosis in dogs, and it may also play a role in thrombotic events in IMHA, and perhaps in PIMA. Therefore, assessment of PS exposure in dogs with immune-mediated anemias is warranted. 15 SUMMARY Immune-mediated anemia is one of the most common causes of anemia in dogs, with IMHA being the most well studied type. IMHA is characterized by evidence of immune-mediated targeting and destruction of mature RBCs, most specifically spherocytosis, RBC agglutination, ghost cells, and/or  Coombs’  test  positivity,  and  it  is   usually accompanied by erythroid regeneration. The underlying cause of IMHA remains unknown, and mortality remains a problem, particularly for dogs that develop thromboembolic disease. In a different condition, dogs with nonregenerative anemia have ineffective erythropoiesis that has occasionally been associated with phagocytosis of erythroid precursors (rubriphagocytosis) or myelofibrosis. Based on some responses to immunosuppressive therapy, relapses after therapy withdrawal, and, in some cases, evidence of concurrent IMHA, these dogs have been considered to have immunemediated nonregenerative anemia, or PIMA. The pathogenesis of PIMA has not yet been determined, but roles for immunoglobulin and/or complement have been suspected. Evidence of involvement of apoptosis-like mechanisms with phosphatidylserine (PS) exposure in immune-mediated anemias of people and dogs also warrants further studies about the role of PS in PIMA and IMHA. Our experience indicates that the diagnosis of PIMA has historically been missed, or only weakly suggested, because patterns of PIMA have not been recognized, rubriphagocytosis has gone undetected, and awareness of PIMA without hemolysis has not been widespread. We hypothesize that PIMA is part of a spectrum of immune-mediated anemias, including IMHA, in which IgG, with or without the contribution of PS, targets and promotes phagocytosis of different stages of erythroid cells, simultaneously or 16 independently. Targeting of epitopes expressed only on erythroid precursors would result in PIMA with ineffective erythropoiesis and without evidence of IMHA, and targeting of epitopes present only on mature RBCs would yield classical IMHA with a typical regenerative presentation. Targeting of epitopes shared by erythroid precursors and mature RBCs would yield concurrent hemolysis (IMHA) and ineffective erythropoiesis (PIMA). In support of this hypothesis is knowledge that 1) erythroid precursors and their mature counterparts express shared and independent cell surface markers (Okumura et al., 1992; Chen et al., 2009; Spivak 2005), 2) antiplatelet antibodies often impair thrombopoiesis and mediate platelet destruction in IMT (Olsson et al., 2005), 3) there is evidence of antibody-mediated reticulocytopenic AIHA associated with erythroid maturation arrest in human patients (Conley et al., 1982; Cazzola et al., 1983; Keefer et al., 1988; Crosby et al., 1956; Wranne et al., 1970; Hedge et al., 1977; Hauke et al., 1983; Jacobs et al., 1985; Liesveld et al., 1987), and 4) increased susceptibility of RBCs and erythroid precursors to phagocytosis has been documented in people with AIHA and thalassemia (Conley et al., 1982; Gallagher et al., 1983; Herron et al., 1986). The research described in this dissertation represents a step toward better characterizing the pathogenesis of PIMA and its association with IMHA, with the ultimate goals of establishing diagnostic criteria, identifying appropriate therapeutic strategies based on knowledge of the pathogenesis of the diseases, and raising veterinary awareness of PIMA. 17 HYPOTHESIS AND SPECIFIC AIMS These studies represent the first steps towards exploring the mechanism behind PIMA. The central hypothesis is that PIMA is part of a spectrum of immune-mediated anemias including IMHA, in which IgG, with or without the contribution of phosphatidylserine (PS), target and promote phagocytosis of different stages of erythroid cells, simultaneously or independently. The rationale is that clarification of the mechanism of PIMA will lead to development of appropriate therapeutic strategies and appropriate diagnostic assays. The specific aims are as follows: Aim 1. Retrospectively characterize clinical and laboratory findings in dogs with rubriphagocytosis and PIMA, with emphasis on bone marrow cytologic and histologic findings in order to better characterize the condition and facilitate its diagnosis (Chapter 2). Aim 2. Determine if erythroid populations in dogs with PIMA have increased surface IgG, which could promote phagocytosis (Chapter 3 and Chapter 4). Hypothesis: Erythroid precursors from dogs with PIMA have increased membrane IgG compared to those from healthy and unhealthy non-PIMA dogs (sick dogs without evidence of immune-mediated anemia). Aim 2 initially included additional assessment of C3 on erythroid precursors of PIMA dogs, but that was not possible. Details about this topic are discussed in Appendix A. 18 Aim 3. Determine if phosphatidylserine (PS) plays a role in dogs with immunemediated anemias (Chapter 4). 3.1. Determine if erythroid populations in dogs with PIMA have increased PS exposure. Hypothesis: Erythroid precursors from dogs with PIMA have increased PS exposure compared to those from healthy and unhealthy non-PIMA dogs. 3.2. Determine if circulating erythrocytes of dogs with IMHA have increased PS exposure. Hypothesis: PS exposure is increased on erythrocytes of dogs with IMHA compared to those from healthy and unhealthy non-IMHA dogs (sick dogs without evidence of immune-mediated anemia). Aim 4. Determine if erythroid precursors in dogs with PIMA have increased susceptibility to phagocytosis (Appendix B). Hypothesis: Erythroid precursors from dogs with PIMA undergo increased phagocytosis compared to those from healthy and unhealthy non-PIMA dogs. 19 OUTLINE OF STUDIES My studies are presented in three chapters followed by a conclusion that discusses a summary of findings, limitations and future directions, and two appendices. The goals and brief descriptions of chapters 2 through 4, and appendices A and B, follow: Chapter 2 Chapter 2 describes a retrospective study of dogs with PIMA that were assessed at the MSU VMC. The main goal of this chapter was to help characterize canine PIMA, with particular focus on bone marrow findings, in order to better understand the condition, facilitate its recognition and diagnosis, and ultimately lead to better therapeutic management and clinical outcomes. Summary of study: All dogs with paired bone marrow aspirate and core biopsy samples collected at the MSU VMC from January 2006 through December 2009 were reviewed, and PIMA dogs were identified. Laboratory and clinical data were collected, and response to treatment was assessed when available. Three bone marrow patterns were identified in PIMA dogs, depending on the stage of erythroid precursor being phagocytized, and a fourth pattern consisted of dogs with severe myelofibrosis. Response to immunosuppressive therapy took a median time of one month, and approximately half the dogs did not respond to treatment. Chapter 3 Chapter 3 describes the development of flow cytometric assays for RBC and erythroid precursor IgG, including a Percoll gradient separation for erythroid populations. 20 The main goal of this study was to develop methods that would be used for isolation of erythroid precursors and their assessment for IgG and PS. Summary of study: An assay for RBC IgG was developed, characterized, and applied to IMHA, non-IMHA, and healthy dogs. This assay was adapted for assessment of erythroid precursors. A Percoll gradient separation method was developed for separation of different stages of erythroid precursors from other bone marrow cells, and a 3-color flow assay was developed and characterized to be used for IgG detection on erythroid precursors. Bone marrow cells from DEA1.1 negative and positive dogs were incubated with anti-DEA1.1 typing serum in order to prove our capacity to detect IgG on the surface of erythroid precursors. An additional finding of this study was the expression of DEA1.1 on canine erythroid precursors from rubriblasts through metarubricytes. Chapter 4 Chapter 4 involves testing of IMHA and PIMA dogs for IgG and PS on erythroid cells. The main goal of this study was to test our hypotheses that IgG and PS play a role in immune-mediated anemias in dogs. Summary of study: IMHA, non-IMHA, and healthy dogs were tested for IgG and PS on RBCs, and results showed significantly higher IgG and PS in IMHA dogs when compared to other groups. A subset of PIMA dogs had increased IgG-positive erythroid precursors when compared to healthy and non-IMHA dogs. Erythroid precursors from most tested PIMA dogs had more PS-positive erythroid precursors than healthy dogs, however no sick dogs without PIMA were tested for comparison. A limitation of this study, especially in regard to PIMA dogs, is the low number of animals that were tested. 21 Appendix A This appendix discusses interesting problems found while trying to develop an assay to assess erythroid cells for canine complement (C3) and IgM. Limitations regarding commercially available reagents and development of a positive control hindered addressing involvement of these mediators as part of Aim 2. Appendix B This appendix describes the phagocytic assay developed with the goal of assessing Aim 4, and discusses limitations found with the method and why it ultimately was not used. 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News Physiol Sci. 2004;19:124-128. 31 CHAPTER 2 Histologic and Cytologic Bone Marrow Findings in Dogs with Ineffective Erythropoiesis and Phagocytosis of Erythroid Precursors – Suspected PrecursorTargeted Immune-Mediated Anemia 32 ABSTRACT Background: Precursor-targeted immune-mediated anemia (PIMA) has been suspected in dogs with nonregenerative anemia and bone marrow findings varying from erythroid hyperplasia to pure red cell aplasia. Phagocytosis of erythroid precursors (rubriphagocytosis) reported in some affected dogs suggests a destructive component to the pathogenesis of PIMA. Objectives: To characterize laboratory and clinical findings in dogs with suspected PIMA and rubriphagocytosis, with emphasis on bone marrow cytologic and histologic findings. Methods: Dogs with PIMA and rubriphagocytosis were identified by review of paired bone marrow aspirate and core biopsy slides collected over a 4-year period. Samples were systematically assessed and characterized along with other pertinent laboratory data and clinical findings. Results: Twenty-five dogs met criteria for PIMA and had rubriphagocytosis that was relatively stage-selective. Erythropoiesis was expanded to the stage of erythroid precursor undergoing most prominent phagocytosis, yielding patterns characterized by a hypo-, normo-, or hypercellular erythroid lineage. A fourth pattern involved severe collagen myelofibrosis, and there was a spectrum of mild to severe collagen myelofibrosis overall. Evidence of immune-mediated hemolysis was rare. Immunosuppressive therapy was associated with remission in 77% of dogs known to be treated for at least the median response time of 2 months. Conclusions: Bone marrow patterns in dogs fulfilling criteria for PIMA were aligned with stage-selective phagocytosis of erythroid precursors and the development of collagen myelofibrosis, a common feature in dogs with PIMA. Recognition of these patterns and 33 detection of rubriphagocytosis facilitates diagnosis of PIMA, and slow response to immunosuppressive therapy warrants further investigation into its pathogenesis. 34 INTRODUCTION Since 1980, nearly 270 dogs have been described with nonregenerative anemia suspected to be caused by an immune-mediated mechanism (Weiss et al., 1982; ScottMoncrieff et al., 1995; McManus 2000; Stokol et al., 2000; Weiss 2002; Jonas et al., 1987: Klag et al., 1993; Kellerman et al., 1995; Burgess et al., 1997; Weiss 2008; Holloway et al., 1990; Weiss 1986; Stockham et al., 1980; Reagan et al., 1992; Villiers et al., 1999), and commonly suggested to involve targeting of erythroid precursors (Klag et al., 1993; Kellerman et al., 1995; Burgess et al., 1997; Jonas et al., 1987; Stockham et al., 1980; Holloway et al., 1990; McManus 2000; Stokol et al., 2000; Weiss 2008; Villiers et al. 1999). Approximately 170 of these dogs had evidence of concurrent erythrocyte  attack,  some  with  a  positive  Coombs’  test,  suggesting  a  relationship  to   immune-mediated hemolytic anemia (IMHA) and leading to use of the term nonregenerative IMHA (NRIMHA) whether hemolysis was detected or not (Klag et al., 1993; Kellerman et al., 1995; Burgess et al., 1987; Jonas et al., 1987; Stokol et al., 2000; Weiss 2008; Scott-Moncrieff et al., 1995; Weiss 1986; Reagan et al., 1992; Villiers et al., 1999). Others have considered dogs with similar findings to have nonregenerative immune-mediated anemia (NRIMA, NIMA), a more general term consistent with the lack of hemolysis in some cases (Weiss et al. 1982; Scott-Moncrieff et al., 1995; McManus 2000; Stokol et al., 2000; Weiss 2002; Jonas et al., 1987; Weiss 1986). Although some of these dogs may have been presented within a few days of becoming acutely anemic, while in a preregenerative stage of IMHA (Kellerman et al., 1995; Burgess et al., 1987; Weiss 2008), dogs in these reports have generally had persistent nonregenerative anemias indicative of impaired erythropoiesis. 35 Comparisons among these cases are complicated by inconsistent inclusion criteria, terminology, and degrees to which bone marrow aspirate and core biopsy findings were reported. Bone marrow findings in dogs with suspected NIMA or NRIMHA have varied markedly from what was considered to be pure red cell aplasia (PRCA) to erythroid hyperplasia often accompanied by erythroid maturation arrest or left shift (Stokol et al., 2000; Weiss 2008; Weiss 1986; Jonas et al., 1987; Holloway et al., 1990; Stockham et al., 1980; Weiss et al. 1982; Scott-Moncrieff et al., 1995; McManus 2000; Weiss 2002). A few reports describe the presence of erythroid precursor phagocytosis (Stockham et al., 1980; Jonas et al., 1987; Holloway et al., 1990; McManus 2000), which we refer to as rubriphagocytosis (RP) to differentiate it from phagocytosis of erythrocytes (erythrophagocytosis)  and  to  reflect  the  “rubri”  component  in  all  stages  of  erythroid   precursors from rubriblasts through metarubricytes. In one of these studies, many of 44 dogs diagnosed with NRIMA, but not dogs with IMHA or hemorrhage-induced anemias, had increased marrow macrophages, RP, and macrophages containing basophilic granules consistent with degraded cellular material (McManus 2000). We, too, have detected RP in the vast majority of dogs with nonregenerative anemia, ineffective erythropoiesis, and an ultimate working diagnosis of what we refer to as precursor-targeted immune-mediated anemia (PIMA). This term  avoids  “hemolytic”   because of the inconsistency and common absence of a hemolysis in these dogs, and reflects the suspected immune-mediated targeting of erythroid precursors based on nonregenerative anemia with 1) concurrent evidence of immune-mediated hemolysis in some dogs (Holloway et al., 1990; Klag et al., 1993; Kellerman et al., 1995; Burgess et al., 1997; Jonas et al., 1987; Stokol et al., 2000; Weiss 2008; Scott-Moncrieff et al., 1995; Weiss 2002), 2) favorable responses to immunosuppressive therapy in some 36 dogs (Klag et al., 1993; Jonas et al., 1987; Stockham et al., 1980; Weiss et al 1982; Holloway et al., 1990; Scott-Moncrieff et al., 1995; Stokol et al., 2000; Weiss 2002), 3) apparent relapses after withdrawal of immunosuppressive therapy (Stockham et al., 1980; Weiss et al 1982; Scott-Moncrieff et al., 1995; Stokol et al., 2000; Weiss 2002), 4) RP, which may reflect immune-targeting of erythroid precursors (Stockham et al., 1980; Jonas et al., 1987; Holloway et al., 1990; McManus 2000), and 5) evidence that some dogs with PRCA have a circulating inhibitor to erythropoiesis that is in the IgGcontaining fraction of plasma (Weiss 1986). However, the pathogenesis of disease in these dogs is unknown, and bone marrow findings have not been comprehensively reviewed, particularly with regard to RP and its relationship to other findings. The major goal of this study was to better characterize clinical and laboratory findings in PIMA dogs, with emphasis on bone marrow cytologic and histologic abnormalities. It is hoped that this will facilitate recognition and diagnosis of this condition, and eventually lead to a better understanding of the disease and its optimal management. 37 MATERIAL AND METHODS Samples PIMA cases were identified through the authors’  review  of  archived  slides  for  all   dogs with paired bone marrow aspirate and core biopsies collected within 24 h of a complete blood count (CBC) at the Michigan State University Veterinary Medical Center (MSU VMC) from January 2006 through June 2009. Wright-stained aspirate smears were reviewed without knowledge of previous descriptions, but with knowledge of CBC results and indications for bone marrow assessment. Additional cases were acquired from July through December 2009 through assessment of marrow reports and review of archived slides for dogs with a possible PIMA component; reportedly inadequate samples were reviewed for adequacy and evidence of PIMA. Thin (3 µm target) histologic sections of B5-fixed tissue stained with hematoxylin and eosin were subsequently reviewed without knowledge of previous core biopsy findings, but with knowledge of aspirate reassessment findings. This process paralleled routine clinical diagnostic service and provided consistent and complete assessments of all cases without the variability inherent in using original reports from multiple pathologists. Each aspirate and core bone marrow sample was assessed for routine features (Table 1) and RP, and findings were systematically recorded. Left shifts were considered maturation arrest if there was a prominent diminution of a lineage at a certain nucleated precursor stage and beyond. Cases were excluded if either sample was considered inadequate and lacking in any useful information regarding the indication for marrow assessment. 38 Table 1: Features and categories for cytologic and histologic assessment of bone marrow aspirate and core specimens. ASPIRATE AND CORE Quality Inadequate Marginal Adequate Low (< 25%) Moderate (25 – 50%) High (> 75%) Decreased Within limits of health Increased Orderly Left shifted Decreased Within limits of health Increased Unclear Orderly and complete Left shifted Maturation arrest Within limits of health Increased Atypical cell populations Dysplastic changes Cytophagia† Absent Small amounts Moderate amounts Large amounts Hematopoietic Cellularity Megakaryocyte Number Megakaryocyte Maturation Myeloid to Erythroid Ratio Maturation of Cell Lineages (Myeloid and Erythroid) Numbers of Other Cell Types* Cell Abnormalities Hemosiderin 39 Table  1  (cont’d) CORE Erythroid Islands (number and size) Decreased Within limits of health Increased Necrosis Absent Present Bone Abnormalities Increased osteoclasts Bone lysis Bone formation Vascular Lesions Sinusoidal dilation Hemorrhage Vasculitis Thrombosis Collagen Fibrosis Mild Moderate Marked * Lymphocytes, plasma cells, macrophages, mast cells, osteoclasts, osteoblasts †  Specific  cell  types  and  stages  noted;;  most  apparent  cytologically Dogs were classified as having PIMA when the following criteria were met: 1) persistent nonregenerative anemia of enough significance to direct the attending clinicians to collect bone marrow (on the day of marrow collection, all had evidence of anemia for  at  least  5  days,  unexplained  Hct  ≤  25%,  and  no  reticulocytosis  (≤76,000/µL)), 2) ineffective erythropoiesis as evidenced by persistent anemia with concurrent expanded or arrested erythroid islands (cores) or increased particle hematopoietic cellularity with decreased myeloid to erythroid ratios (aspirates), 3) lack of significant dysplasia, and 4) clear selective phagocytosis of intact erythroid precursors by intact normal-appearing macrophages (Figure 1). Although PIMA was diagnosed in several other dogs over this time period when RP was suspected by cytoplasmic fragments of macrophages containing intact erythroid precursors or by increased degraded cellular material within macrophage cytoplasm (Figure 2), these cases were excluded from this 40 study to avoid any concerns about whether RP was actually present. Dogs with suspected nonselective cytophagic disorders and concurrent phagocytosis of neutrophils or other nucleated cell types, e.g., hemophagocytic syndrome or histiocytic sarcoma, were excluded. Figure 1: Cytophagia in aspirates of canine bone marrow. Rubriphagocytosis (RP) of intact early- (rubriblast, A), mid- (rubricyte, B), and late-stage (C) erythroid precursors by normal-appearing macrophages in dogs with PIMA. Although frequently present with intact RP, dogs whose macrophages (D) or macrophage cytoplasmic fragments (E) contained only degraded cells with indistinct limiting membranes and chromatin patterns were excluded, even when the nuclear material formed too large an inclusion to have been an extruded metarubricyte nucleus (D, E). Dogs with phagocytosis only of cells with apoptotic features were also excluded, though this was rarely seen and was not seen in dogs considered to have PIMA (F). Wright-Giemsa stain, 100× magnification. 41 Figure 2: Macrophage cytoplasmic fragments containing erythroid precursors. Although treated clinically as PIMA cases, cases were excluded if the evidence of RP was restricted to cytoplasmic fragments of macrophages containing intact erythroid precursors (A-E), in case there was any question as to the nature of these fragments. The arrows point to the outer edge of the erythroid precursor within the macrophage fragments, several of which contain other inclusions typical of macrophages. These fragments are contrasted by fragments containing only degraded cellular material or extruded metarubricyte nuclei (F). Wright-Giemsa stain, 100× magnification. Clinical and laboratory data For each case, clinical and laboratory data were retrospectively collected from patient files. Results for CBC at admission and serum color were recorded for all dogs, and results for the following initial tests were recorded when generated:  Coombs’  test,   42 serum protein and bilirubin concentrations, hemostasis profile, and serologic and molecular tests for Babesia canis, Borrelia burgdorferi, Anaplasma phagocytophilum, Ehrlichia canis, and Rickettsia rickettsii. These results were generated by the MSU Diagnostic Center for Population and Animal Health. Radiographic, ultrasonographic, cytologic, and histologic results were also recorded when available. Other recorded clinical information for each dog included signalment, transfusion and disease history, recent or current medications, presenting complaint, days after beginning immunosuppressive therapy at MSU until response and until remission, achievement of second remission, and survival days based on the last known information obtained from the owner or referring veterinarian. Response to therapy was considered an increase in the reticulocyte concentration to exceed the upper reference limit or an increase in Hct to within reference interval (WRI). Remission was defined as a return of Hct to WRI. When there was a relapse of anemia after remission, a second remission was defined by either resolution of anemia or by owner-reported resolution of all clinical signs. Data analysis Dogs with PIMA were grouped by the stage of erythroid precursor undergoing RP combined with cellularity of the erythroid lineage and the presence of severe myelofibrosis.  Groups  were  tested  for  normality  using  D’Agostino-Pearson test. Comparisons between two groups were done using the Mann Whitney test, and comparisons among three groups or more were done using the Kruskal-Wallis test; p<0.05 was considered significant. Prism 6 (GraphPad Software Inc, La Jolla, CA, USA) was used for all statistics. 43 RESULTS Paired bone marrow aspirate and core biopsy samples were examined for 121 patients, and 16 dogs were excluded because of at least one inadequate sample. Inclusion criteria for PIMA were fulfilled for 25 of the remaining 105 dogs. Indications for marrow assessment in these 25 patients were either isolated nonregenerative anemia (17, 68%) or nonregenerative anemia with one other cytopenia (8, 32%). PIMA dogs accounted for 17 of the 32 dogs (53%) assessed for nonregenerative anemia alone, and 8 of the 24 dogs (33%) assessed for nonregenerative anemia and neutropenia (8) or nonregenerative anemia and thrombocytopenia (16). Of the 25 PIMA dogs, 11 were male (2 intact) and 14 were female (1 intact), ranging in age from 1 to 15 years (median = 7 years). Four were of mixed breeding, two were Labrador retrievers, two were Pembroke Welsh corgis, and the other 17 were from 17 other distinct breeds. At the time of admission, six dogs were receiving immunosuppressive therapy, four for their current anemias and two for IMHA diagnosed 5 months or 1 year prior. One other dog had a history of relapsing immune-mediated thrombocytopenia (IMT). Presenting clinical complaints were lethargy (23/25), anorexia/hyporexia (13/25), weight loss (9/25), collapse (7/25), exercise intolerance (6/25), vomiting (3/25), seizures (1/25, on phenobarbital and potassium bromide), and pica (1/25, also vomiting). One patient had received a blood transfusion eight days prior to admission, and two had transfusions less than 24 hours before a CBC was obtained, raising their Hcts to 23% and 31%. The latter dog had undergone an exploratory laparotomy for vomiting the previous day, and a surgical biopsy yielded a diagnosis of moderate chronic-active lymphoplasmacytic hepatitis with fibrosis. 44 CBC findings Other than nonregenerative anemia, hematologic abnormalities were variable (Table 2). Anemia was moderate to severe in the majority of dogs, and clearly nonregenerative in all dogs at the time of bone marrow evaluation. Ten dogs had decreased reticulocyte concentrations; these dogs had Hcts of 8 to 23%. Most anemias were normocytic normochromic (6 of 25), macrocytic normochromic (5 of 25), or macrocytic hypochromic (7 of 25). Nucleated RBCs (nRBCs) were reported in six dogs, with a median for those six dogs of 0.4×103/µL (low-high: 0.1-1.0×103/µL). Features common in IMHA were rare in these PIMA dogs. No erythrocyte agglutination was observed in blood tubes, in blood films, or on the ADVIA 120 analyzer scatterplot of any PIMA patient. Mild spherocytosis was reported for one dog, but it was not  confirmed  on  later  review.  A  polyvalent  direct  Coombs’  test  was  positive  in  two  of  the   nine dogs that were tested. Leukocyte findings were variable, with leukocytosis in 40% (10/25) of the dogs, mild leukopenia with neutropenia in three dogs, and mild neutropenia in two others (Table 2). Thrombocytosis (low-high: 500,000-882,000/µL; median = 610,000/µL) was present in five dogs, and thrombocytopenia (11,000/µL; RI: 155,000-393,000) was present in one dog suspected of having concurrent IMT. 45 Table 2: Hematologic findings in the 25 dogs fulfilling diagnostic criteria for PIMA. Unit Median Low - High < LRL WRI > URL RI Hct % 18† 8 - 31†,  + n = 25 n=0 n=0 40 - 55 Reticulocytes /µL 17,000 <5,000 - 102,000+ n = 10 n = 14 n = 1+ 12,000 - 76,000 MCV fL 71 61 - 100 n=2 n = 11 n = 12 62 - 71 CHCM g/dL 34.2 27.4 - 38.1 n =11 n = 12 n=2 33.5 - 36.4 WBCs /µL 10,260 5,240 - 55,950 n=3 n = 12 n = 10 5,900 - 11,600 Neutrophils /µL 7,090 2,600 - 38,430 n=5 n=8 n = 12 4,000 - 8,200* †  Includes  two  dogs  with  transfusions  less  than  24  h  before  obtaining  Hcts  of  23%  and  31%. + The dog with highest Hct and reticulocyte concentration at admission had decreased values one day prior to bone marrow collection (3 days after admission) – Hct = 21%, reticulocyte concentration = 42,900, and on the day of bone marrow collection (4 days after admission) – Hct = 19%, reticulocyte concentration = 29,200. * RI for microscopic differentials (18 of 25 dogs); RI for automated differentials is 3,800-7,800/µL (7 of 25 dogs) LRL = lower reference limit; URL = upper reference limit; WRI = within reference interval. 46 Bone marrow findings Selective RP was detected in bone marrow aspirates from 23 dogs, in only cytologic smears of the spleen for one dog, in only histologic sections of marrow for one dog, and in histologic sections and cytologic smears for 8 dogs. RP was typically less prominent than indirect evidence of RP, including cytoplasmic fragments containing morphologically intact precursors and increased numbers of macrophages containing basophilic inclusions consistent with degraded cells (Figure 2). These findings were present in the marrow and spleen of the dog with clear RP only in the spleen. RP was prominent in some cases, but in other cases it was detected only after careful examination of one or several smears. It was often best found along or near the side edges of aspirate smears, where nucleated cells were somewhat concentrated, relatively well spread, and intact. In tissue sections, RP was less clear because of high cell density and diminished cellular detail, but it was detectable when the plane of sectioning included the macrophage nucleus along with the phagocytized cell surrounded by macrophage cytoplasm containing hemosiderin or other inclusions. Although all PIMA dogs had ineffective erythropoiesis and RP, four patterns emerged from their combined cytologic and histologic bone marrow samples. These patterns were defined by differences in erythroid cellularity, maturation stage of cells undergoing phagocytosis, and degree of collagen myelofibrosis (Table 3; Figures 3 and 4). With all patterns, expanded erythroid islands on histologic sections provided evidence of stimulated erythropoiesis up to the stage of most prominent RP, which corresponded with the stage of maturation arrest, when present. 47 Table 3: Features of the four bone marrow patterns apparent in the 25 dogs with PIMA. PIMA Pattern Predominant Cells Undergoing RP Erythroid Cellularity M:E Ratio Collagen MF Number Early-stage Rubriblasts, prorubricytes Low* High Absent 3 Mid-stage Prorubricytes, early rubricytes Moderate Moderate Absent 3 Late-stage Late rubricytes, metarubricytes High Low Absent to moderate 14 Severe MF Rubricytes to metarubricytes Low Low Severe 5 MF = myelofibrosis; M:E = myeloid to erythroid ratio * with expanded erythroid islands 48 Figure 3: Four histologic patterns identified in the 25 dogs with PIMA. Low erythroid cellularity with a marked erythroid left shift (A), moderate erythroid cellularity with a moderate erythroid left shift or maturation arrest (B), high erythroid cellularity with many mid- and late-stage erythroid precursors (C), and severe collagen fibrosis combined with erythroid hypocellularity but expanded and typically left-shifted erythroid islands (D). In all cases, erythropoiesis was ineffective and characterized by expanded erythropoiesis in active areas. B5 fixation, H&E stain, 50× magnification (A-C), 20× magnification for D to better show the extent of fibrosis. 49 Figure 4: Mild (A), moderate (B), and severe (C) collagen myelofibrosis in dogs with late-stage PIMA. Fibrosis progressed from small, indistinct linear eosinophilic regions (A) to narrow and moderately wide intertrabecular and/or paratrabecular bands of fibroblasts and collagen (B), to effacing fibrosis with new bone formation (C). B5 fixation, H&E stain, 20× (A) and 10× (B, C) magnification. Early-PIMA dogs (3/25) had erythroid hypocellularity associated with early-stage RP and early maturation arrest at the rubriblast (2) or prorubricyte (1) stage (Figure 3A). Rubriblasts varied from rare to increased. Overall hematopoietic cellularity was moderate or high because of active myelopoiesis. Concurrent ineffective neutropoiesis was suspected in the one dog with high hematopoietic cellularity. All three dogs in this group had mild increases in lymphocytes and/or plasma cells. Mid-PIMA dogs (3/25) had no clear increase or decrease in erythroid cellularity, but they had mostly mid-stage RP (Figure 3B) associated with erythroid maturation arrest or left shift and decreased metarubricytes. Although RP of prorubricytes and rubricytes 50 predominated, there was additional phagocytosis of metarubricytes in one dog. One dog had a mild increase in plasma cells. Late-PIMA dogs (14/25) had increased hematopoietic cellularity with erythroid hypercellularity and mostly late-stage RP (Figure 3C), but phagocytosis of basophilic rubricytes was additionally seen in two dogs. Myeloid to erythroid ratios were consistently decreased because of erythroid hyperplasia. Maturation arrest or left shift in the nucleated erythroid series was detected in 9 of 14 dogs (64.3 %). Seven of these 14 dogs had mild (3) to moderate (4) collagen myelofibrosis characterized by narrow to moderately wide intertrabecular or paratrabecular bands of fibroblasts and collagen in histologic sections (Figure 4). SMF-PIMA dogs (5/25) had severe myelofibrosis effacing most medullary tissue (Figure 3D), with bone remodeling and new bone formation within some fibrous bands. Remaining foci of hematopoietic tissue contained expanded islands of erythropoiesis, and had decreased myeloid to erythroid ratios with late-stage RP in three dogs, midand late-stage RP in one dog, and only mid-stage RP in one dog. Altogether, 12 of the 25 PIMA dogs had mild to severe collagen myelofibrosis (Table 4), all with mostly mid- to late-stage RP. When collectively compared to latePIMA dogs without collagen myelofibrosis, late-PIMA dogs with any degree of myelofibrosis were older (p=0.03). SMF-PIMA dogs had greater MCV than dogs with no myelofibrosis (p=0.04). 51 Table 4: Selected findings in late-stage PIMA dogs grouped by degree of collagen myelofibrosis. No MF Any MF Mild MF Moderate MF Severe MF RI Number 7 12 3 4 5 - Age (yr) 5.0 8.0* 8.0 9.5 7.0 - Hct (%) 20.0 17.0 18.0 16.0 16.0 40-55 MCV (fL) Number < or > RI 71 3H, 1L 72 7H 69 1H 65 1H 76* 5H 62-71 CHCM (g/dL) Number < or > RI 33.1 5L 34.0 2H, 5L 33.2 2L 35.6 2H, 1L 33.7 2L 33.5-36.4 Reticulocytes (×104/µL) 5.7 1.8* 0.5 1.8 1.8 1.2-7.6 Number < or > RI 2L 4L 2L 1L 1L All values are medians. RI = reference interval. H = high and L = low with respect to RI. *Significantly different from the No MF group. 52 Hemosiderin was present in the bone marrow of all 25 PIMA dogs, with moderate to large amounts in all but three dogs (two with late-PIMA and one with early-PIMA). Large amounts were noted in 6 of 14 late-PIMA dogs, in 1 of 3 early-PIMA dogs (multifocal dense accumulations), and in 2 of 5 SMF-PIMA dogs (one with multifocal dense accumulations). Macrophages subjectively appeared increased in six dogs, four late-PIMA dogs, one early-PIMA dog, and one SMF-PIMA dog. Eight PIMA dogs had megakaryocytic hyperplasia; given the association between megakaryocytes and fibrosis in people, it is noteworthy that seven of these eight dogs lacked collagen myelofibrosis, and no megakaryocyte dysplasia was detected. No necrosis, vascular disease, inflammation, or atypical cell populations were detected in any of the 25 dogs. Other findings One  of  the  Coombs’-positive dogs had mild hyperbilirubinemia, with a total bilirubin concentration of 1.4 mg/dL (RI = 0.1-0.4), most of which was indirect (1.1 mg/dL; RI = 0.1-0.4). Four other dogs had slight hyperbilirubinemia (0.5-0.6 mg/dL) of questionable significance. Of the 19 dogs that underwent abdominal imaging, five had splenomegaly, and splenic tissue was cytologically evaluated for two of them and for three other dogs with suspected splenic abnormalities. Increased splenic hematopoiesis was reported in four of the five dogs, one of which also had erythroid maturation arrest and destruction of erythroid precursors in the spleen. Similar findings were not reported in the other dogs, but their slides were not available for review. Of the 14 dogs assessed for coagulation abnormalities, four had decreased antithrombin III, increased D-dimers, and increased 53 fibrin degradation products. None of the 11 dogs assessed for tick-borne disease had results indicative of disease. Clinical outcomes Follow-up information was available for all but one of the 25 dogs, a dog with SMFPIMA that was lost to follow-up upon discharge. All other dogs were treated with immunosuppressive therapy and, when indicated, packed erythrocyte transfusions. Dogs were initially treated with prednisone alone (7) or in combination with cyclosporine (4) or azathioprine (13); azathioprine or cyclosporine were added later in five dogs. There was no apparent relationship between initial treatment regimen and outcome. Of the 24 dogs for which follow-up information was available, 12 had evidence of response, although two were lost to follow-up before a response might be expected (lost at 6 and 20 days after admission). The median time from the beginning of immunosuppressive therapy at MSU until response was 23 (1-48) days for the 10 dogs with follow-up reticulocyte concentrations, and 27 (1-48) days when excluding the three dogs on immunosuppressive therapy at admission. After further exclusion of two dogs with early (days 1 and 7) transient reticulocytosis that may not have been an appropriate indicator of response, the remaining five dogs had a similar median response time of 32 (21-48) days. Ten of the twelve known responders reached clinical remission, and immunotherapy was stopped in at least eight of them. The median time from beginning immunosuppressive therapy at MSU until documented remission for the six dogs with Hct follow-up data was 67 (31-123) days. Eight of the ten remission-achieving dogs lived at least 1 year after initial presentation; the two dying sooner were a dog that underwent 54 euthanasia for aggression and a dog whose owners declined recommended therapy at the time of relapse. Five of the eight dogs lived at least 3 years and two more lived at least 2 years after initial presentation. The eighth died of pneumonia 14 months after presentation. The two responders not reaching remission had increases in reticulocyte concentrations from < 20,000/µL to greater than 200,000/µL within 5-7 weeks after therapy was initiated, but died within two months, one with a portal vein thrombosis and acute renal failure, and the other of unknown cause. Five of the ten known remission-achieving dogs had relapses of PIMA, three did not, and relapse information was not available for two. Three of the five dogs that relapsed had a second remission; one died from PIMA when the relapse occurred and recommended therapy was not accepted, and one was still on treatment at last followup. An additional two dogs were treated for IMT after remission of PIMA. Of the 12 dogs with some follow-up information and no evidence of response to therapy, one died on day 17 and six underwent euthanasia within 45 days because of no response (3), economic reasons (1), septic peritonitis (1), or for unknown reasons (1). Another underwent euthanasia 120-150 days after presentation. The other four dogs were lost to follow-up after not responding to initial or altered therapeutic protocols for 6, 20, 90, or 270 days. Overall, at least four of these dogs lived 45 days and at least three lived three months without response. Eight of the twelve dogs had collagen myelofibrosis, including three with SMF-PIMA; five of the eight were known to have undergone euthanasia 11 days to 5 months after admission. Group differences 55 Dogs with early-PIMA were younger than dogs in other groups (p=0.04), and all three were female and had reticulocytopenia. Moreover, in contrast to all but one of the other PIMA dogs, they all had increased lymphoid cells in the bone marrow (increases that did not appear to be just relative). When comparing early-PIMA dogs to all other PIMA dogs combined, early-PIMA dogs had lower reticulocyte concentrations (p=0.01) and tended to have lower Hcts (p=0.05). The  two  Coombs’-positive dogs had late-stage RP, one with SMF-PIMA and one with late-PIMA. Two late-PIMA dogs were known to have had previous episodes of spherocytic, regenerative IMHA (5 months or 1 year prior to presentation for PIMA). Both dogs that developed IMT after PIMA also had late-PIMA, as did four of the five dogs that relapsed. Four of the seven dogs that survived the disease to last follow-up had late-PIMA; the other three belonged one each to the other three groups. One dog each with mild, moderate, and severe collagen myelofibrosis was in the group of responders, and one achieved remission and lived for at least 33 months after admission; the other two died within 2 months, one with PIMA, and one with acute renal failure and portal vein thrombosis. There was no difference in days until response among groups. 56 DISCUSSION PIMA was prevalent in this MSU VMC population, being the single most common diagnosis in dogs evaluated for nonregenerative anemia alone, and common in dogs assessed for nonregenerative anemia accompanied by either neutropenia or thrombocytopenia. There was no apparent breed or sex predisposition, and clinical signs were nonspecific or related to anemia. Immunosuppressive therapy was associated with apparent responses in 50% of the dogs, though the response rate might have been higher with greater therapeutic efforts and complete follow-ups. Responders had median times to response and remission of approximately 1 and 2 months, respectively. Some dogs lived years after remission and discontinuation of therapy, while at least 40% relapsed one or more times after steroid withdrawal or tapering, and other dogs did not respond despite months of immunosuppressive therapy. Bone marrow findings formed a spectrum of four patterns, none of which predicted outcome. Each pattern included evidence of stimulated erythropoiesis up to the stage of erythroid precursor undergoing phagocytosis. When rubriblasts and prorubricyte phagocytosis was primarily detected, the pattern mimicked severe erythroid hypoplasia or pure red cell aplasia. However, the presence or expansion of early erythroid stages together with their phagocytosis supports an erythroid stimulus and response accompanied by arrested maturation, not hypoplasia in the sense of a decreased erythropoietin stimulus or decreased marrow response. If that were the case, one would expect a more proportionate decrease in all erythroid stages without early expansion or RP. In contrast, most PIMA dogs had hypercellular marrows with erythroid hyperplasia and usually a maturation arrest or more subtle left-shift associated with mostly late-stage 57 RP. In dogs without obvious left shifts, the presence of RP and a markedly expanded and coalescing erythroid islands in the face of persistent nonregenerative anemia allowed differentiation from early pre-reticulocytosis responses to anemia. Five dogs with evidence of mid- or late-stage PIMA were classified separately because they had severe collagen myelofibrosis and an overall decrease in the erythroid lineage associated with marrow effacement; however, stimulated erythropoiesis was evidenced by decreased myeloid to erythroid ratios and expanded foci of erythropoiesis in core sections. In the fourth pattern characterized by mostly mid-stage RP and mid-stage maturation arrest, erythroid islands were not clearly expanded or reduced, hence there was erythroid normocellularity, but early-stage precursors were increased, again reflecting an erythropoietic stimulus. In all cases, whether a cause or an effect of ineffective erythropoiesis, RP appeared to be relatively stage-restricted, and it was typically aligned with the most mature stage that was well represented. This may have occurred because of relatively stage-specific targeting or because the most abundant stage would be phagocytized most often with RP that was not stage restricted. In either case, the cells undergoing phagocytosis appeared intact and lacked morphologic features that would identify them as apoptotic cells. If antibodies or complement mediate RP in PIMA, as they mediate IMHA, restricted phagocytosis may relate to restricted expression of target epitopes on different stages of development, and concurrent evidence of hemolysis in some cases may relate to shared epitopes on developing and mature cells (Okumura et al., 1992; Spivak 2005) or to multiple distinct antibodies (Mangan et al., 1984). RP associated with maturation arrest in the spleen of one study dog, as we have seen in others (Lucidi et al., 2011), supports that PIMA is a systemic disorder not restricted to the marrow. 58 If RP mediates PIMA, the question arises as to why it has been so uncommonly reported. This might relate to varied and sometimes more limited degrees of attack, a rapid rate of phagolysosomal degradation (Oh et al., 1996), and difficulties in detecting RP when present. Cytologic detection requires a sensitized eye, evaluation in the most optimal regions of smears, and a directed search. Because the cells undergoing phagocytosis are intact, they blend into their surroundings until being too degraded to be recognized, though the presence of large nuclear masses suggests something other than physiologic clearance of extruded metarubricyte nuclei. Additionally, macrophages containing phagocytized cells commonly break into cytoplasmic fragments, which are more difficult to recognize (Figure 2). A more intense search for RP is warranted when there is an increase in the number of macrophages containing basophilic material consistent with degraded cells (McManus 2000). In our experience, RP is seen in most PIMA dogs, but is rarely present in dogs with no evidence of PIMA; its detection adds confidence to a clinical diagnosis of PIMA. Of note, RP was detected even in the hemodiluted and poorly cellular smears from the five SMF-PIMA dogs, and together with decreased myeloid to erythroid ratios and erythroid left shifts, supports PIMA as an underlying disorder in dogs that might otherwise be diagnosed as having idiopathic myelofibrosis. The presence of mild or moderate collagen myelofibrosis in seven other study dogs, with 48% of the dogs affected overall, is evidence that fibrosis may develop secondarily over time in dogs with PIMA. This aligns with previous reports of reticulin (Stokol et al., 2000) or collagen (Scott-Moncrieff et al., 1995; Stokol et al., 2000; Reagan 1993; Villiers et al., 1999) myelofibrosis in dogs with suspected immune-mediated anemia, and with reports of some  Coombs’-positive or corticosteroid-responsive anemia 59 in dogs with severe myelofibrosis (Hoff et al., 1991). The mechanism of fibrosis in these dogs is unknown, but, as has been suggested by others (Villiers et al., 1999), it may be similar to that of autoimmune myelofibrosis (AIMF) reported in human patients with such conditions as systemic lupus erythematosus and autoimmune hemolytic anemia (Vergara-Lluri et al., 2014; Pullarkat et al., 2003). Alternatively, myelofibrosis in PIMA dogs may be related to a state of chronic inflammation in the bone marrow tissue, which can increase fibrotic signaling and lead to organ fibrosis (Ueha et al., 2012). Whatever the mechanism, myelofibrosis may have further impaired erythropoiesis and hematopoiesis in general, thus affecting treatment responses, but it may also have simply limited treatment because of concerns about prognosis in dogs with myelofibrosis. Of the 12 nonresponders, 67% had some degree of collagen myelofibrosis in contrast to only 25% of the responders, and four underwent euthanasia within 45 days of diagnosis. It is noteworthy that one of the dogs with collagen myelofibrosis lived at least 33 months after diagnosis. However, it is not known if fibrosis resolved in any dog, because bone marrows were not reassessed. Recovery from presumed PIMA with collagen myelofibrosis has been described, though again without follow-up marrow assessment (Villiers et al., 1999; Hoff et al., 1991; Reagan et al., 1992), and resolution of reticulin fibrosis has been reported in PIMA dogs (Stokol et al., 2000). In people, including those with AIMF, reticulin myelofibrosis is often reversible (Kuter et al., 2007; Vergara-Lluri et al., 2014; Pullarkat et al., 2003), while collagen myelofibrosis is considered less likely reversible and perhaps irreversible (Kuter et al., 2007). However, findings indicate that dogs with some degree of collagen myelofibrosis may respond and achieve remission from PIMA, so clinical pessimism related to treating dogs with collagen myelofibrosis may be excessive. 60 While severe collagen myelofibrosis may have contributed to neutropenia in two of the five dogs with neutropenia, ineffective neutropoiesis was suspected in two of the other three dogs because of ample to increased neutropoiesis and no evidence of overwhelming inflammatory lesions. Two other dogs were excluded from the PIMA group because of neutrophil phagocytosis accompanied by evidence of ineffective neutropoiesis in addition to findings typical of early-PIMA. Although excluded because of the potential for nonselective phagocytosis, we suspect that phagocytosis in these and similar dogs (Lucidi et al., 2011) is selective, with selective targeting of more than one cell type. This is not surprising, as multiple (Michel et al., 2009; Martino et al., 1995) or shifting (Day 2010; Taniguchi et al., 1988; Meyer et al., 1978; Mangan et al., 1984; Tohda et al., 1992; Crosby et al., 1956) targets in immune-mediated hematologic disorders are not uncommon and may be further evidenced in this study population by two dogs that had recovered from IMHA prior to developing PIMA, by one dog with a history of IMT, by two dogs that developed IMT after remission from PIMA, similar to a dog in a previous study (Stokol et al., 2000), and by one study dog with a clinical diagnosis of concurrent IMT. Although PIMA appears to be part of a spectrum of cytopenic disorders, pathogeneses may vary. The early-PIMA group differed from mid- and late-stage PIMA by an over-representation of females and younger dogs, lower reticulocyte concentrations, the absence of collagen myelofibrosis, and by mild increases in bone marrow lymphocytes and/or plasma cells, which were identified in only one other dog. Group numbers were low, but these findings mirror those reported for dogs with PRCA (Weiss 2002; Weiss 2008), a similar condition, and suggest that the pathogenesis of early-PIMA and PRCA may differ from that of mid- and late-PIMA. We currently classify 61 the pattern of early maturation arrest with a predominance of rubriblasts accompanied by early-stage RP as early-PIMA, whereas we apply the PRCA label to cases with an absence or marked paucity of erythroid precursors without a left shift or RP. This may or may not be an artificial division. Several factors may explain why evidence of hemolysis in dogs with PIMA has varied from common (Stokol et al., 2000) to uncommon (Scott-Moncrieff et al., 1995), the latter being the case in this study. In our hospital, dogs with nonregenerative and possibly preregenerative anemia but obvious signs of hemolytic disease are typically treated for IMHA without marrow examination, and therefore would not have entered our study group unless they failed to respond to treatment. In contrast, such dogs may have been included in other studies of nonregenerative immune-mediated anemia, some of which actually required evidence of hemolysis and erythrocyte targeting for inclusion (Jonas et al., 1987; Klag et al., 1993; Kellerman et al., 1995; Burgess et al., 1997; Weiss 2008). Interestingly, we have detected selective late-stage RP in dogs with IMHA when bone marrow was sampled despite classic presentations and regenerative anemias, but because such samples from classic IMHA patients are rare in our hospital, it is unknown how frequently this occurs and therefore the degree to which impaired erythropoiesis may potentially contribute to anemia in dogs with acute, regenerative IMHA. Other reasons for less hemolysis in our PIMA population may relate to varied diagnostic interpretations. Dogs with no hemolytic signs but with persistent nonregenerative anemia and evidence of ineffective erythropoiesis may be interpreted as having erythroid hypoplasia (early- and mid-PIMA), myelodysplasia, or, in the case of latePIMA, erythroid hyperplasia without a provided cause. Additionally, our population differed from others by the requirement for RP, which may have selected a different set 62 of dogs, including those with severe collagen myelofibrosis that might otherwise have been considered to have idiopathic myelofibrosis. Dogs with IMHA are prothrombotic and prone to thromboembolic disease that is associated with increased mortality (Carr et al., 2002; Klein et al., 1989; Fenty et al., 2011), and thrombotic disease has also been reported in a few dogs with nonregenerative immune-mediated anemia (Jonas et al., 1987; Stokol et al., 2000). However, these dogs may have had a hemolytic component to their anemias, whereas, in our study, one dog lacking evidence of hemolysis developed portal vein thrombosis and had increased FDPs and D-dimers, while four other dogs, including one the day after exploratory surgery, had decreased antithrombin III, increased D-dimers, and increased FDPs without detected thrombosis. Further studies are warranted to determine if thromboembolic events are a significant complication in dogs with PIMA as they are in dogs with IMHA. In conclusion, we evaluated 25 dogs with PIMA, defined by persistent nonregenerative anemia and evidence of ineffective erythropoiesis with selective RP. Three bone marrow patterns were identified based on early-, mid-, or late-stage RP and maturation arrest, and a fourth pattern was based on the presence of concurrent severe collagen myelofibrosis. Response to immunosuppressive therapy, and in some cases relapse upon withdrawal of therapy, supports an immunologic condition, as does detection of RP in bone marrow and spleen. However, remission required weeks to months and evidence of hemolysis was rare, supporting that PIMA should be distinguished from IMHA. Mild to severe myelofibrosis is common in dogs with PIMA, and does not prevent clinical remission. If severe collagen myelofibrosis is detected in canine bone marrow cores, PIMA should be a differential diagnosis and further evidence 63 of PIMA should be sought (i.e., expanded erythroid islands, erythroid left shift, selective RP). Further investigations to determine the pathogenesis of PIMA, the cause of RP, and the best therapeutic approach for managing affected patients are warranted. 64 REFERENCES 65 REFERENCES Burgess KE, Rand W, Moore A. Immune mediated hemolytic anemia in dogs: a retrospective study of 60 cases treated with cyclophosphamide [abstract]. J Vet Intern Med. 1997;11:143. Carr AP, Panciera DL, Kidd L. Prognostic factors for mortality and thromboembolism in canine immune-mediated hemolytic anemia: a retrospective study of 72 dogs. J Vet Intern Med. 2002;16:504-509. Crosby WH, Rappaport H. Reticulocytopenia in autoimmune hemolytic anemia. Blood. 1956;11:929-936. Day MJ. Immune-mediated anemias in the dog. In: Weiss DJ, Wardrop KJ, eds. 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Primary autoimmune myelofibrosis: definition of a distinct clinicopathologic syndrome. Am J Hematol. 2003;72:8-12. Reagan W, Scott-Moncrieff C, DeNicola D, et al. Immune mediated non-regenerative anemia and myelofibrosis in female beagle dogs [abstract]. Vet Clin Pathol. 1992;21:127. Reagan WJ. A review of myelofibrosis in dogs. Toxicol Pathol. 1993;21:164-169. Scott-Moncrieff JCR, Reagan WJ, Glickman LT, et al. Treatment of nonregenerative anemia with human  γ-globulin in dogs. J Am Vet Med Assoc. 1995;206:1895-1900. Spivak JL. The anaemia of cancer: death by a thousand cuts. Nat Rev Cancer. 2005;5:543-555. Stockham SL, Ford RB, Weiss DJ. Canine autoimmune hemolytic disease with a delayed erythroid regeneration. J Am Anim Hosp Assoc. 1980;16:927-931. 67 Stokol T, Blue JT, French TW. Idiopathic pure red cell aplasia and nonregenerative immune-mediated anemia in dogs: 43 cases (1988-1999). J Am Vet Med Assoc. 2000;216:1429-1436. Taniguchi S, Shibuya T, Morioka E, et al. Demonstration of three distinct immunological disorders on erythropoiesis in a patient with pure red cell aplasia and autoimmune haemolytic anaemia associated with thymoma. Br J Haematol. 1988;68:473-477. Tohda S, Nara N, Tanikawa S, et al. Pure red cell aplasia following autoimmune haemolytic anaemia. Cell-mediated suppression of erythropoiesis as a possible pathogenesis of pure red cell aplasia. Acta Haematol. 1992;87:98-102. Ueha S, Shand FH, Matsushima K. Cellular and molecular mechanisms of chronic inflammation-associated organ fibrosis. Front Immunol. 2012;3:71. Vergara-Lluri ME, Piatek CI, Pullarkat V, et al. Autoimmune myelofibrosis: an update on morphologic features in 29 cases and review of the literature. Hum Pathol. 2014; 45:2183-2191. Villiers EJ, Dunn JK. Clinicopathological features of seven cases of canine myelofibrosis and the possible relationship between the histological findings and prognosis. Vet Rec. 1999;145:222-228. Weiss DJ, Stockham SL, Willard MD, et al. Transient erythroid hypoplasia in the dog: report of five cases. J Am Anim Hosp Assoc. 1982;18:353-359. Weiss DJ. Antibody-mediated suppression of erythropoiesis in dogs with red blood cell aplasia. Am J Vet Res. 1986;47:2646-2648. Weiss DJ. Bone marrow pathology in dogs and cats with non-regenerative immunemediated haemolytic anaemia and pure red cell aplasia. J Comp Pathol. 2008;138:4653. Weiss DJ. Primary pure red cell aplasia in dogs: 13 cases (1996-2000). J Am Vet Med Assoc. 2002;221:93-95. 68 CHAPTER 3 Flow Cytometric Assays for Canine Immune-mediated Anemias; Detection of DEA1.1 on Canine Erythroid Precursors 69 ABSTRACT Objective: To develop and characterize flow cytometric assays for IgG on erythroid populations from the blood and bone marrow of dogs, ultimately to explore the pathogenesis of suspected precursor-targeted immune-mediated anemia. Samples: Blood from 20 healthy and 61 sick dogs with (33) or without (28) immunemediated hemolytic anemia, and bone marrow from 39 healthy or sick dogs. Procedures: A flow cytometric assay for RBC IgG and a positive control were developed,  and  the  assay’s  analytical  and  diagnostic  performance  were  characterized.   The assay was combined with Percoll fractionation of marrow cells and a 2-color erythroid precursor (nRBC) assay to detect nRBC IgG. Sorting and cytology were used to confirm target populations, and anti-DEA1.1 blood typing serum was used to make IgG-positive nRBCs. Results: The RBC IgG assay had within-run and between-run coefficients of variation of 0.1-13.9%, and >90% of spiked positive cells were detected. Clinical assessment yielded diagnostic sensitivity, specificity, and accuracy of 87.9%, 92.9%, and 90.2%, respectively. Cytologic assessment of cell-sorted early-, mid-, and late-stage nRBC regions from 3 healthy dogs showed 89-98% nRBC purity. IgG was detected on nRBCs from DEA1.1-positive dogs, but not DEA1.1-negative dogs, after IgG coating with antiDEA1.1 typing serum. Conclusions and Clinical Relevance: The RBC IgG assay had favorable analytical and diagnostic performance and can be used as a diagnostic or research tool for immunemediated erythrocyte destruction. The assay was successfully applied to nRBCs isolated from canine bone marrow, and IgG-coated nRBCs were detected. In addition, the findings support the presence of DEA1.1 on nRBC. 70 INTRODUCTION Precursor-targeted immune-mediated anemia (PIMA) has been reported in several studies (Weiss et at., 1982; McManus 2000; Scott-Moncrieff et al., 1995; Stockham et al., 1980; Stokol et al., 2000; Jonas et al., 1987; Weiss 2008; Holloway et al., 1990), and accounts for approximately 25% of the canine bone marrow diagnoses at the Michigan State University Veterinary Medical Center (MSU VMC). However, its pathogenesis is unclear. The condition is characterized by nonregenerative anemia, ineffective erythropoiesis, and, in most MSU VMC cases, by rubriphagocytosis – phagocytosis of intact erythroid precursors (nucleated red blood cells, nRBCs). We have found that rubriphagocytosis in PIMA dogs is quite stage-selective, and that the stage of erythroid precursor being phagocytized (i.e., early-, mid-, or late-stage nRBCs) aligns with a spectrum of cytologic and histologic bone marrow patterns that commonly include mild to severe myelofibrosis (Lucidi et al., 2010). An immune-mediated mechanism has been proposed, but not proven, based on phagocytosis of erythroid precursors (Stockham et al., 1980; Jonas et al., 1987; Holloway et al., 1990; McManus 2000), responses to immunosuppressive therapy (Jonas et al., 1987; Stockham et al., 1980; Weiss et al., 1982; Holloway et al., 1990; Scott-Moncrieff et al., 1995; Stokol et al., 2000), apparent relapses after withdrawal of immunosuppressive therapy (Stockham et al., 1980; Weiss et al., 1982; Scott-Moncrieff et al., 1995; Stokol et al., 2000), and evidence of concurrent immune-mediated hemolytic anemia (IMHA) in some cases (Holloway et al., 1990; Jonas et al., 1987; Stokol et al., 2000; Weiss 2008; Scott-Moncrieff et al., 1995). However, responses to immunosuppressive drugs are slow, relapses are common, and many of the affected dogs die or undergo euthanasia due to clinical pessimism or financial constraints. 71 Flow cytometry has been used to diagnose and characterize immune-mediated hemolytic anemias in dogs (Wilkerson et al., 2000; Quigley et al., 2001; Kucinskiene et al., 2005; Morley et al., 2008) and people (Chaudhary et al., 2006; Lin et al., 2009; Wang et al., 2001), and the method has been shown to be more sensitive than standard agglutination tests for IgG detection (Wilkerson et al., 2000; Quigley et al., 2001; Kucinskiene et al., 2005; Chaudhary et al., 2006; Lin et al., 2009; Wang et al., 2001). A potential PIMA counterpart which may be mediated by IgG directed against nRBCs has been identified in human patients (Mangan et al., 1984; Meyer et al., 1978; van de Loosdrecht et al., 2000), and flow cytometry has been used to identify IgG on bone marrow-derived nRBCs in one of these cases (van de Loosdrecht et al., 2000). These studies indicate that flow cytometry could also be used to assess PIMA dogs for increased nRBC IgG. The goal of this work was to develop a method that would allow isolation and flow cytometric assessment of IgG on different stages of nRBCs from the bone marrow of dogs. To this end, an assay for red blood cells (RBC) IgG was first developed and validated, and this method was applied to marrow-derived nRBCs. These assays will facilitate a variety of blood and stage-selective marrow assessments in dogs with PIMA, as well as other disorders, and help determine if there is a role for IgG in the pathogenesis of canine PIMA. Better understanding of the mechanism of PIMA may lead to improved management strategies and therapeutic responses in affected dogs. 72 MATERIAL AND METHODS Dogs For assessment of IgG on RBCs, blood was collected by routine venipuncture into K2EDTA from 20 dogs enrolled in the MSU VMC Blood Donor Program and deemed clinically and hematologically healthy based on annual physical exam, CBC, serum chemistry, and screening for infectious diseases (heartworm disease, babesiosis, ehrlichiosis, leishmaniasis, and hemotropic mycoplasmosis). Blood was similarly collected from 61 MSU VMC patients as part of their routine diagnostic evaluation. These patients included 33 dogs with IMHA and 28 dogs with non-IMHA conditions. Dogs were classified as having IMHA if they had a clinical diagnosis of IMHA associated with regenerative anemia (all had hematocrit below 33%) and at least one of the following with no other explanation: 1) spherocytosis, 2) erythrocyte agglutination, or 3) Coombs’  test  positivity.  The  unhealthy  non-IMHA group comprised sick dogs with no clinical suspicion or hematologic evidence of immune-mediated anemia. For development and evaluation of a flow cytometric assay for IgG on nRBCs, bone marrow was collected from 39 dogs: 21 clinically healthy colony dogs and 18 MSU VMC patients. Some colony dogs received 1-2 physical examinations per month, while the others received physical examinations twice per year. All dog and sample use was in compliance with the Michigan State University and MPI Research Institutional Animal Care and Use Committees. Bone marrow collection Approximately 2-5 mL of bone marrow was aspirated from the proximal humerus or wing of the ilium of anesthetized dogs using a 15 or 18 gauge Illinois bone marrow 73 needle. Marrow was collected into a syringe containing 0.5 mL of 3% K 2EDTA in saline, and then dispensed into a Petri dish for particle harvesting and preparation of cytologic smears; the remaining sample was used for flow cytometric analysis. RBC sample preparation Blood samples collected routinely into EDTA-anticoagulated vacuum tubes were used on the day of collection or stored at 4°C and used the next day, and always processed at room temperature (20 to 22°C). Whole blood was centrifuged at 1200 g for 5 minutes, and after aspiration of the plasma and buffy coat, 10 µL of packed RBCs were withdrawn and washed twice by resuspension in 1.5 mL PBS-0.3%BSA (PBS: 137 mM NaCl, 2.7 mM KCl, 9.6 mM NaH2PO4, 1.5 mM KH2PO4, containing 0.3% bovine serum albumin and 0.025% sodium azide, pH 7.3) followed by centrifugation at 1200 g for  1  minute.  The  washed  pellet  was  resuspended  in  800  μL  PBS-0.3%BSA. Flow cytometric assay for RBC IgG The following procedure was applied after optimization studies that included assessment  of  signal  to  noise  ratios  and  number  of  wash  steps.  For  each  dog,  50  μL  of   washed  RBC  suspension  were  incubated  with  15  μL  of  fluorescein  isothiocyanate   (FITC)-conjugated caprine anti-[dog  IgGγ]  IgG  (IgG  test  antibody,  KPL,  Gaithersburg,   MD) in the dark for 30 minutes. RBCs were washed once as described above, resuspended in 1.5 mL PBS-0.3%BSA, and analyzed on a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA) by acquiring 10,000 events and using CellQuest Pro software (Becton-Dickinson, San Jose, CA). The electronic settings for RBC analysis were: forward scatter (FSC) voltage = E00, AmpGain = 2.25; side scatter (SSC) voltage 74 = 304, AmpGain = 1.32, FL1 voltage = 703; and FSC threshold = 100. RBCs were recognized and gated based on FSC and SSC, as previously described (Lucidi et al., 2011). Positive control samples were developed and negative and positive control samples were assessed with each test sample. Negative controls were made by incubating washed RBCs from healthy dogs with IgG test antibody (negative control for IgG), and by incubating test RBCs from each tested dog with FITC-conjugated caprine anti-[chicken IgG-heavy and light chain] IgG (nonsense antibody, KPL, Gaithersburg, MD) as a control for nonspecific binding. Positive control samples consisted of dog erythrocyte antigen (DEA)1.1-positive RBCs treated with a polyclonal anti-DEA1.1 typing serum (Animal Blood Resources International, Stockbridge, MI) to yield IgG-coated RBCs. More specifically, positive control cells were made by a 30 minute room temperature  incubation  of  800  μL  washed  RBC  suspension  of  DEA1.1-positive RBCs with  800  μL  of  anti-DEA1.1 typing serum diluted with PBS-0.3%BSA. Cells were washed two times by resuspension in 1.5 mL PBS-0.3%BSA followed by centrifugation at 1200 g for  1  minute,  and  the  washed  pellet  was  resuspended  in  800  μL  PBS-0.3%BSA. This positive control was assessed for stability, and new batches were made approximately once a month, as needed. IgG positivity of control cells varied from moderate to strong, depending on the cells and serum dilution used. Various methods of determining IgG positivity were assessed, and it was concluded that results would be reported as % positive events, with the cutoff for positive fluorescence of each test sample being at the level defining the top 2% of events for the nonsense antibody (nonspecific binding). When RBC agglutinates were identified on flow plots based on RBC FSC and SSC (Figure 5), they were gated out and 75 excluded from the analysis in order to prevent falsely decreased IgG positivity caused by setting a higher 2% fluorescence cutoff to accommodate the increased fluorescence of agglutinates with the nonspecific control (Arndt et al., 2010). Figure 5: Effect of RBC agglutinates on IgG detection. When included in the gate of interest (A), RBC agglutinates result in a higher 2% fluorescence cutoff based on the nonsense antibody (B), and falsely lower IgG positivity based on the test antibody (C, 5.7% positive events), compared to when agglutinates are excluded from the gate of interest (D), thereby lowering the cutoff (E) and allowing more accurate detection of IgG positivity (F, 18.7% positive events). Analytical precision of negative and positive samples was assessed, and the final coefficients of variation (CVs) were calculated based on % positive events. For withinrun precision, 20 different RBC suspensions were prepared from a single negative blood sample, and 40 µL of each was spiked with 10 µL of positive control cells to yield 20% 76 positive cells. Negative control cells were similarly tested in parallel. For assessment of between-run precision and stability over time, IgG negative and positive control RBCs were  stored  in  Alsever’s  solution  (Sigma-Aldrich, Saint Louis, MO) with 0.5% BSA (Sigma-Aldrich, Saint Louis, MO) at 4°C, and RBCs were tested for 10 consecutive weekdays,  followed  by  weekly  testing  for  3  weeks.  Prior  to  each  assessment,  50  μL  of   RBC suspension were washed with 1.5 mL PBS-0.3%BSA,  resuspended  in  50  μL  of  the   same buffer, and tested for IgG positivity with test and nonsense antibodies as described above. Analytical accuracy was assessed in triplicate by a mixing experiment in which IgG negative control RBCs were spiked with IgG positive control RBCs to target 0, 20, 40, 60, 80, and 100% positive events. The percentage of targeted positivity that was detected was determined. To assess changes in % positive events and IgG signal (median fluorescence intensity of test minus nonsense antibody) with decreasing amount of IgG per RBC, DEA1.1-positive RBCs incubated with doubling dilutions of antiDEA1.1 typing serum from 1/16 to 1/512 in PBS-0.3%BSA were assessed. Clinical assessment of the RBC IgG assay RBCs from healthy dogs, dogs with IMHA, and unhealthy dogs without IMHA were tested, and diagnostic sensitivity, specificity, and accuracy were calculated. The mean % positive events + 3 standard deviations of samples from 20 healthy dogs was used as a cutoff to classify IMHA and unhealthy non-IMHA dogs into IgG-negative (below cutoff) and IgG-positive (above cutoff) groups. To assess the effects of testing day-old samples, and to generate comparison values if they differed, the values for healthy dogs 77 were determined for fresh and day-old samples. Pertinent clinical and laboratory information was gathered for each of these dogs. Fractionation of bone marrow cells Bone marrow samples were transported to the laboratory in a cooled container and processed at room temperature within 30 minutes of collection in order to minimize changes in cell membrane or density, which might promote nonspecific binding or affect Percoll gradient separation, respectively. However, we did not evaluate if such changes would in fact occur with storage. Samples were mixed with 40 mL PBS-1%BSA (PBS containing 1% bovine serum albumin and 0.025% sodium azide, pH 7.3) and centrifuged at 500 g for 10 minutes before supernatant was removed and cell pellets were resuspended in 5 mL PBS-1%BSA. Cell suspensions were applied to a 3-layer Percoll (Sigma-Aldrich, Saint Louis, MO) gradient (3 mL each of 52%, 64%, 68% Percoll in PBS-1%BSA) modified from previous studies (Weiss et al., 2000). After centrifugation at 500 g for 30 minutes, the three cell fractions that will be referred to as top, middle, and bottom fractions were harvested from top to bottom using plastic Pasteur pipettes. Each was dispensed into a 5 mL tube, washed twice with alternating PBS-1%BSA (10 mL and then 3 mL) and centrifugation at 500 g. Washed pellets were resuspended in 0.5-3 mL PBS-1%BSA depending on pellet size, and nucleated cell concentrations were determined by hemocytometer. Desired cell separation was confirmed by microscopic examination of stained (Wright-stain), air-dried, stop-flow preparations of cells from each layer. Preparation of bone marrow fractions for flow cytometry 78 A 2-color assay was developed using the nucleic acid stain LDS751 (Life Technologies, Grand Island, NY) in combination with labeling for CD18 on non-erythroid cells. The following processing procedure was adopted after trial assessments and optimization steps. For each sample, 106 bone  marrow  cells  in  100  μL  PBS-1%BSA were  incubated  with  2  μL  of  murine  anti-[dog CD18] monoclonal antibody (clone CA1.4E9, isotype IgG1, AbD Serotec, Raleigh, NC) under mechanical rotation for 30 minutes, washed twice with 1.5 mL PBS-0.3%BSA,  and  resuspended  in  100  μL  of  the   same  buffer.  Cells  were  then  incubated  with  5  μL  of  polyclonal  phycoerythrin  (PE)conjugated goat anti-[mouse IgG] (AbD Serotec, Raleigh, NC ) antibody under mechanical rotation for 30 minutes in the dark, washed twice with 1.5 mL PBS0.3%BSA,  resuspended  in  400  μL  of  the  same  buffer,  and  transferred  to  a  flow   cytometer analysis tube. These cells were then incubated for at least 20 minutes (progressively longer for samples analyzed later in a batch) in the dark with 160 µL of LDS751  working  solution  prepared  by  diluting  2  μL  stock  into  1.5  mL  PBS-0.3%BSA (Saad et al., 2000). Flow cytometric identification of bone marrow nRBCs nRBCs from each of the top, middle, and bottom Percoll fractions were expected to stain strongly with LDS751 and not label for CD18. This was assessed by processing bone marrow samples from eight dogs as described above, and then sorting them with a BD Influx cell sorter (Becton-Dickinson, San Jose, CA) using BD FACS Sortware sorter software (Becton-Dickinson, San Jose, CA). Sorted fractions of interest were collected into buffered saline solution to which was immediately added bovine fetal serum (SigmaAldrich, Saint Louis, MO) at a ratio  of  50  μL  serum  per 1 mL of saline. The cell content of 79 each erythroid fraction of three dogs was assessed by differential cell counts on modified Wright-stained cytocentrifuge preparations. Certain other sorted fractions from these and other dogs were also assessed cytologically to determine the location of potentially confounding cell populations. IgG detection on nRBCs Top, middle, and bottom Percoll fractions of bone marrow were assessed for IgGpositive nRBCs by essentially combining the assay for RBC IgG with the 2-color bone marrow assay described above to yield a 3-color assay for nRBC IgG. For each fraction, 106 nucleated cells were processed as described for the 2-color assay with the addition of test anti-IgG and nonsense antibodies at the time of incubation with goat anti-[mouse IgG]-PE. Assay optimization studies showed no false positive IgG signal caused by binding of IgG test antibody to anti-CD18 antibodies. Cells were analyzed on a FACSCalibur flow cytometer by acquiring 10,000 events, using CellQuest software. The electronic settings for analysis of erythroid populations were: FSC voltage = E00, AmpGain = 2.0; SSC voltage = 400, AmpGain = 1.0, FL1 voltage = 673, FL2 voltage = 610, FL3 voltage = 540; FSC threshold = 100, and compensation settings of FL1 - 15% FL2; FL2 - 21%FL1; FL2 - 6.7%FL3; FL3 - 6.7%FL2. As for the RBC assay, nRBC IgG results were reported as % positive events in the gates of interest. To assess the ability of the assay to detect IgG on nRBCs, we used an approach parallel to the RBC IgG assay positive control and explored the generation of IgGpositive DEA1.1-positive nRBCs by incubation with anti-DEA1.1 typing serum. For 3 DEA1.1-positive dogs, 100 µL containing 106 cells from each bone marrow fraction were incubated with  100  μL  of  anti-DEA1.1 typing serum at low (1/16) or high (1/128) dilutions 80 (v/v) in PBS-0.3%BSA for 30 minutes. Low and high dilutions were used to assess detection of stronger and weaker signals. Cells were washed twice with 1.5 mL PBS0.3%BSA and resuspended  in  100  μL  of  the  same  buffer  prior  to  3-color testing as described above. Bone marrow fractions of 3 DEA1.1-negative dogs were processed similarly, and samples from 10 healthy dogs were also assessed for nRBC IgG (without the DEA antiserum steps). Hematologic health of these colony dogs was based on bone marrow aspirate evaluation, semi-annual CBCs in some dogs, and CBCs at the time of marrow collection in the others. DEA1.1-positive and negative dogs were identified by flow cytometric blood typing (Lucidi et al., 2011). The same controls used for the RBC IgG assay were used for each bone marrow sample. Statistical analysis Significant differences (p<0.05) in RBC and nRBC IgG signal among groups were assessed  by  Kruskal  Wallis  and  Dunn’s  multiple comparison post hoc tests using a statistical software (GraphPad Prism, GraphPad Software Inc, San Diego, CA). 81 RESULTS RBC IgG assay characterization The CV for within-run precision was 6.8% for a modestly positive sample with a mean of 20.9% positive events, and 13.4% for negative control cells with a mean of only 6.8% positive events. Between-run precision of negative (mean of 4.4% positive events) and strongly positive (mean of 99.9% positive events) control cells yielded 10-day CVs of 13.9% and 0.1%, respectively. A 10-day assessment is reported because mild shifts in positivity and progressive hemolysis were apparent beyond that time, but control cells were clearly negative and positive for at least one month, with the negative control IgG positivity varying from 4.9% (day 1) to 2.8% (day 34), and that of the positive control varying from 100% (day 1) to 99.8% (day 34). IgG-uncoated RBCs spiked with positive cells targeting 0, 20, 40, 60, 80, and 100% positive events yielded 0, 18, 37, 56, 78, and 100% positive cells, with the percentage of detected to targeted cells varying from 90% to 100%. When using samples with decreasing amounts of canine IgG per RBC, a constant number of 100% positive events was detected, but median fluorescence intensity decreased curvilinearly with IgG signal, ranging from 2,372 to 190 (Figure 6). 82 Figure 6: Dose-response curve for RBC IgG. DEA1.1-positive RBCs incubated with doubling dilutions of anti-DEA1.1 typing serum yielded a curvilinear reduction in signal with reduction in IgG. Clinical assessment of the RBC IgG assay The mean RBC IgG positivity for fresh and day-old samples from 20 healthy dogs, reported as % positive events, was 4.8% and 5.2%, respectively, yielding similar cutoffs for IgG positivity of 9.4% and 8.9% for fresh and day-old samples, respectively. Results were positive in 88% of IMHA dogs and in 7% of unhealthy non-IMHA dogs (Table 5), and results were the same whether matched fresh and day-old cutoffs were used or not. Differences in IgG positivity were significant between the IMHA group and each other group, but not between unhealthy non-IMHA and healthy groups. Based on these results, the diagnostic sensitivity, specificity, and accuracy for detecting IMHA were 87.9%, 92.9%, and 90.2%, respectively. 83 Table 5: Number of IMHA and unhealthy non-IMHA dogs with negative and positive RBC IgG results. IgG Result Negative Positive Number of IMHA dogs (median % positive, low-high) 4 (3.2%, 2.6-6.7%) Number of non-IMHA dogs (median % positive, low-high) 26 (4.9%, 1.2-8.2%) 29 (82.5%, 10.4-100%) 2 (17.4%, 10.5-24.2%) Bone marrow fractions and detection of nRBCs Percoll separation yielded a top fraction enriched in early-stage erythroid precursors, with fewer mid-stage erythroid precursors, many early to mid-stage myeloid cells, and few megakaryocytes, macrophages, and lymphocytes. The middle fraction was enriched in mid-stage (with few late-stage) erythroid precursors, and also contained a mixture of mid- to late-stage granulocytes, few lymphocytes, and rare plasma cells. The bottom fraction contained mostly late-stage (with few mid-stage) erythroid precursors and segmented neutrophils (Figure 7). All three fractions also contained low to moderate numbers of mature erythrocytes and reticulocytes, with greater numbers in the bottom fraction. 84 Figure 7: Canine bone marrow fractions after separation using a Percoll gradient. Percoll separation yielded top (A), middle (B), and bottom (C) fractions enriched in early, mid-, and late-stage erythroid precursors, respectively. Other cell types such as granulocytes, macrophages, lymphocytes, mature erythrocytes and reticulocytes were also present. Erythroid populations were identified as CD18-negative (FL2 channel) and LDS751-positive (FL3 channel) events (Figure 8A-C, gate R1), which were shown by cell sorting and cytologic examination to have little contamination with other cell types (Figure 9B-D). Cytologic assessment of cell-sorted nRBC regions from 3 healthy dogs showed  ≥  89%  nRBCs  in  all  samples,  with  89-99% in the early-stage nRBC region and 98-100% nRBCs in the mid- and late-stage nRBC regions. Additionally, populations that were CD18-negative and LDS751-moderate, and CD18-negative and LDS751-negative, consisted of reticulocytes (Figure 8A-C, gate R2; and 9E) and mature erythrocytes (Figure 8A-C, gate R3; and 9F), respectively, making it possible to assess erythroid precursors, reticulocytes, and erythrocytes simultaneously if enough cells were acquired. 85 Most of the contaminating cells in the nRBC regions appeared lymphoid, and because of the greater contamination in the nRBC gate of top Percoll fractions, this region was further assessed by sorting and cytologic evaluation after splitting it into top and bottom sections. The top and bottom sections had similar numbers of lymphocytes, and the bottom was found to include many mid-stage nRBCs in mitosis. Consequently, to maximize interrogation of early-stage nRBCs, only the top section of the nRBC region from the top Percoll fraction was used to assess for IgG on nRBC. Additional sorting of non-erythroid regions of selected clinical samples demonstrated where signals of other potentially contaminating populations fell, including plasma cells, macrophages, lymphocytes, and myeloid cells (Figure 8A-C). Figure 8: Flow cytometric identification of potentially contaminating cell populations based on cell sorting of non-erythroid regions. Representative dot plots of bone marrow cells in the top (A), middle (B), and bottom (C) Percoll fractions show different positivity patterns for CD18 (horizontal axis) and LDS751 (vertical axis). Erythroid precursors (R1), reticulocytes (R2), and mature erythrocytes (R3) were CD18-negative and LDS751-positive, CD18-negative and LDS751-moderate, and CD18-negative and LDS751-negative, respectively. Additionally, the following non-erythroid cell populations 86 Figure  8  (cont’d): were identified in the labeled regions: * lymphocytes (A-B),  †  myeloid cells (A-C), ¶ macrophages (A, not present in this sample), and ** plasma cells (B). Figure 9: Cytologic findings in sorted samples from erythroid regions of LDS751 versus CD18 dot plots. A) Representative dot plot of bone marrow cells in the middle Percoll fraction with CD18 labeling (horizontal axis) and LDS751 staining (vertical axis) showing erythroid precursor (R1), reticulocyte (R2), and mature erythrocyte (R3) gates, respectively. Cell sorting of top, middle, and bottom fractions confirmed R1 gates of each to be enriched in early- (B), mid- (C), and late-stage (D) erythroid precursors, respectively, with little contamination of other cell types, and R2 and R3 gates (A) to consist mostly of reticulocytes (E) and mature erythrocytes (F), respectively. 87 IgG detection on nRBCs The median percentages of IgG positive events in the nRBC regions of 10 healthy dogs were 5.7% (range, 1.6-12.3%) for the early nRBCs, 8.6% (range, 2.5-15.2%) for mid-stage nRBCs, and 6.8% (range, 4.2-17.5%) for the late-stage nRBCs. Reticulocytes were abundant enough in the bottom Percoll fractions of each dog to assess, and they had a median of 5% positive IgG events (range, 2.1-15.9%). Mature RBCs were not generally acquired in enough concentration to assess for IgG; they were instead assessed using blood samples. Median IgG signals for the 3 DEA1.1-negative dogs after incubation with antiDEA1.1 typing serum were 4.2% (range, 3.6-7.3%), 6.0% (range, 5.3-10.2%), 16.1% (range, 13.1-16.3%), and 12.6% (range, 11.8-25.6%) positive events for the top, middle, and bottom Percoll fractions and the bottom-fraction reticulocytes, respectively. These cell populations from the 3 DEA1.1-positive dogs after sample incubation with antiDEA1.1 typing serum yielded median IgG results of 71.1% (range, 49.1-90.1%), 98.1% (range, 95.7-99.3%), 99.4% (range, 99.3-99.5%), and 100% positive events, respectively. There were significant differences in % positive events for all nRBC fractions and bottom-fraction reticulocytes when comparing DEA1.1-positive dogs and healthy dogs. Although median fluorescence (not shown) and % positivity results were clearly different for nRBCs from DEA1.1-negative and DEA1.1-positive dogs (Figure 10), the differences were not statistically significant. 88 Figure 10: nRBC IgG detection using DEA1.1-positive cells coated with anti-DEA1.1 IgG. Erythroid precursors from the top (A), middle (B) and bottom (C) Percoll fractions (i.e., early-, mid-, and late-stage precursors, respectively), and reticulocytes (D) from the bottom Percoll fraction had strong signals for IgG in DEA1.1-positive dogs in contrast to those from healthy (*, p<0.05) and DEA1.1-negative dogs after cells from DEA1.1positive or -negative dogs had been incubated with anti-DEA1.1 typing serum. Median, minimum, and maximum values are shown for each group. 89 DISCUSSION In order to investigate the role of IgG in canine immune-mediated anemias, including IMHA and PIMA, we first developed and herein characterized a flow cytometric assay for RBC IgG using a heavy-chain specific antiglobulin reagent and positive and negative control cells. The assay proved to have good analytical precision and accuracy, and its diagnostic accuracy was good when applied to a population of sick dogs with or without IMHA. This RBC IgG assay was then applied to early-, mid-, and late-stage nRBCs after developing a process to harvest them from bone marrow and identify them by a 2-color flow cytometric assay. This nRBC assay was shown to reliably detect IgG on early-, mid- and late-stage nRBCs, and reticulocytes. Additionally, we describe for the first time, based on immunologic detection, the expression of DEA1.1 on early- to late-stage canine nRBCs, and reticulocytes. During initial stages of assay development, we attempted to detect IgM and C3 on erythroid cells, in addition to IgG, as IgM and C3 may also play roles in mediating cell destruction. However, the IgM assay was hindered by difficulty in developing a reliable positive control solely for IgM, and despite development of a reliable positive control for canine complement, extensive testing of dogs with IMHA yielded only one questionable positive result for canine C3. These findings together with differences in canine and human complement results in vitro (data not shown) and the negative findings of others with similar assays (Wilkerson et al., 2000; Quigley et al., 2001), led us to question the reliability of commercially available anti-[dog C3] antibodies to detect complementmediated opsonization of canine erythrocytes. Therefore, assay development was limited to detection of IgG, further ensured by use of a heavy-chain specific antiglobulin 90 reagent, which was selected to minimize cross reactivity with other classes of immunoglobulins. Although there are different approaches to defining positivity in flow cytometric assays for cell-associated antibody (Kucinskiene et al., 2005; Lucidi et al., 2011; Quigley et al., 2001; Morley et al., 2008; Wilkerson et al., 2000), we used % positive events after accounting for nonspecific binding of a nonsense antibody because this approach appeared to be most sensitive and reliable. It was more sensitive than median or mean fluorescence intensity because many dogs with IMHA had only a subpopulation of detectably positive cells, and the positive events could be clearly increased without significantly shifting the median fluorescence (Figure 11). Regarding nonspecific binding, amounts varied among individuals, and increased amounts of autologous canine IgG on the cell surface was associated with greater nonspecific binding of the nonsense antibody and thus presumably the test antibody (Figure 11). Therefore, use of the nonsense antibody binding to set the cutoff for each sample was important. One particularly noteworthy dog with zinc toxicity had nonspecific binding with the nonsense antibody that was as great as a strong positive reaction, so it was impossible to assess IgG positivity in this sample and the dog was excluded from the study. Similar to other dogs with zinc toxicosis (Gurnee et al., 2007), this dog had a severe regenerative anemia (hematocrit = 13%) associated with marked hemolysis, Heinz bodies, moderate spherocytosis (not pyknocytosis), many ghost cells, and schizocytosis. Although this dog may truly have been positive for RBC IgG, the strong nonspecific signal made analysis impossible, perhaps because of high autofluorescence or membrane changes causing increased adherence of nonsense and test antibodies. 91 Figure 11: Median fluorescence intensity (MdFI) versus % positive events for assessing IgG positivity. Among different ways to assess RBC IgG positivity, % positive events was the most sensitive as represented here by a 2-fold increase in MdFI, but a 10-fold increase in % positive events (% pos) when comparing RBCs incubated with IgG test antibody (bottom row) from a weakly positive IMHA dog to a healthy dog. An IgG nonsense antibody was used to set a cutoff for IgG positivity for every dog in order to account for nonspecific binding (top row), and nonspecific binding tended to be greater for individuals with more IgG – IMHA (strong) – than for those with lower amounts of autologous canine IgG on the cell surface – Healthy and IMHA (weak). Clinical evaluation of the RBC IgG assay in dogs with and without IMHA were favorable, with few (4 of 33) negative results in dogs with IMHA and few (2 of 28) positive results in unhealthy dogs without a clinical diagnosis of IMHA. The four negative 92 results in IMHA dogs may have been related to mediation by IgM (C3) or IgA without involvement of IgG (Piek et al., 2012; Wilkerson et al., 2000; Slappendel 1979; Day 1996; Segel et al., 2014), or, in one dog, to 4 days of immunosuppressive therapy prior to testing, as IgG positivity by flow cytometry has been shown to decrease with treatment (Wilkerson et al., 2000). Low amounts of cell-bound IgG (Sachs et al., 2006; Segel et al., 2014) or insufficient assay sensitivity may also have yielded negative results, but flow cytometric assays have been described as having greater diagnostic sensitivity  for  IMHA  than  the  Coombs’  test  in  both  human  and  canine patients (Wilkerson et al., 2000; Quigley et al., 2001; Kucinskiene et al., 2005; Wang et al., 2001; Chaudhary et al., 2006; Lin et al., 2009). Finally, false-negative results could have been caused by low affinity IgG removed by cell washes not conducted at 4°C or with low ionic strength buffers (Segel et al. 2014). Regarding the two positive results for RBC IgG in the 28 unhealthy non-IMHA dogs, one result was very weak in a nonanemic dog with a pheochromocytoma, and one was mildly increased in an anemic dog (hematocrit = 33%) with hypoadrenocorticism and hypothyroidism. Further evaluation of the anemia in this dog was not done. Although it has been reported that storage of blood samples for up to 7 days did not  affect  Coombs’  test  results  when  compared to fresh samples (Caviezel et al., 2014), effects on a flow cytometric assay, which may be more sensitive, were not assessed. Our results from 20 healthy dogs showed that testing blood-derived RBCs for IgG on the day of collection versus the day after collection following storage at 4°C did not affect results or clinical interpretation, and we have not seen clinically significant differences with 1-2 day storage in sporadic paired assessments, but we have not systematically 93 assessed effects of storage over longer periods of time or on samples from unhealthy or positive dogs. Favorable analytical and diagnostic performance of the RBC IgG assay led to efforts to apply it to nRBCs from the bone marrow. Initial attempts involved lysis of RBCs to remove them as interfering events from bone marrow samples. However, pre- and post-lysis cytologic preparations indicated that tris ammonium chloride, ammonium chloride, and a commercial erythrocyte lysing buffer (Erythrolyse Red Blood Cell Lysing Buffer, AbD Serotec, Raleigh, NC) all resulted in lysis of nRBCs in addition to mature RBCs (data not shown). By combining a Percoll gradient separation method with a 2color flow cytometric assay, concentrations of mature RBCs and reticulocytes were markedly reduced and easily discriminated from nRBCs, and omission of a lysis step decreased potential cell damage and loss. Initial studies also explored an anti-canine CD45 (Rat anti dog CD45:RPE monoclonal antibody, AbD Serotec, Raleigh, NC) antibody to separate erythroid cells from other populations (Cobbold et al., 1994; Weiss 2004), but it yielded poorer discrimination (results not shown) of cell populations than did the anti-[dog-CD18] antibody, so its use was discontinued. If anti-[dog nRBC] antibodies (e.g. anti-[dog transferrin receptor], anti-[dog glycophorin]) become available, they should be explored as better labor- and time-saving options that would mirror human methods for nRBC analysis (Loken et al., 1987; Civin et al., 1987; Chen et al., 2009; Bony et al., 1999). As  finally  developed,  the  assay’s  CD18-negative and LDS751-positive populations contained very few non-erythroid cells, and combined use of Percoll fractionation and 2color separation allowed independent analysis of largely early-, mid-, and late-stage nRBCs. All three of these nRBC fractions from DEA1.1-positive dogs had strong signals 94 for IgG after incubation with anti-DEA1.1 typing serum. This was in contrast to the nRBC fractions from DEA1.1-negative dogs submitted to the same treatment, all of which had results similar to those of 10 healthy dogs. Detection of this IgG on the DEA1.1-positive nRBCs demonstrated the ability of the assay to detect nRBC IgG, and supports its application to dogs with PIMA. Additionally, it provided evidence that DEA1.1 is expressed on early through late stages of nRBCs in DEA1.1 positive dogs, something not  reported  to  date,  to  the  authors’  knowledge.  Further  evidence  of  DEA1.1  expression   on canine nRBCs could be sought by nonimmunologic approaches such as mass spectrometry of RBC membranes or detection of DEA1.1 mRNA. The presence of DEA1.1 on precursors could be of interest given that certain blood group antigens expressed on human erythroid precursors (Blacklock et al., 1984; Bony et al., 1999; Wada et al., 1990) and human RBC-incompatible allogeneic bone marrow transplantations can have deleterious consequences such as immediate and delayed hemolytic transfusion reactions, delayed RBC recovery, and pure red cell aplasia (Blacklock et al., 1984; Rowley et al., 2011). Even though current donor bone marrow conditioning and immunosuppressive regimens appear to overcome these deleterious effects, crossmatching is still warranted, and RBC-compatible bone marrow transplants are preferable (Rowley et al., 2011). DEA1.1 expression on canine erythroid precursors may be relevant to allogeneic bone marrow transplantations in dogs. In conclusion, we have developed and described a new method for isolation and identification of early-, mid-, and late-stage nRBCs from the bone marrow of dogs, and demonstrated that we can detect IgG on all erythroid populations by applying the fundamentals of a flow cytometric assay for IgG detection on RBCs. In the process, we obtained evidence that DEA1.1 is expressed on rubriblasts through erythrocytes of 95 DEA1.1-positive dogs. 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Weiss DJ, Stockham SL, Willard MD, et al. Transient erythroid hypoplasia in the dog: report of five cases. J Am Anim Hosp Assoc. 1982;18:353-359. Weiss DJ. Bone marrow pathology in dogs and cats with non-regenerative immunemediated haemolytic anaemia and pure red cell aplasia. J Comp Pathol. 2008;138:4653. Weiss DJ. Flow cytometric evaluation of canine bone marrow based on intracytoplasmic complexity and CD45 expression. Vet Clin Pathol. 2004;33:96-101. Wilkerson MJ, Davis E, Shuman W, et al. Isotype-specific antibodies in horses and dogs with immune-mediated hemolytic anemia. J Vet Intern Med. 2000;14:190-196. 100 CHAPTER 4 Exploring the Roles of IgG and Phosphatidylserine in Canine Regenerative and Nonregenerative Immune-Mediated Anemias 101 ABSTRACT Background: Many cases of suspected immune-mediated precursor-targeted anemia (PIMA) have been described and suggested to be caused by immune targeting of erythroid precursors, but the pathogenic mechanism of anemia is unknown. Objective: To test the hypothesis that IgG and phosphatidylserine (PS) are involved in mediating PIMA, and that PS is involved in mediating IMHA. Animals: Blood and bone marrow samples from healthy colony dogs, and client-owned dogs with IMHA, PIMA, or other (non-IMHA, non-PIMA) conditions were assessed prospectively. Methods: A Percoll gradient separation method was used to separate different stages of erythroid precursors from bone marrow samples. Flow cytometric assays were used to assess for IgG and PS on erythrocytes (RBCs) and erythroid precursors. Results: RBC IgG was increased on 9 of 11 and 0 of 9 IMHA and non-IMHA dogs, respectively; 10 of 11 and 2 of 9 IMHA and non-IMHA dogs were positive for PS on RBCs, respectively. Erythroid precursor IgG was not statistically different between PIMA and non-PIMA dogs, but 5 of 17 and 0 of 7 PIMA and non-PIMA dogs, respectively, were positive based on values from healthy dogs. Erythroid precursor PS was significantly increased in PIMA dogs compared to healthy dogs, with positivity in 5 of 6 PIMA dogs. Conclusions and Clinical Importance: Our data suggests a role for PS in canine IMHA, and for IgG and PS in canine PIMA. Additional studies are warranted to further assess multiple mechanisms and optimal therapy. 102 INTRODUCTION Precursor-targeted immune-mediated anemia (PIMA) has been described in dogs with nonregenerative anemia and evidence of ineffective erythropoiesis, and although it has been suggested that the pathogenesis may relate to immune targeting of erythroid precursors (Jonas et al., 1987; Stockham et al., 1980; Weiss et al., 1982; Holloway et al., 1990; Scott-Moncrieff et al., 1995; McManus 2000; Stokol et al., 2000; Weiss 2008), this has not been demonstrated. Suspicion of an immune-mediated mechanism is based on: 1) apparent responses to steroid treatment (Jonas et al., 1987; Stockham et al., 1980; Weiss et al., 1982; Holloway et al., 1990; Scott-Moncrieff et al., 1995; Stokol et al., 2000) and relapses when tapering it (Stockham et al., 1980; Weiss et al., 1982; ScottMoncrieff et al., 1995; Stokol et al., 2000), 2) phagocytosis of intact erythroid precursors (Jonas et al., 1987; Stockham et al., 1980; Holloway et al., 1990; McManus 2000), and 3) association with concurrent evidence of immune-mediated hemolytic anemia (IMHA), such  as  spherocytosis,  red  blood  cell  (RBC)  agglutination,  or  Coombs’  positivity  (Jonas   et al., 1987; Stockham et al., 1980; Holloway et al., 1990; Scott-Moncrieff et al., 1995; Stokol et al., 2000; Weiss 2008), or with other immune-mediated disease such as immune-mediated thrombocytopenia (IMT) (Holloway et al., 1990; Scott-Moncrieff et al., 1995; Stokol et al., 2000). However, dogs diagnosed with PIMA typically respond slowly to immunosuppressive therapy (Jonas et al., 1987; Stockham et al., 1980; Weiss et al., 1982; Holloway et al., 1990; Scott-Moncrieff et al., 1995; Stokol et al., 2000), some do not respond at all (Jonas et al., 1987; Stokol et al., 2000), and many have no evidence of concurrent IMHA (Weiss et al., 1982; Scott-Moncrieff et al., 1995; Stokol et al., 2000), leaving unanswered questions about the mechanism(s) of anemia in these dogs. 103 Dogs with an ultimate diagnosis of PIMA account for approximately 25% of canine bone marrow submissions to the Diagnostic Center for Population and Animal Health (DCPAH) at the Michigan State University Veterinary Medical Center (MSU VMC), and for approximately 50% of those submissions for dogs with the main complaint of nonregenerative anemia (Chapter 2). In our experience, phagocytosis of intact erythroid precursors, rubriphagocytosis (RP), by normal-appearing macrophages is found in bone marrow samples from most dogs with PIMA. This RP is usually selective for the erythroid lineage and for selective stages of erythroid development, with RP of different stages associated with different bone marrow patterns (Lucidi et al., 2010; Chapter 2). This suggests a role for phagocytosis in the ineffective erythropoiesis and nonregenerative anemia in these dogs. The different bone marrow patterns seen in these dogs may reflect a spectrum of disease caused by a single mechanism, or they may result from multiple mechanisms. Phagocytosis by macrophages may proceed through recognition of several cell membrane mediators, including IgG, complement, and phosphatidylserine (PS). Although the role of antibodies in the pathogenesis of IMHA is well demonstrated, membrane exposure of PS through oxidative stress (Pesillo et al., 2004; Tan et al., 2012) and an apoptotic-like mechanism (Tan et al., 2012) has been suggested to contribute to RBC destruction in dogs and mice (Lee et al., 2004). Eryptosis, which is apoptosis of mature RBCs, is characterized by cell shrinkage, cell membrane scrambling, and PS exposure triggering phagocytosis by macrophages (Lang et al., 2015). Apoptosis of erythroid precursors may occur as in other nucleated cells, and increased PS exposure of bone marrow-derived erythroid precursors has been 104 demonstrated in association with IgG positivity in one human patient with reticulocytopenic immune-mediated anemia (van de Loosdrecht et al., 2000). The goal of this study was to explore the potential roles of IgG and PS in mediating canine immune-mediated anemias. We hypothesized that PS exposure on RBCs and erythroid precursors plays a role in canine IMHA and PIMA, respectively, and that IgG on erythroid precursors contributes to PIMA as it does to IMHA. 105 MATERIAL AND METHODS Animals RBCs from suspected IMHA dogs admitted to the MSU VMC from October 2013 through November 2014 were assessed for IgG and PS. RBCs from unhealthy dogs thought to lack immune-mediated anemia and collected within 24 h of samples from suspected IMHA samples were also tested for IgG and PS in order to evaluate the specificity of results for IMHA. Some dogs from each group were excluded after testing if they were later found to not fulfill criteria. Inclusion criteria for IMHA required a clinical diagnosis of IMHA associated with regenerative anemia (all had hematocrit [Hct] below 33%) and at least one of the following with no other explanation: 1) spherocytosis, 2) erythrocyte  agglutination,  and  3)  Coombs’  test  positivity.  Unhealthy  non-IMHA dogs were defined as any sick dogs with no clinical suspicion or hematologic evidence of immune-mediated anemia, including the absence of spherocytosis, RBC agglutination, and  Coombs’  positivity. Dogs from which a bone marrow aspirate sample was collected for cytologic examination at the MSU VMC from April 2012 through December 2014 were assessed for IgG on erythroid precursors and RBCs. Samples of dogs assessed after October 2013 were also tested for PS. The following data were collected: signalment, clinical history and presenting complaint, bone marrow aspirate (and core, when available) findings, relevant diagnostic test results, clinical diagnosis and therapeutic regimen, and clinical outcome with emphasis on hematologic response. Clinical and diagnostic data were reviewed in order to identify dogs with clear diagnoses of PIMA and dogs that clearly did not have PIMA (non-PIMA dogs). Inclusion criteria for PIMA dogs were as follows: 1) unexplained persistent nonregenerative or inappropriately regenerative 106 anemia  (on  the  day  of  bone  marrow  collection,  all  had  Hct  ≤  29%,  inadequate  erythroid regeneration for the degree of anemia, and documented anemia or anemia-related signs for at least 5 days), 2) ineffective erythropoiesis, as evidenced by persistent anemia with erythroid hyperplasia or by erythroid maturation arrest or left-shift with RP, 3) lack of erythroid dysplasia, and 4) a clinical diagnosis of PIMA. Inclusion criteria for non-PIMA dogs were the same as described for unhealthy non-IMHA dogs, with the addition of 1) no evidence of erythroid precursor targeting, including absence of ineffective erythropoiesis, and 2) a clear clinical diagnosis of a condition other than PIMA. Dogs were excluded from the study if they had any evidence of neoplastic cells in bone marrow samples, or if the cell yield from bone marrow aspirates was insufficient for flow cytometric assessment. A control population for IgG and PS on RBCs consisted of 20 clinically and hematologically healthy adult dogs enrolled in the MSU VMC Blood Donor Program (Chapter 3). Similarly, IgG was assessed on erythroid precursors from 10 clinically and hematologically healthy adult colony dogs (Chapter 3), and PS was assessed for 5 of these dogs. Use of leftover blood and bone marrow specimens from clinical patients, and sample collection procedures from all dogs were performed in compliance with the MSU and MPI Research Institutional Animal Care and Use Committees. RBC samples and isolation RBCs from clinical patients were isolated from ethylenediaminetetraacetic acid (EDTA)-anticoagulated  blood  submitted  to  the  DCPAH  laboratory  for  a  CBC  or  Coombs’   test. Blood was tested on the day of collection or stored at 4°C and tested on the next day. RBCs  were  isolated  as  described  in  a  previous  study  (Chapter  3).  Briefly,  10  μL  of   107 packed  RBCs  were  washed  twice  and  resuspended  in  800  μL  phosphate  buffered  saline   (PBS) containing 0.3% bovine serum albumin (BSA) to make a RBC suspension. Bone marrow samples and erythroid precursor isolation Erythroid precursors of bone marrow-tested dogs were isolated from samples collected into 0.5 mL of 3% EDTA for cytologic assessment. After collection, bone marrow material was transferred to a Petri dish for particle collection and slide preparation, transported to the laboratory in a cooled container, and sample processing was initiated within 60 minutes of collection. Erythroid precursors were isolated as described in a previous study (Chapter 3). Briefly, 2-5 mL of bone marrow aspirate was washed with PBS-1%BSA, cells were applied to a Percoll density gradient with 68, 64, and 52% Percoll layers, and centrifuged for 30 minutes at 500 g. The three layers of cells formed by centrifugation were harvested from top to bottom into individual tubes. Cells were washed, and nucleated cell concentration was determined by hemocytometer. The mixtures of cells in these top, mid, and bottom Percoll fractions were shown by cell sorting (Chapter 3) to be enriched in early (rubriblasts and prorubricytes), mid (early- to mid-rubricytes), and late (mid- to late-stage rubricytes and metarubricytes) erythroid precursors, respectively. This separation allowed for IgG and PS assessment of distinct stages of erythroid development. For every sample, stop-flow cytologic preparations of each fraction were prepared and assessed microscopically in order to ensure proper separation. For 5 healthy dogs, bone marrow samples were transported in a cooled container from MPI Research in Mattawan, MI to MSU after harvesting the Percoll layers and before continuation of the protocol for IgG testing. 108 RBC testing for IgG As described previously (Chapter 3), RBC suspensions were incubated with a fluorescein isothiocyanate (FITC) conjugated anti-[dog  IgGγ]  antibody (IgG test antibody, KPL, Gaithersburg, MD) and analyzed on a FACSCalibur flow cytometer (BectonDickinson, San Jose, CA) with CellQuest Pro software (Becton-Dickinson, San Jose, CA). For each sample, 10,000 events with target FSC and SSC properties (Lucidi et al., 2011) were acquired, and RBC agglutinates were excluded by gating (Arndt et al., 2010). Controls for every patient sample were: 1) RBCs from a healthy dog incubated with IgG test antibody (negative control for IgG), 2) dog erythrocyte antigen (DEA)1.1positive RBCs treated with a polyclonal anti-DEA1.1 typing serum (Animal Blood Resources International, Stockbridge, MI) and then incubated with IgG test antibody (positive control for IgG), and 3) RBCs from each tested patient incubated with an anti[chicken IgG-heavy and light chain]-FITC-labeled antibody (nonsense antibody, KPL, Gaithersburg, MD) to control for nonspecific binding. IgG positivity was determined by % positive events as follows: RBCs from each tested dog incubated with nonsense antibody were used to set a marker for 2% positive events, based on a dot plot distribution. RBCs incubated with IgG test antibody that fell above this marker were considered positive events for IgG. The same strategy was applied to results of 20 healthy dogs, and cutoffs for IgG positivity were calculated based on the mean percent positive events plus 3 standard deviations (SDs). Patients with greater % positivity were considered positive for RBC IgG (Figure 12). 109 Figure 12: Representative dot plots of flow cytometric IgG fluorescence (vertical axis) versus forward scatter (horizontal axis) of red blood cells (RBCs). Markers for positivity were set at 2% positive events for each tested dog after incubation with nonsense antibody (A, C), and RBCs falling above this marker after incubation with IgG test antibody were used to determine positivity (B, D). Examples are of a healthy dog, 3.2% positive events (A, B), and an IgG-positive dog, 96.2% positive events (C, D). RBC testing for PS RBCs  were  prepared  for  PS  analysis  by  washing  20  μL  of  RBC  suspension  with   annexin buffer (140 mM NaCl, 5 mM CaCl2, and 10 mM HEPES, pH 7.4; filter sterilized) and  then  resuspending  and  incubating  the  RBCs  in  the  dark  for  15  minutes  in  100  μL  of   110 incubation  buffer  mixed  with  2  μL  of  5(6)-carboxyfluorescein-N-hydroxysuccinimide ester-conjugated annexin V (annexin V-FLUOS, Roche Diagnostics, Indianapolis, IN) in the presence of calcium (AnV-Ca).  RBCs  were  then  resuspended  into  400  μL  of  annexin   buffer and analyzed by flow cytometry. Controls consisted of: 1) RBCs from a healthy dog incubated with AnV-Ca (negative control for PS), 2) PS-positive control RBCs incubated with AnV-Ca (positive control for PS), 3) RBCs from each tested patient incubated as described above, but in the absence of annexin V-FLUOS (control for RBC autofluorescence), and 4) RBCs from each tested patient incubated as described above, but with a calcium-free annexin control buffer (annexin buffer without CaCl2) used in every step (AnV-Ca-free) to control for nonspecific binding of annexin, since binding to PS requires calcium (Tait et al., 1992). PS-positive  control  cells  were  made  by  twice  washing  10  μL  of  packed  RBCs   from a healthy dog with a BSA-NaCl solution (147.5 mM NaCl, 2.5 mM KCl, 5 mM Dglucose, 20 mM HEPES, 0.1% BSA, and 0.025% sodium azide; pH 7.5), resuspending the  cell  pellet  into  800  μL  of  the  same  solution  without  BSA  or  sodium  azide,  and   incubating  200  μL  of  this  RBC  suspension  with  2  μL  of  10  mM  2′(3′)-O-(4benzoylbenzoyl)adenosine  5′-triphosphate triethylammonium (BzATP, Sigma-Aldrich, St. Louis, MO) stock solution at 37°C for 4 hours (Sluyter et al., 2007). RBCs were then washed  and  resuspended  in  200  μL  of  annexin  buffer  for  use. PS positivity was determined by percent positive events as follows: control RBCs from each tested dog were incubated with AnV-Ca-free and used to set a visual marker to include the main population of RBCs in the fluorescence histogram. This resulted in 0.1-0.7% positive events outside the main peak. Those test RBCs incubated with AnVCa that had greater fluorescence were considered positive PS events. This strategy was 111 applied to results of 20 healthy dogs, and cutoffs for PS positivity were calculated based on the mean % positive events plus 3 standard deviations (SD). Patients with % positive events above the cutoff determined based on healthy dogs were considered positive for PS on RBCs (Figure 13). IgG and PS results from same-day and next-day patient samples were interpreted using same-day and next-day reference cutoffs, respectively. Figure 13: Representative frequency histograms of flow cytometric phosphatidylserine (PS) fluorescence (horizontal axis) on red blood cells (RBCs). Markers for positivity were set at the end of the main population for each tested dog after incubation with annexin V-FLUOS in the absence of calcium (A, C), and RBCs with fluorescence greater than this marker after incubation with annexin V-FLUOS in the presence of calcium were 112 Figure 13 (cont’d): used to determine positivity (B, D). Examples are of a healthy dog, 0.3% positive events (A, B), and a PS-positive dog with IMHA and 5.4% positive events C, D). Erythroid precursor testing for IgG and PS Assessment of IgG on erythroid precursors was performed as previously described (Chapter 3). Briefly, nucleated bone marrow cells from each Percoll fraction were incubated with a monoclonal murine anti-[dog CD18] antibody (AbD Serotec, Raleigh, NC) and then with a phycoerythrin (PE) conjugated polyclonal goat anti-[mouse IgG] antibody (AbD Serotec, Raleigh, NC) and an IgG test antibody. Cells were then incubated for 20 minutes to approximately 1 hour (longer times for samples analyzed later in a batch) with the nucleic acid stain LDS751 (Life Technologies, Grand Island, NY) as previously described (Saad et al., 2000) immediately prior to flow cytometric analysis. At least 10,000 total events were acquired, depending on erythroid precursor concentrations, and erythroid precursors were identified as CD18-low, LDS751-high events. Negative and positive IgG controls were the same as used for RBC testing, with the addition of Percoll fractions of test-dog bone marrow cells incubated with nonsense antibody. Percent positive results for IgG were determined based on IgG nonsense and test antibodies in the same manner as for RBCs (Figure 14). 113 Figure 14: Representative dot plots of flow cytometric IgG fluorescence (vertical axis) versus forward scatter (horizontal axis) of erythroid precursors (nRBCs). Markers for positivity were set at 2% positive events for each tested dog after incubation with nonsense antibody (A, C), and events falling above this marker after incubation with IgG test antibody were used to determine positivity (B, D). Examples are of nRBCs from the mid Percoll fractions of a healthy dog, 2.5% positive events (A, B), and an IgG-positive PIMA dog, 57.6% positive events (C, D). Erythroid precursor testing for PS was done by applying the same method used for RBCs, but assessing 106 bone marrow-derived nucleated cells from each Percoll fraction.  After  resuspending  cells  in  400  μL  of  annexin  buffer,  cells  were  mixed  with  160   μL  of  LDS751 working solution (Saad et al., 2000) and incubated for 20 minutes to 114 approximately 1 hour in the dark before flow cytometric analysis. Erythroid precursors were identified based on CD18 labeling and LDS751 staining. Negative and positive PS controls were the same as those used for RBCs, and nucleated cells from Percoll fractions were additionally incubated with LDS751 to test for autofluorescence and nonspecific annexin binding. PS positivity was determined by % positive events as follows: erythroid precursors from each Percoll fraction of each dog were incubated with AnV-Ca-free and used to set a marker with 2% positive events based on dot plot distributions. All test sample events with fluorescence greater than this cutoff were considered positive (Figure 15). Because PS positivity was high on erythroid precursors compared to RBCs, effects of processing and time were individually assessed on unfractionated marrow nucleated cells by testing them immediately and 5.5 hours later, the duration of the assay. No effects of processing (i.e., Percoll fractionation, washing, or time) on PS results were detected, but nucleated erythroid populations could not be assessed alone, as incubations with CD18 and LDS751 reagents would have been required, thus adding additional variables of time and washing. 115 Figure 15: Representative dot plots of flow cytometric phosphatidylserine (PS) fluorescence (vertical axis) versus forward scatter (horizontal axis) of erythroid precursors (nRBCs). Markers for positivity were set at 2% positive events for each tested dog after incubation with annexin V-FLUOS in the absence of calcium (A, C), and events falling above this marker after incubation with annexin V-FLUOS in the presence of calcium were used to determine positivity (B, D). Examples are of nRBCs from the mid Percoll fractions of a healthy dog, 7.5% positive events (A, B), and a PS-positive PIMA dog, 53.1% positive events (C, D). Statistical analysis Comparisons among 3 or more groups were performed using Kruskall-Wallis and Dunn’s  multiple  comparison  tests.  Comparisons between healthy dogs tested for IgG 116 and PS on same-day or next-day were done using the paired t-test, and the two-tailed Mann-Whitney test was used to assess differences in erythroid precursor PS between PIMA and healthy dogs. Statistical analyses were performed using statistical software (GraphPad Prism, GraphPad Software Inc., San Diego, CA), and differences were considered to be significant at p<0.05. 117 RESULTS RBC IgG and PS in IMHA Eleven IMHA and 9 non-IMHA dogs were assessed. Of the non-IMHA dogs, 1 was not anemic, and the other 8 had regenerative (4 of 8, Hct range: 30-35%) and nonregenerative (4 of 8, Hct range: 28-39%) anemias. Flow cytometric results revealed 9 of 11 IMHA and 0 of 9 non-IMHA dogs positive for IgG (Figure 16). The median % positive events for IgG in IMHA and non-IMHA dogs were 46.8% (range: 2.6-100%) and 4.4% (range: 2.2-8.2%), respectively. One of the two IgG-negative IMHA dogs had been treated with prednisone for 4 days before blood was collected. There was no significant difference in RBC IgG for healthy dogs tested on the day of collection or the day after collection, so same-day results were used for group comparisons with sick animals. RBC IgG results in dogs with IMHA were significantly different from those of healthy and non-IMHA dogs, but no statistical difference was found between healthy and non-IMHA dogs. 118 Figure 16: IgG positivity on RBCs from healthy dogs (n = 20) and dogs with IMHA (n = 11) or without IMHA (non-IMHA, n = 9). RBCs from healthy dogs were tested on the day of and 1 day following sample collection. Horizontal bars represent group medians and the dashed line represents the cutoff for IgG positivity based on healthy dogs tested on the day of collection. a No significant difference, and b Significantly different (p<0.05). RBC PS positivity was found on 10 of 11 IMHA dogs and 2 of 9 non-IMHA dogs (Figure 17). The median % positive events for PS in IMHA and non-IMHA dogs were 1.9% (range: 0.8-5.4%) and 0.6% (range: 0.2-1.8%), respectively. One of the two PSpositive non-IMHA dogs had 0.9% positive events, no anemia, and a diagnosis of chronic renal disease, while the other had 1.8% positive events, mild nonregenerative anemia (Hct = 35%), and a diagnosis of immune-mediated neutropenia. There was no 119 significant difference in RBC PS for healthy dogs tested on the day of collection or the day after, so same-day results were used for group comparisons with sick animals. PS results for dogs with IMHA were significantly different from those of healthy dogs, but PS results for non-IMHA dogs were not significantly different from those of either IMHA dogs or healthy dogs. Figure 17: Phosphatidylserine (PS) positivity on RBCs from healthy dogs (n = 20) and dogs with IMHA (n = 11) or without IMHA (non-IMHA, n = 9). RBCs from healthy dogs were tested on the day of and 1 day following sample collection, and no significant difference was found in PS positivity. Horizontal bars represent group medians and the dashed line represents the cutoff for PS positivity based on healthy dogs tested on the day of collection. * Significantly different (p<0.05). 120 Erythroid precursor IgG and PS in PIMA Bone marrow samples from 40 dogs were analyzed, and after review of case materials for each, 17 PIMA and 7 non-PIMA dogs were identified. Of the 16 remaining dogs, one was excluded because of high nonspecific binding of erythroid precursors, one had diagnostically inadequate bone marrow samples, 3 were excluded because of suspected cytopenias secondary to phenobarbital therapy (Plumb 2015), the mechanism of which has not been demonstrated, and 11 were classified as unclear cases because they did not clearly fulfill criteria for either PIMA or non-PIMA groups. Unclear cases consisted of dogs with moderate to severe anemia (Hct < 30%) and erythroid left-shift and/or RP, which suggested a possible immune-mediated component against erythroid precursors, but PIMA criteria were not met for a variety of reasons. For some of these dogs,  anemia  was  too  mild  (Hct  ≥  30%)  or  not  persistent,  or  a  clinical   diagnosis of PIMA was not reached by the attending clinician. For others, there was nonselective phagocytosis involving the erythroid lineage, or unexplained anemia and erythroid hypoplasia, both of which could have involved an immune-mediated component against erythroid precursors. Clinical diagnoses for unclear dogs were immune-mediated neutropenia (IMN) (2 of 11), IMT (1 of 11), hemophagocytic syndrome secondary to ehrlichiosis (1 of 11), chronic large bowel diarrhea (1 of 11), and unknown (6 of 11); the dogs with unknown diagnoses had suspected immune-mediated cytopenias (3), suspected hemophagocytic syndrome or inflammatory/infectious disease (2), or pancytopenia that resolved without therapy (1). All 35 dogs classified as PIMA, non-PIMA, or unclear were tested for IgG, and 10 of those were also tested for PS on erythroid precursors (6 PIMA, 2 non-PIMA, and 2 unclear). Clean gating of early erythroid precursors in the top Percoll fraction was 121 problematic in certain clinical samples with a relatively large number of lymphocytes or early myeloid cells, so only the mid and bottom Percoll fractions were used to assess IgG and PS. Because of insufficient cells, not every fraction could be assessed for each dog. Of the 35 IgG-tested dogs, the bottom fraction was assessed in 34, and of the 10 PS tested dogs, the mid and bottom fractions were assessed in 9 and 8 dogs, respectively. No statistical differences were found among dogs classified as healthy, PIMA, nonPIMA, or unclear regarding IgG positivity on nRBCs. However, compared to fractionspecific cutoffs based on the median percent positive events plus 3 SD for 10 healthy dogs, 5 of 17 PIMA dogs (Figure 18) were positive for IgG on erythroid precursors from at least one of the two Percoll layers (range: 19.0-59.5% positive events), while IgG positivity was not found in any of the 7 non-PIMA dogs. Two of the 12 IgG-negative PIMA dogs had been on immunosuppressive therapy for 8 (prednisone) to 24 (mycophenolate) months, and two had received prednisone in the 5 days prior to bone marrow collection. Additionally, 4 of 11 unclear cases were positive for IgG with one or both fractions (range: 21.2-36.4% positive events). One of these dogs had pancytopenia and RP with potential immune-mediated destruction of all cell lineages. RP was not detected in the other 3 dogs, one with pancytopenia suspected to be secondary to recent bone marrow injury, one with leptospirosis but evidence of secondary hemophagocytic syndrome, and one with an unexplained nonregenerative anemia associated with IMT. 122 Figure 18: IgG positivity on erythroid precursors from the mid (A) and bottom (B) Percoll fractions of bone marrow from healthy dogs (n = 10) and dogs with PIMA (n = 17 for mid, n = 16 for bottom), without PIMA (non-PIMA, n = 7), or with unclear diagnoses (n = 11). Horizontal bars represent group medians and dashed lines represent cutoffs for IgG positivity based on healthy dogs. No statistically significant differences were detected, but increased values were present in some dogs with PIMA or unclear diagnoses that may have had an immunologic component. When comparing groups for PS on erythroid precursors, PIMA dogs (n = 5) were statistically different from healthy dogs (n = 5) for both the mid and bottom Percoll fractions, but there were too few (n = 2) non-PIMA dogs to compare statistically (Figure 19). When compared to fraction-specific cutoff values based on median % positive events plus 3 SD for 5 healthy dogs, 5 of 6 tested PIMA dogs were positive, four in the mid fraction (range: 36.9-77.8% positive events) and three in the bottom fraction (range: 64.6-70.0% positive events). Neither of the 2 non-PIMA dogs was positive for PS; 1 of the 2 unclear dogs was positive for PS (33.7% positive events) in the mid fraction. This dog had pancytopenia suspected to be secondary to recent bone marrow injury. 123 Figure 19: PS positivity on erythroid precursors from the mid (A) and bottom (B) Percoll fractions of bone marrow from healthy dogs (n = 5) and dogs with PIMA (n = 5 for mid and bottom), without PIMA (non-PIMA, n = 2 for mid, n = 1 for bottom), or with unclear diagnoses (n = 2). Horizontal bars represent group medians and dashed lines represent cutoffs for PS positivity based on healthy dogs. Dogs with PIMA had greater PS positivity than healthy dogs (*, p<0.05), but dogs without PIMA were too few for statistical comparisons. IgG was assessed on RBCs of 14 of 17 PIMA dogs, 6 of 7 non-PIMA dogs, and in 9 dogs with unclear diagnoses; positivity was found for 5 of 14 PIMA (9.5, 10.2, 16.0, 76.9, and 99.4% positive events), 2 of 6 non-PIMA (9.9 and 23.1% positive events), and 2 of 9 unclear (11.0 and 23.8% positive events) dogs (Figure 20). Three of the 5 PIMA dogs with IgG positivity on RBCs were also IgG positive on erythroid precursors, and one  of  them  was  also  Coombs’  test  positive.  PS  was  assessed  on  the  RBCs of 7 PIMA, 2 non-PIMA, and 4 unclear dogs, and all of them yielded negative results (Figure 20), 124 including one PIMA and one non-PIMA dog that each had IgG-positive RBCs (76.9 and 23.1% positive events, respectively). Figure 20: IgG (A) and PS (B) positivity on RBCs from healthy dogs (n = 20) and dogs with PIMA (n = 14 for IgG, n = 7 for PS), without PIMA (non-PIMA, n = 6 for IgG, n = 2 for PS), or with unclear diagnoses (n = 9 for IgG, n = 4 for PS). RBCs from healthy dogs were tested on the day of and 1 day following sample collection, and no significant difference was found in IgG or PS positivity. Horizontal bars represent group medians and dashed lines represent cutoffs for IgG or PS positivity based on healthy dogs tested on the day of collection. RP was seen in all 17 PIMA dogs, which, combined with bone marrow findings as previously described (Chapter 2), allowed characterization of  these  dogs’  conditions  as early (n = 2), mid (n = 4), or late PIMA (n = 11). The relationship between the Percoll fractions with IgG and/or PS positivity and stage of RP in PIMA dogs is shown in Table 6. 125 Table 6: Relationship between the stage of rubriphagocytosis (RP) reported on bone marrow examination and the Percoll fractions of bone marrow with IgG and/or phosphatidylserine (PS) positivity in PIMA dogs. Stage of RP Fraction with IgG Fraction with PS positivity positivity Late Mid, bottom ND Late Bottom ND Late Mid, bottom ND Late Mid, bottom Bottom* Late Bottom None Mid None Mid Late None Mid, bottom Late None Mid** Mid None Mid and bottom *Mid fraction not tested because of insufficient cells. **Bottom fraction not tested because of insufficient cells. ND = not done Clinical and diagnostic findings in PIMA dogs Nine PIMA dogs were spayed females, 7 were castrated males, and 1 was an intact male. Their ages ranged from 4 to 13 years old, with most dogs (n = 13) being middle-aged (5-10 years old). Dogs were of the following breeds: mixed (n = 5), dachshund (n = 3), miniature dachshund (n = 1), Labrador retriever (n = 2), Chihuahua (n = 1), German shepherd (n = 1), fox terrier (n = 1), Boston terrier (n = 1), Pembroke Welsh corgi (n = 1), and Havanese (n = 1). One dog had evidence of previous immunologic disease, having been diagnosed with PIMA 25 months prior to assessment for this study. Evidence of concurrent RBC targeting was seen in 2 of 17 PIMA dogs: mild spherocytosis in one mid-PIMA  dog,  and  Coombs’  positivity  at  20-21°C (but not 37°C) in one dog with late-PIMA.  Only  3  dogs  were  Coombs’  tested. 126 Mild to severe collagen myelofibrosis (MF) was detected in core biopsies of 4 of 17 PIMA dogs. All 4 dogs with MF were negative for IgG on erythroid precursors, and the only MF dog tested for PS was positive in both fractions. None of the 12 non-PIMA or unclear dogs assessed had MF, but 6 could not be assessed for MF because bone marrow core biopsy samples were inadequate (3) or not collected (3). All PIMA dogs were treated with immunosuppressive doses of prednisone, with or without other immunosuppressive drugs. Fourteen out of 17 PIMA dogs achieved remission (10) or responded (4), with remission based on achieving a Hct > 35%, and response based on either an increase in reticulocyte concentration to above the upper reference  limit  (76,000/μL)  or  a  Hct  increment  of  at  least  5  percentage  points  without   transfusion. Three dogs did not respond to treatment, one dying 90 days after diagnosis and two undergoing euthanasia 35 and 39 days after diagnosis because of clinical deterioration and suspected neoplasia in one of them. Of the 5 IgG-positive PIMA dogs, all responded and at least 4 achieved remission. Of the 5 dogs with increased PS, 4 of 5 responded, with at least one of the four achieving remission. The only PIMA dog that was positive for both IgG and PS achieved remission. 127 DISCUSSION These findings suggest that IgG contributes to PIMA by binding to erythroid precursors in at least a subset of dogs, and that PS exposure on erythrocytes and erythroid precursors may contribute to erythroid cell destruction in canine IMHA and PIMA. Further testing in a greater number of dogs is indicated. Increased RBC PS exposure in IMHA dogs supports a potential role for eryptosis in IMHA. Eryptosis has been associated with a variety of anemia-inducing conditions and can be triggered by cell injury from hyperosmolarity, oxidative stress, energy depletion, and hyperthermia (Lang et al., 2015). Increased oxidative stress has been suggested in some IMHA dogs based on their increased plasma malondialdehyde, decreased serum vitamin E (Pesillo et al., 2004), and increased RBC-associated Prx 2, an enzyme involved in antioxidant defense in RBCs (Tan et al., 2012). Additionally, calpain 1, a protease involved in apoptosis (Lang et al., 2015), was increased in RBC membranes of IMHA dogs, further supporting contributions of an apoptosis-like mechanism in these dogs. Alternatively, increased oxidative injury of RBC membranes and PS exposure may be secondary to free iron due to hemolysis, as has been previously demonstrated in people with thalassemia (Musallam et al. 2012) and considered in dogs with IMHA (Pesillo et al. 2004). Although oxidative stress has not been demonstrated to play an important role in human patients with IMHA (Garratty 2008), there is evidence that increased oxidative stress can exacerbate IMHA in mice with decreased antioxidant capacity (Konno et al., 2013). IgG binding to RBCs with concurrent increase in oxidative stress or PS exposure has been documented in vitro with murine and human cells (Lee et al., 2004; Konno et al., 2013; Attanasio et al., 2007). It is controversial if increased oxidative stress and PS 128 exposure trigger immunoglobulin binding to RBCs (Lee et al., 2004) and an autoimmune reaction (Konno et al., 2013), or if, conversely, immunoglobulin binding comes first and promotes PS exposure on RBCs (Attanasio et al., 2007). Increased PS positivity in RBCs of 4 IgG-negative dogs in our study, 2 IMHA and 2 non-IMHA dogs, suggests that PS exposure can occur independently of IgG binding. Although IgG levels may have been too low to detect, or another class of immunoglobulin may have been present in these two IMHA dogs, an additional IMHA dog that was strongly positive for IgG (100% positive events) was PS negative, and two bone marrow-tested dogs had positive IgG but negative PS results on RBCs, suggesting that IgG binding does not necessarily cause PS exposure. To test this hypothesis further, and to exclude the possibility of nonspecific binding of annexin V-FLUOS to RBC-bound IgG molecules resulting in false PS-positive results in IgG-positive dogs, we tested DEA1.1-positive RBCs from a healthy dog for both IgG and PS before and after incubation with anti-DEA1.1 typing serum. PS did not increase and remained below the cutoff based on 20 healthy dogs, while IgG shifted from negative to strongly positive (100% positive events with 50-140× increases in MdFI) after incubation with typing serum. Therefore, IgG binding did not promote PS exposure or nonspecific binding of annexin V-FLUOS. Increased RBC PS can likely occur for multiple reasons, so it is not surprising that RBC PS was increased in two non-IMHA dogs in our study, one with chronic renal failure and one with immune-mediated neutropenia. It is possible that PS exposure accelerated erythrocyte destruction in these dogs. Chronic renal disease has been associated with increased oxidative stress in dogs (Buranakarl et al., 2009), as well as with increased oxidative stress and PS exposure on human RBCs (Bonomini et al., 129 1999; Lang et al., 2015). However, there is no clear association between immunemediated neutropenia and eryptosis. Positivity for erythroid precursor IgG in a subset of PIMA dogs and negativity in all non-PIMA dogs suggests that IgG may play a role in the pathogenesis of PIMA in at least some dogs. This is further supported indirectly by the finding that all 5 IgG-positive dogs appeared to respond to immunosuppressive therapy, with four of them achieving remission, and that 3 of the 4 IgG-positive dogs that were tested were also positive for RBC IgG. These findings align with the previous demonstration of a circulating inhibitor of erythropoiesis in the IgG containing plasma fraction of some dogs with nonregenerative anemia associated with pure red cell aplasia (Weiss 1986). Involvement of IgG has also been documented in a few human patients with reticulocytopenic immune-mediated anemia, either by detecting plasma or RBC-eluate IgG that was reactive with RBCs and erythroid precursors (Mangan et al., 1984; Meyer et al., 1978), or by detecting surface-bound IgG on erythroid precursors by flow cytometry (van de Loosdrecht et al., 2000). Although only 2 early-PIMA dogs were tested and IgG results were not reported for the top Percoll fractions, the stage of erythroid precursor with IgG positivity matched the stage of RP in all IgG-positive dogs (all late PIMA), with additional positivity of the mid Percoll fraction for 3 of 5 dogs. Positivity in both fractions could have been caused by imperfect separation of erythroid stages among Percoll fractions or by targeting of an epitope common to both mid and later erythroid precursors. Additionally, the stage of PS positivity also matched the stage of erythroid precursor attack in PS-positive dogs, although the bottom fraction was not tested (insufficient cells) in one late-PIMA dog that was PS positive in the mid fraction. 130 Negative results for IgG on erythroid precursors of 12 PIMA dogs may have occurred for several reasons. Four PIMA dogs had received immunosuppressive therapy, which could have been associated with decreased IgG positivity if treatment was effective. As in IMHA, IgM, C3, or IgA rather than IgG may have mediated PIMA in some dogs (Piek et al., 2012; Wilkerson et al., 2000; Slappendel 1979; Day 1996); we attempted to develop RBC and erythroid precursor assays for IgM and C3, but, despite being able to develop a positive control RBCs for canine C3, testing was unsuccessful due to lack of proper IgM control cells and our continued inability to clearly detect C3 in clinical IMHA patients. Negative IgG results may also have occurred in some PIMA dogs because of inadequate assay sensitivity for low amounts of IgG. Additionally, cellmediated destruction or involvement of unknown mediators cannot be excluded as potential mediators of PIMA in some dogs. nRBC IgG positivity in the 4 dogs with unclear diagnoses is difficult to interpret, but several of these dogs could certainly have had an immunologic component to their anemias. Phagocytosis of multiple cell lines was present in the bone marrow of two of these dogs, and it may have reflected immune-mediated disease. However, nonselective phagocytosis may also occur through nonimmunologic mechanisms. A third dog had concurrent IMT and may have had additional immunologic disease directed at erythroid precursors. In agreement with our previous findings (Chapter 2), PIMA dogs in this study had rare evidence of  RBC  targeting  (e.g.,  spherocytosis,  Coombs’  test  positivity) based on routine testing; however, 5 of 14 tested PIMA dogs were positive for RBC IgG by flow cytometry. This indirectly supports an IgG-mediated targeting of erythroid precursors as well, and raises the question of what erythroid epitopes are being attacked in PIMA. 131 Although it is tempting to suspect antibodies targeting a single epitope shared by RBCs and precursors, potentially via the same antibodies (Meyer et al., 1978), distinctly different concurrent antibodies to RBCs and erythroid precursors have been documented in a human patient (Mangan et al., 1984). The increase in erythroid precursor PS, but not RBC PS, in PIMA dogs compared to healthy dogs suggests that PS may contribute to RP in at least some dogs. However, RP in PIMA dogs is characterized by morphologically normal cells within macrophages, and this contrasts with phagocytosis of apoptotic-appearing cells as is seen, for example, in the bone marrow of some dogs with myelodysplastic syndrome. Interestingly, in the only such report found, one person with reticulocytopenic IMHA and increased erythroid precursor IgG and PS had no morphologic abnormalities of erythroid precursors by light microscopic examination (van de Loosdrecht et al., 2000). These findings may in part be explained by reports that PS exposure occurred before morphologic changes on cytologic preparations and before detection of cell death using propidium iodide (Martin et al., 1995; Shiratsuchi et al., 1998). Externalized PS by itself can induce phagocytosis of cells in early apoptosis (Shiratsuchi et al., 1998), supporting the idea that PS exposure could have mediated RP of intact-appearing erythroid precursors in PIMA. However, it is unclear what might cause increased PS exposure in PIMA dogs. No relationship between IgG and PS positivity was apparent for PIMA dogs, either on erythroid precursors or RBCs. This suggests that IgG and PS could be involved independently of each other, in different subsets of affected dogs. Additionally, no evidence was found to suggest a relationship between IgG or PS positivity and MF, which was present only in PIMA dogs of this study, supporting our previous findings 132 (Chapter 2) of an association between MF and PIMA, and further suggesting that MF may be secondary to the pathogenic mechanism(s) causing anemia (Villiers et al., 1999; Stokol et al., 2000). Limited availability of bone marrow samples from healthy and non-PIMA dogs was a limitation of this study, warranting further studies using larger populations. Unfortunately, bone marrow collection, especially of core samples, is not without some risk, so collection is difficult to justify in dogs that do not require bone marrow evaluation for their diagnostic work-up. 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Isotype-specific antibodies in horses and dogs with immune-mediated hemolytic anemia. J Vet Intern Med. 2000;14:190-196. 137 CHAPTER 5 Summary and Conclusions 138 SUMMARY OF FINDINGS The primary focus of my dissertation research was to characterize and explore the bone marrow findings in dogs with suspected precursor-targeted immune-mediated anemia (PIMA). Methods were developed and studies were done to test the hypotheses that PIMA is mediated by IgG and PS, and that PS, in addition to immunoglobulins, plays a role in IMHA by promoting erythroid cell destruction and anemia. In characterizing 25 clinical cases of PIMA with rubriphagocytosis (RP), we found three different bone marrow patterns reflecting ineffective erythropoiesis at different stages of erythroid attack. When late-stage precursors are attacked, there is erythroid hyperplasia, typically with a left shift (i.e., an increased ratio of immature to mature erythroid precursors). When mid-stage erythroid precursors are targeted, there is a normocellular erythroid series with mid-stage maturation arrest (i.e., a paucity of erythroid precursors beyond the point of mid-stage of development). And when earlystage erythroid precursors are phagocytized, we see a reduced erythroid lineage with early-stage maturation arrest (i.e., few erythroid precursors beyond the early-stage of development). A fourth pattern was characterized by severe, effacing collagen myelofibrosis with foci of left-shifted erythropoiesis associated with mostly late-stage rubriphagocytosis. We also documented that PIMA is not a rare condition in our bone marrow submission population, accounting for approximately 25% of all canine cases, and for most of the dogs presenting with a poorly regenerative or nonregenerative anemia. Evidence of concurrent IMHA was rare in these dogs, and mild to severe myelofibrosis was common, being found in up to 48% of the studied canine populations. Many dogs had slow or no apparent response to therapy, and one third of the dogs that appeared to 139 respond to therapy relapsed after a few weeks or months. Most of the patients with a clinical follow-up (13/20) died or underwent euthanasia within days to weeks after diagnosis, often apparently because of sluggish regenerative responses, relapses, or clinical pessimism about a successful outcome. However, many dogs (7) lived for years. In order to test if PIMA is mediated by IgG binding to erythroid precursors, a onecolor flow cytometric assay was first developed to assess RBCs of IMHA dogs (Chapter 3). The assay had high diagnostic sensitivity (88%), specificity (93%), and accuracy (90%) for IgG detection on RBCs of IMHA dogs when compared to a variety of sick dogs not affected by IMHA. Validation of the RBC assay showed good within- and betweenday precision; coefficients of variation for negative control cells with negligible IgG were 13% and 14%, respectively, and they were 7% and 0.1% for positive control cells, respectively, using modest positivity for the within-day testing and markedly positive cells for the between-day positivity. Stability of negative and positive IgG control RBCs was good for up to one month, and accuracy was high based on mixing experiments yielding recoveries of 0, 18, 37, 56, 78, and 100% IgG-positive RBCs after spiking with 0, 20, 40, 60, 80, and 100% IgG-positive cells. Initial plans to assess the role of C3 as a mediator of erythroid precursor destruction in PIMA were thwarted by unsuccessful concerted attempts to develop an assay for detection of complement (C3) on RBCs of IMHA dogs. In vitro experiments and clinical testing resulted in the conclusion that commercially available anti-[dog C3] antibodies could not reliably detect the appropriate canine C3 fragment on canine RBCs, as discussed on Appendix A. IgM testing was considered an alternative to C3, but the relative importance of IgG over IgM in most immune-mediated cytopenias, and 140 difficulties in developing a positive control for IgM discouraged continuation of these studies (Appendix A). To assess erythroid precursors for IgG and PS, a method was developed to isolate different stages of erythroid precursors from canine bone marrow samples, and to separate them from other cell types (Chapter 3). We aimed to separate different stages of erythroid cells in order to assess if potential IgG and/or PS positivity could have any relationship with the stage of erythroid precursor undergoing RP in PIMA dogs. That goal was based on our retrospective study findings of early-, mid-, and late-stage RP associated with distinct bone marrow patterns, suggesting that attacks may occur at different stages of development in different dogs (Chapter 2). Percoll gradient separation allowed fractionation of bone marrow samples into a top fraction enriched in rubriblasts and prorubricytes (early stages), a middle fraction enriched in early- to mid-stage rubricytes (mid stages), and a bottom fraction enriched in late-stage rubricytes and metarubricytes (late stages). Regarding identification of erythroid precursors, sorting experiments showed good separation of erythroid cells from other cell populations in canine bone marrow samples from three different dogs, by using a two-color flow assay. Gating strategy relied on CD18 antibody labeling and LDS751 staining. Gated erythroid precursor populations from the top, mid, and bottom Percoll fractions contained, respectively,  ≥  89%,  98%,  and  98%  erythroid  precursors. Using this flow cytometric assay, we were also able to gate on reticulocytes and RBCs. However, we opted to assess IgG and PS on RBCs from blood because of inconsistent RBC concentrations in Percoll fractions of bone marrow samples. This flow cytometric bone marrow assay was then applied, with the addition of IgG antibodies used for RBCs, to assess IgG binding to erythroid precursors (Chapter 3). 141 The first step was to assess the  assay’s ability to detect IgG on erythroid precursors. To that end, erythroid precursors from DEA1.1 negative and positive dogs were incubated with anti-DEA1.1 typing serum, in the same fashion as was done for IgG control RBCs (Chapter 3). Results from this experiment showed clear differences in median IgG signals for 3 DEA1.1 negative dogs (4.2%, 6.0%, and 16.1% positive events for the top, mid and bottom Percoll fractions, respectively) compared to 3 DEA1.1 positive dogs (71.1%, 98.1%, and 99.4% positive events for the top, mid and bottom Percoll fractions, respectively) and compared to healthy dogs. Expression of certain blood type antigens on erythroid precursors has been described in humans, but to the author’s knowledge, this was the first time that DEA1.1 expression has been reported on canine erythroid precursors, from rubriblasts through metarubricytes. The 3-color flow cytometric assay was then used to prospectively test bone marrow samples of PIMA, sick non-PIMA, and healthy dogs, in order to assess if IgG binding to erythroid precursors is involved in PIMA (Chapter 4). Based on cutoffs generated from 10 healthy dogs, 5 of 17 PIMA dogs were positive for IgG on erythroid precursors from the middle or bottom Percoll layers, while non-PIMA dogs were negative. Additionally, 4 of 11 dogs with unclear diagnoses were positive for IgG, and 3 of these had findings that could have reflected immune targeting of erythroid precursors. These results offer support for the hypothesis that PIMA is mediated by IgG, at least in some dogs, but further assessment is required. PS positivity was increased on erythroid precursors from 5 of 6 of the tested PIMA dogs compared to 5 healthy animals, and these differences were significantly different (Chapter 4). This supports a potential role for PS in PIMA. 142 In addition, the stage of erythroid precursor undergoing RP generally matched the Percoll fraction with IgG and/or PS positivity in most positive dogs (Chapter 4), indicating that RP may be mediated by Fc receptor or PS receptor signaling. However, only two early-PIMA dogs were tested, and we were unable to assess erythroid precursors in the top Percoll fraction, so the role of IgG and PS in early-stage RP of early-PIMA dogs was not assessed. Involvement of an apoptotic-like mechanism has been previously suggested in IMHA, in addition to an immune-mediated attack. Our results support this idea and our hypothesis that PS plays a role in IMHA, by showing that 10 of 11 IMHA dogs but only 2 of 9 non-IMHA dogs were positive for RBC PS (Chapter 4). Statistical analysis confirmed significant differences between PS results of IMHA versus healthy dogs. Our data from client-owned dogs and experiment results suggest that IgG and PS positivity are independent events, and warrant further studies to clarify the mechanism and potential consequences of PS exposure in IMHA dogs. Despite our repeated attempts to optimize and apply an assay to assess for susceptibility of nRBC to phagocytosis, we found several limitations that hindered these studies (Appendix B). However, our data suggested that in vitro phagocytosis and extensive degradation of IgG-coated nRBCs by macrophages can happen in as little as 15 minutes, suggesting that rapid clearance of abnormal cells by macrophages may limit detection of RP in the bone marrow of PIMA dogs. 143 LIMITATIONS AND UNANSWERED QUESTIONS The main limitation of this work was the availability of bone marrow samples from healthy dogs and from dogs with clear diagnoses of PIMA or non-PIMA, particularly after the PS assay was developed. Obtaining samples from healthy dogs was especially problematic, because owners are understandably unlikely to give consent for a procedure with very low, but real risk, of bone fracture, infection, or, in the case of core biopsies, anesthetic complications, and purchase and conditioning of healthy dogs for just bone marrow collection was cost prohibitive. Consequently, sample sizes for earlyPIMA, non-PIMA, and healthy dogs were low for IgG and especially PS assessment on erythroid precursors. Despite testing all but two bone marrow samples that qualified for this study and that were collected at the Michigan State University Veterinary Medical Center over 32 months, only 40 usable dogs were identified, and those included 17 PIMA and 7 clear non-PIMA cases. Most (11) of the remaining 16 dogs had unclear diagnoses, and could not be classified as either PIMA or non-PIMA. The retrospective study included 25 PIMA cases over a period of 4 years, which proportionally matches the frequency of 17 PIMA cases identified in a period of 32 months, resulting in approximately 6 to 7 clear PIMA cases identified in our hospital per year. A prospective study with longer duration, with recruitment of clinical cases from other clinics, and increased numbers of healthy dogs would potentially allow for larger sample sizes and stronger statistical results. Only 2 early-PIMA dogs were tested for erythroid precursor IgG, and both were negative, but neither could be tested for PS at the time. In addition, the top Percoll fraction could not be reliably assessed for some of the dogs in this study because of imperfect flow cytometric separation between erythroid precursors and other cell types. 144 These two complications left open an important question, proposed in chapter 2, regarding the pathogenesis of early-PIMA. Early-PIMA dogs differed from mid- and latePIMA dogs in their bone marrow, laboratory, and signalment findings, raising the idea that the pathogenesis of the former may differ from that of mid- and late-PIMA. Additionally, 4 of 17 PIMA and 1 of 7 non-PIMA dogs could not be assessed for IgG and/or PS in at least one of the Percoll fractions because of insufficient numbers of cells or high nonspecific binding. The protocol used for these studies required 0.5-1×106 cells to be tested per tube, ultimately requiring three to six million cells per Percoll fraction to allow for both IgG and PS testing when including all controls. 145 FUTURE DIRECTIONS This work provides direct evidence for IgG involvement in at least a subset of PIMA dogs, but 12 of 17 were negative for IgG on erythroid precursors. Of these 12, all that were tested for PS were positive (4 of 4) suggesting PS involvement in a different subset of PIMA dogs. Larger sample sizes are needed to confirm these findings, and future studies are warranted to explore alternative mechanisms for PIMA, including the involvement of C3 and cell-mediated targeting, particularly for early-PIMA, which is typically characterized by a marrow lymphocytosis not seen in mid-and late-PIMA. Our results support involvement of PS in IMHA as well as in PIMA, but it is unclear what promotes PS exposure in immune-mediated anemias, if PS exposure is associated with apoptotic events, if oxidative stress occurs and if it is a primary or secondary event, if and how increased PS exposure may be involved in thromboembolic events in these dogs, and how this may impact therapeutic approach. The following could be explored to help address these issues: 1) a possible correlation of PS exposure and positivity for canine C3 in erythroid cells, 2) an association between positivity for necrotic markers (such as propidium iodide) and PS positivity in PIMA, 3) assessment of caspase (i.e., caspases 5, 6) activation to explore if PS exposure is a consequence of apoptotic/eryptotic mechanisms or if it is a membrane event independent of apoptosis/eryptosis, 4) the levels of expression of antioxidant proteins by erythroid cells in dogs with immune-mediated anemia versus control dogs, and 5) the potential induction of erythroid oxidative stress in vitro by incubating plasma of dogs with immunemediated anemia and erythroid cells of control dogs. Candidate erythroid epitopes have been identified in canine IMHA, but no information is currently available regarding which epitopes may be involved in canine 146 PIMA. Our results show that targeting of erythroid cells by IgG in PIMA dogs may be directed to precursors, RBCs, or both concurrently, suggesting an immune-mediated attack of epitopes that are sometimes shared by erythroid cells in different stages of development. It is also unknown if attacked epitopes are public (i.e., common to all dogs) or specific to individuals. Further studies to help identify the targeted epitopes in IgG-positive PIMA dogs could involve identification of IgG-bound membrane proteins by methods such as Western blot and immunoprecipitation. Additionally, incubation of eluates from nRBCs or plasma from IgG-positive PIMA dogs with erythroid precursors from healthy dogs or sick dogs not affected with PIMA could be performed to explore if nRBC-reactive antibodies are public. The data suggest that increased membrane-associated IgG and PS exposure may mediate RP in PIMA. We had hypothesized that erythroid precursors in PIMA undergo RP because of their increased susceptibility to phagocytosis, be it from IgG, PS, or other mechanisms. Attempts to demonstrate increased susceptibility of PIMA erythroid precursors to phagocytosis by using a cell culture-based phagocytic assay were unsuccessful because of the high numbers of cells needed for assessment, and the low assay sensitivity (Appendix B). However, we demonstrated rapid phagocytosis of IgGcoated erythroid precursors, which is detected as early as 15 minutes after incubation, with intact-looking erythroid precursors inside macrophages (Appendix B). This mirrors the appearance of phagocytized cells in clinical samples. If RP is mediated by PS receptors on macrophages in PS-positive PIMA dogs, a question that remains unanswered is why phagocytized erythroid precursors look intact rather than apoptotic inside macrophages prior to their degradation. This may be because PS-positive cells are in early stages of apoptosis, and phagocytosis takes place rapidly, before later 147 apoptotic morphologic changes occur. Further mechanistic experiments could be directed at assessing other markers of apoptosis and phagocytosis of PS-positive canine nRBCs. Rapid removal of these cells may also help explain the lack of appreciable apoptotic-appearing cells in the bone marrow of PIMA dogs. Autoimmune MF is a recently characterized condition in humans, but it has not been described, as such, in dogs. In our studies (Chapters 2 and 4), collagen MF was common in PIMA dogs, and the spectrum of MF suggests progression over time in affected dogs. The mechanism that induces MF in these dogs is unknown. There is some evidence that dogs with even severe collagen MF may improve with treatment, but a better understanding of the pathogenesis of MF in these dogs may improve treatment responses. PIMA and control dogs could be assessed for fibrosis-related cytokines and growth factors such as TGF-β  in  the  serum  and  bone  marrow,  and  for  the  potential   involvement of peripheral monocytes and CD4-positive lymphocytes, as these cells have been proposed to be sources for increased serum TGF-β  and substance P in autoimmune MF in people. Lastly, further studies could be performed to confirm DEA expression on canine erythroid precursors, including mass spectrometry of RBC membrane proteins and detection of DEA1.1 mRNA. Additionally, more studies may be warranted to determine if RBC-incompatible allogeneic bone marrow transplantations in dogs can have the same deleterious consequences as demonstrated in human patients. 148 APPENDICES 149 APPENDIX A Assessment of Canine C3 150 Complement involvement in immune-mediated anemias Briefly, in human autoimmune hemolytic anemia (AIHA), the classical complement pathway is activated by IgM or doublets of IgG molecules bound to the erythrocyte, ultimately leading to C3 activation and fixation to the cell membrane as C3b. Cell-bound C3b can lead to cleavage and fixation of C5, C6, C7, C8, and C9 to the erythrocyte membrane promoting the formation of the membrane attack complex (MAC) and lysis of the erythrocyte, or C3b can be cleaved into C3d and C3c. In the latter case, C3c is released into the plasma and C3d remains bound to the circulating erythrocytes (Garratty 1984). Human macrophages express receptors for C3b (CR1), but not for C3d (CR2), which are expressed only by lymphocytes (Fearon 1985). It has been shown that C3bcoated erythrocytes are recognized by C3b receptor-bearing macrophages and removed from the circulation, while C3d-coated erythrocytes remain in the blood stream and have a normal survival time (Brown et al., 1970). Therefore, the C3 fragment that predominates on erythrocytes in AIHA is C3d, and this is the fragment that should be detected  in  human  Coombs’  tests  (Garratty  1984;;  Engelfriet  et  al.,  1970).  Polyvalent   antiglobulin reagents are commonly used to screen patients for AIHA, and monovalent reagents (i.e., anti-IgG and anti-C3d) are used later for further characterization of the disease in patients with positive results for the polyvalent reagents (Leger 2011). The complement fragment (C3b versus C3d) bound to erythrocyte membrane in canine IMHA has not yet been characterized. Detection of complement fragment C3 in dogs In order to address Aim 2 and explore the involvement of complement in canine 151 immune-mediated anemias, we developed a flow cytometric assay for detection of canine IgG and C3 using antibodies from Bethyl Laboratories. Bethyl antibodies for IgG detection were FITC-labeled nonspecific sheep IgG and sheep FITC-anti-[dog whole IgG] test antibodies. FITC labeled nonspecific goat IgG and goat FITC-anti-[dog C3] test antibodies were used for C3, and goat FITC-anti-[dog IgM] was used for IgM detection. Antibodies used for IgG detection were later purchased from Kirkegaard & Perry Laboratories for studies described in chapters 3 and 4, because of their specificity for the gamma chain of the IgG molecule, minimizing the chances of heavy chain cross reactivity and detection of other immunoglobulin types. Bethyl antibodies were used to assess erythrocytes from left over EDTAanticoagulated CBC samples of 29 dogs with suspected immune-mediated hemolytic anemia (IMHA). Fifteen of 29 IMHA suspects had a final clinical diagnosis of IMHA, based on the presence of regenerative anemia with one or more of the following: spherocytosis,  erythrocyte  agglutination,  and  Coombs’  test  positivity.  At  the  time  of   testing, 13 IMHA dogs were not undergoing any immunosuppressive treatment, and two dogs received one dose of dexamethasone within 24 hours of blood collection; one of the latter two was also receiving cyclosporine. Six healthy blood donor dogs were tested to assess IgG and C3 binding in health. Positivity for IgG and C3 was defined by net signal or net median fluorescence intensity (median fluorescence intensity of test minus nonsense antibody) as compared to the median plus 3 standard deviations of IgG or C3 net signals in 6 healthy dogs: 3.9 and 2.9 for IgG and C3, respectively. Preliminary results showed IgG positivity in none of the healthy dogs and in 14 of 15 IMHA dogs: 7 of 14 had weak positivity (range of IgG net signal = 5.2 – 16.2), and 7 of 14 had strong positivity (range of net IgG signal = 32.3 – 352). Both of the dogs that received 152 immunosuppressive drugs prior to blood collection had strong IgG positivity. In contrast, only one of the 15 IMHA dogs had a positive signal for surfaceassociated C3, while all other 14 IMHA dogs were negative for C3, all with C3 net signals below 1.9. C3 positivity in the one C3-positive patient was weak (net signal = 6.2), and it had the highest IgG (352) net signal of all tested dogs. IgM was also assessed, and the C3-positive dog had the highest IgM net signal (76.1), while the 14 C3-negative dogs had low IgM net signals (< 5.1). High IgM net signal supported the presence of C3 in this patient, but the C3 signal was too weak to be a clear and reliable positive. In every run, a positive control for C3 (sucrose-treated canine erythrocytes) yielded strong flow cytometric positivity for the test antibody (median fluorescence intensity = 176 – 457), while erythrocytes washed with buffer (PBS, 0.3% BSA) yielded negative results (median fluorescence intensity < 18). Despite the low numbers of healthy dogs tested for comparison, it was clear that none of the 15 IMHA dogs had clear and strong C3 positivity. Five of the 15 IMHA dogs had ghost cells reported, and 7 had hemolyzed samples (mild to marked), increasing the likelihood of complementmediated intravascular hemolysis in these dogs. Similar to these findings, most studies reported over the last 30 years show that assessment of C3 in IMHA dogs usually yielded negative or only weakly positive results (Jackson et al., 1985; Jones et al., 1987; Barker et al., 1992; Jones et al, 1992; Day 1996;;  Overmann  et  al.,  2007;;  Piek  et  al,  2012)  using  either  Coombs’- or ELISA-based methods and various sources of antibodies: rabbit anti-[dog C3] made in-house (Jones et al, 1987; Jones et al., 1992), goat anti-[dog C3] from Nordic Laboratories (Barker et al., 1992; Day 1996; Piek et al., 2012; Warman et al., 2008) and goat anti-[dog C3] from MP Biomedicals (Overmann et al., 2007). Only in one report are many strong positive 153 C3 results reported for dogs, and those data were generated with a rabbit anti-[dog C3b] from the Netherlands Red Cross Blood Transfusion Service (Slappendel 1979). In some studies, positivity for canine C3 most often occurred at 4°C rather than at room temperature or 37°C (Warman et al., 2008; Quigley et al., 2001), but the significance of positive results at only 4°C is questionable, since the clinical relevance of C3 bound to cells at nonphysiologic temperatures is not clear. In human patients, the direct antiglobulin test (DAT) is usually performed at room temperature (Roback 2011), and C3 is involved in most cases of autoimmune hemolytic anemia (AIHA) (Leger 2011). Flow cytometry has been used to detect canine C3 in two studies (Wilkerson et al., 2000; Quigley et al., 2001), both of which used the same goat anti-[dog C3] from Bethyl Laboratories that was initially used for our samples. In one study, none of the 12 IMHA dogs was positive for C3, but all were positive for IgG and/or IgM (Wilkerson et al., 2000). Negativity for C3 could have been due to true negativity, inappropriate reactivity of the anti-C3 antibody, or to analytical error, since C3-bound positive control cells were not used. In a different flow cytometric study (Quigley et al., 2001), 6 of 13 dogs with IMHA tested positive for C3; however, none were strong positives, and the accuracy of C3 detection in this study was questionable because positivity was defined by values greater than the mean plus 2 standard deviations (SD) for 13 clinically normal dogs. Using 3 SD or the greatest healthy value to set a cutoff would have been a more conservative approach that may have resulted in reclassification of weak positive results as negative. Few and weak positive results for canine C3 in IMHA, based on our data and that of others, has led us to the following questions: 1) Are the commercially available anti[dog C3] antibodies reliable for detection of the C3 fragment fixed on canine 154 erythrocytes? 2) What is the fragment of C3 fixed on canine erythrocytes? 3) How is the complement system involved in canine IMHA? 4) Can complement be detected on canine erythrocytes as a mark of opsonization in immune-mediated anemias? Experiments designed to address some of these questions are summarized below. C3-coated positive control cells Several methods have been described for preparing C3-coated erythrocytes for use in experimental studies or as positive controls for diagnostic tests. Well accepted protocols are the sucrose and the Fruitstone methods, which fix complement to the erythrocyte membrane through the classical and alternative complement pathways, respectively. Briefly, in the sucrose method, whole blood is incubated with a low-ionic strength sucrose solution at 37°C, allowing immunoglobulins from the plasma to fix C3b and C4b on the erythrocytes (Garratty et al, 1975). In the Fruitstone method, whole blood is incubated with a sucrose solution containing EDTA, for inhibition of the classical pathway, and magnesium chloride is added to accelerate C3 coating through the alternative pathway. This is carried out at low pH (5.1) and temperature (0°C), and results in erythrocytes coated with only C3b (Chaplin et al., 1980; Fruitstone 1978). A few methods are also available for cleavage of C3b into C3d, including trypsinization (Morton et al., 1947; Issitt et al., 1976) and treatment of cells with heat-inactivated serum (Ross et al., 1982). Table 7 shows a summary of in vitro complement fixing methods, and the complement fragment(s) expected to be bound to erythrocytes. Anticipation that complement-fixation methods used for humans would work for dogs was based on evidence that canine and human C3 molecules and their cleavage fragments have similar structure and molecular weight (Johnson et al., 1985; Kay et al., 1985; 155 Ameratunga et al., 1998). Table 7: Methods of complement fixation to erythrocytes, and complement fragments expected to be fixed as a result. Complement fragment Sucrose treatment Fruitstone treatment Trypsinization C3b/C4b C3b only C3d (C4d) Reactivity of anti-[dog C3] antibodies to canine C3b and/or C3d In an attempt to investigate if the FITC-labeled anti-[dog C3] antibody from Bethyl Laboratories can detect C3d (presumably the C3 fragment on canine erythrocytes in IMHA), erythrocytes were sucrose-treated and then trypsinized. Human erythrocytes were tested in parallel with canine cells to ensure that the methods of C3 fixation and cleavage were performed correctly. The Fruitstone method was also performed to assure that antibody signal was not a result of C4b detection. A primary polyclonal chicken anti-[human C3d] antibody from Quidel Laboratories, and a secondary caprine anti-[chicken IgG]-FITC labeled antibody from Bethyl Laboratories were used for human C3 detection. Sucrose- and Fruitstone-treated human erythrocytes yielded strong positive results, and remained positive after trypsinization (Table 8), though with some loss of signal, which could have occurred due to damage to the C3 molecule, or because the polyclonal anti-[human C3] antibody detects epitopes on C3b which are not present on the C3d molecule. Communication with the technical support at Quidel Laboratories supported that their anti-[human C3d] antibody may yield stronger reactivity to C3b than to purified C3d. For further characterization, sucrose-, Fruitstone- and trypsin-treated human erythrocytes could be tested with anti-[human C3c] and anti- 156 [human C3d] monoclonal antibodies in order to prove loss of signal for C3c and stability of signal for C3d after trypsinization (Lachmann et al., 1983), but we did not pursue these studies. The FITC-anti-[dog C3] antibody from Bethyl Laboratories yielded strong flow cytometric signals on sucrose-treated canine erythrocytes (expected to bear C3b) but weak signals on trypsinized sucrose-treated canine cells (expected to bear C3d), suggesting either lack of reactivity of the antibody with canine C3d, or lack of C3d generation with the trypsinization technique (Table 8). The Fruitstone method did not work for the dog (Table 8), despite performing the test separately with four different concentrations of magnesium chloride (i.e., the recommended concentration for the human protocol and three higher concentrations; data not shown for higher concentrations). Greater concentrations were tested because of species differences in magnesium requirements for alternative pathway activation (Gorman 1984; McConnell et al., 1978; Gorman 1983). C3-coated canine and human erythrocytes incubated with nonsense (for canine) or only secondary (for human) antibodies, and negative control washed canine and washed human erythrocytes incubated with their respective speciesspecific anti-C3  test  antibodies  all  had  low  mean  fluorescence  intensity  (≤  7). 157 Table 8: C3 detection on canine and human erythrocytes after various treatments for C3b and C3d fixation. RBC  Treatment  (C’  fragment) Complement Signal (Canine, MFI)* 487.0 6.3 8.2 ≤  7 Sucrose (C3b+C4b) Sucrose/trypsin (C3d+C4d) Fruitstone (C3b) Fruitstone/Trypsin (C3d) None MFI = mean fluorescence intensity *Bethyl FITC-anti-[dog C3] **Quidel chicken anti-[human C3d] + FITC-anti-[chicken IgG] All  results  with  negative  control  antibodies  were  ≤  7.   Complement Signal (Human, MFI)** 100.9 31.1 305.1 55.2 ≤  7 We also tested the anti-[dog C3]-FITC labeled antibody from Nordic Laboratories, which has been used in other studies of canine IMHA (Barker et al., 1992; Day 1996; Piek et al., 2012; Warman et al., 2008). This antibody yielded strong flow cytometric positivity for sucrose-treated canine erythrocytes, but most of the signal was lost after trypsinization (Table 9). Even if the remaining signal was real, its weakness under experimental conditions to promote a strong positive suggested that the antibody would have little utility in clinical samples. Each of these experiments was repeated on different days and with different samples, some with modifications of trypsin incubation times, and the findings were always similar, though the magnitude of signals differed because of differing degrees of complement fixation. 158 Table 9: C3 detection on canine erythrocytes using the Nordic anti-[dog C3c] antibody. Complement Signal (MFI) Sucrose (C3b+C4b) 128.6 Sucrose/Trypsin (C3d+C4d) 17.9 None 7.6 MFI = mean fluorescence intensity RBC Treatment Conclusions and future directions Experiments with human erythrocytes suggested appropriate generation of complement fragments with each technique, and the sucrose method fixed C3 on canine erythrocytes as expected. However, C3 was not detected via the Fruitstone fixation method or after trypsinization. This suggests either that these methods do not work for canine C3 because of differences between the C3 molecule of humans and dogs, or that the antibodies used for canine C3 detection cannot accurately detect canine C3b and C3d molecules fixed on erythrocytes by these methods. Therefore, it is still unclear if the tested and reported anti-[dog C3] antibodies can detect C3d, and if C3d is formed from C3b in dogs. The canine C3 gene is predicted to produce a pre-pro-C3 protein, which is identical to the pre-pro-C3 protein in people (Ameratunga et al., 1998). Critical structures in the C3 molecule seem to have high homology between dogs and humans, including amino acid sequences involved in: 1) the thioester bond formation between the erythrocyte and the C3 molecule, 2) ligand binding (including binding sites for CR1 and CR2), 3) C3 inactivation (including the two factor I cleavage sites), and 4) terminal cleavage of C3dg into C3d and C3g (such as the trypsin cleavage site). However, the third factor I (I3) 159 cleavage site in dogs is not conserved with respect to the human sequence, instead being similar to that of guinea pigs and mice (Ameratunga et al., 1998). Factor  I,  also  known  as  C3b/C4b  inactivator,  cleaves  C3b  (the  “active”  form)  into   C3bi  (the  “inactive”  form),  and  then  into  C3c  (which  is  released  into  the  plasma),  leaving   the remaining C3dg fragment bound to the cell (Lachmann 2009). Soluble (e.g., factor H) or cell membrane-bound (e.g., CR1) cofactors are required for these actions of factor I (Minta et al., 1996; Ross et al., 1982). It has been shown that factor I has a divergent segment in mice, human, chicken, and Xenopus (Minta et al., 1996). The lack of homology of the I3 cleavage site in dogs with respect to people, the knowledge gap in characterization of CR1 on canine erythrocytes, and the absence of complete homology between the factor I molecule in mice and humans indicate that the cleavage of C3b into C3bi, and further cleavage into C3c and C3dg, mediated by factor I, may be different among these species, and therefore the erythrocyte-bound C3 fragment may not be the same in dogs and people with IMHA. These findings impeded the pursuit of a role for C3 in canine immune-mediated anemias, and led to the idea that currently available monospecific anti-[dog C3] antibodies are not useful for detecting C3 fragments on circulating canine erythrocytes in IMHA. Identification of the C3 cleavage products in canine IMHA, and assessment of accuracy of currently commercially available anti-[dog C3] antibodies would prevent use of suboptimal or inadequate antibodies in diagnostic tests, and potentially lead to development of more accurate reagents for the diagnosis of canine IMHA. In future studies, our hypothesis may be addressed as described below: Aim 1. Determine if Bethyl and Nordic anti-[dog C3] antibodies can detect the C3 160 fragment on canine erythrocytes after cleavage promoted by serum proteins. Hypothesis: Currently available anti-[dog C3] antibodies cannot detect the canine erythrocyte-bound C3 fragment after cleavage. An attempt to convert canine C3b into C3d would be done by treating C3b-coated cells with canine heat-inactivated serum (i.e., in the presence of canine factor I and its cofactors). The same would be done using human erythrocytes and heat-inactivated serum as a control. If, after species-specific serum treatment of sucrose-treated canine and human erythrocytes, the positive signal for C3 on canine cells and C3d on human cells remained strong, and the signal for human C3c decreased, by using speciesspecific anti-[dog C3], anti-[human C3d], and anti-[human C3c] antibodies, respectively, we would be able to suggest that: 1) treatment with heat-inactivated serum cleaves human C3b into C3d, but does not cleave the C3b molecule bound to canine erythrocytes, or 2) canine C3b is cleaved into C3d, and the tested antibodies are able to detect C3d. Negative results for canine cells after treatment with heat-inactivated serum would likely be more useful, and would suggest that the anti-[dog C3] antibodies cannot detect canine C3d (or the natural degradation product of canine C3b). Destruction of the canine C3b molecule after treatment with heat-inactivated serum is unlikely, since this is a more physiologic approach, and it is in theory less damaging to the cells and the C3 molecules than treatment with trypsin. Ideally these cleavage experiments would be done with cells that were C3b-coated by the Fruitstone method, to ensure coating by C3b only. For that, a modified Fruitstone method using EGTA instead of EDTA could be applied if, as expected, it is shown to work on canine erythrocytes in addition to human erythrocytes (Gorman 1983). 161 Aim 2. Characterize canine C3 degradation. Hypothesis: Canine C3 fragments differ from human fragments and are not detected by currently available monospecific anti[dog C3] antibodies. We have tried to elute C3 from human sucrose- and sucrose/trypsin-treated erythrocytes by using a high ionic strength solution (0.5 M NaCl), with the goal of characterizing the eluted C3 fragment(s) by SDS-PAGE, and reproducing the method with canine erythrocytes. However, elution was unsuccessful because of covalent binding of C3 to erythrocytes through a thioester bond. Characterization of canine C3 cleavage products may be pursued by purification of C3 from plasma, followed by in vitro cleavage of C3, and characterization of C3 fragments by SDS-PAGE and immunoblots (Johnson et al., 1985; Minta et al., 1977; Eggertsen et al., 1985; Ross et al., 1983). However, development of monoclonal antibodies against canine C3 fragments might be necessary for detection and full characterization of C3 cleavage products in the dog (Lachmann et al., 1980). 162 REFERENCES 163 REFERENCES Ameratunga R, Winkelstein JA, Brody L, et al. 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Vet Immunol Immunopathol. 1979;1:49-59. Warman  SM,  Murray  JK,  Ridyard  A,  et  al.  Pattern  of  Coombs’  test  reactivity  has   diagnostic significance in dogs with immune-mediated haemolytic anaemia. J Small Anim Pract. 2008;49:525-530. Wilkerson MJ, Davis E, Shuman W, et al. Isotype-specific antibodies in horses and dogs with immune-mediated hemolytic anemia. J Vet Intern Med. 2000;14:190-196. 166 APPENDIX B Development and Characterization of a Phagocytic Assay for Canine Erythroid Cells 167 Introduction Aim 4 was to determine if erythroid precursors in dogs with suspected precursortargeted immune-mediated anemia (PIMA) have increased susceptibility to phagocytosis, whether or not they have increased surface IgG or PS. The hypothesis was that erythroid precursors from dogs with PIMA undergo increased phagocytosis compared to those from healthy dogs and from sick non-PIMA dogs. This hypothesis was based on the presence of rubriphagocytosis in most dogs with PIMA, and the idea that rubriphagocytosis may mediate anemia in this condition. The paragraphs to follow include: 1) a review of literature to further support this hypothesis, 2) information on a phagocytic assay we developed to test this hypothesis, and 3) limitations we found with the assay and proposed future studies that could help achieve this aim. Mediators of phagocytosis Macrophages use several mechanisms to internalize particles, depending mostly on particle size and signals. Pinocytosis and endocytosis are the mechanisms used for internalization of fluid and small particles, respectively. Phagocytosis is the mechanism used  for  internalization  of  larger  particles  (>  0.5  μm)  and  targeted  cells,  and  through  this   process macrophages participate in important homeostatic events involved with development, remodeling, inflammation, and immune responses (Aderem et al., 1999). The main mediators of phagocytosis on target cells are antibodies, the third component of the complement system (C3), and phosphatidylserine (Aderem et al., 1999), which are recognized by macrophage Fc (Shaw et al., 1981), C3 (Shaw et al., 1981; Carrol 1998), and phosphatidylserine (Fadok et al., 2000) receptors, respectively. Macrophages also have a mannose receptor, which mediates engulfment and removal 168 of some microorganisms (Stahl et al., 1998). Phagocytosis is a complex event, in which most particles are recognized by more than one receptor (Aderem et al., 1999; Shaw, et al., 1981). Antibodies can be found on erythroid cells as a consequence of targeting by the immune system because of surface senescence antigens (Bratosin et al., 1998), autoantigens, or secondary molecules bound or expressed on the erythroid membrane secondary to infectious agents, neoplasia, or drug administration (McCullough 2003). Proteins of the complement system can be fixed on the erythroid surface through membrane-bound antibodies, especially IgM, but also by IgG (Ravetch et al., 1998). Phosphatidylserine, a phospholipid found in the inner leaflet of the plasma membrane of erythroid precursors and erythrocytes, can be flipped to the outer leaflet secondary to senescence (Bratosin et al., 1998; Lang et al., 2015) or apoptotic signals (Mathias et al., 2000; Merchant et al., 2001; Matthes et al., 2000; Lang et al., 2015). Through recognition of these mediators of phagocytosis, macrophages play a critical role in the uptake and removal of senescent or damaged erythroid cells. Macrophages also play a critical role in engulfing extruded nuclei from metarubricytes in the bone marrow during erythropoiesis, thus preventing erythroid nuclear cell remnants from being free in the tissue and triggering inflammation (Yoshida et al., 2005). An alternative mechanism of macrophage recognition of target cells is decreased levels of CD47 on the target cell. CD47, considered a membrane marker of self, is decreased in aging erythrocytes (Oldenborg et al., 2000; Ishikawa-Sekigami et al., 2006), triggering macrophage activation and erythrophagocytosis (Olsson et al., 2006). Even though a role for decreased CD47 has been proposed in human autoimmune 169 hemolytic anemia (AIHA) (Olsson et al., 2006), several studies refute this idea (Arndt et al., 2004; Ahrens et al., 2006; Barros et al., 2009). Erythroid precursor phagocytosis in PIMA As described in chapters 1, 2, and 4, phagocytosis of erythroid precursors (rubriphagocytosis) can be found in the bone marrow of most PIMA dogs at the Michigan State University Veterinary Medical Center. Rubriphagocytosis has also been reported in PIMA by others (Jonas et al., 1987; Stockham et al., 1980; Holloway et al., 1990; McManus 2000). It is important to notice that rubriphagocytosis can also be found in the spleen of affected dogs (Lucidi et al. 2010), supporting a systemic process rather than one localized to the bone marrow. Phagocytosis of erythroid precursors has also been reported in human patients with reticulocytopenic AIHA, a potential counterpart of canine PIMA (Keefer et al., 1988). Phagocytosis of erythroid precursors in PIMA is selective of the erythroid lineage, and relatively restricted to a specific stage of development (early-, mid-, or late-stage erythroid precursors), suggesting an abnormality of the erythroid cells, rather than the macrophages. This is in contrast to certain other hematologic disorders involving the monocyte-macrophage lineage in the bone marrow. Hemophagocytic syndrome and histiocytic sarcoma (Brown et al., 1994; Moore et al., 2006; Weiss 2007) are associated with nonselective phagocytosis of multiple lineages and multiple stages of development, because the phagocytosis is related to abnormal phagocyte behavior rather than abnormal or tagged target cells. In the case of histiocytic sarcoma, atypical cell features may be present (Brown et al., 1994; Weiss 2001; Moore et al., 2006). 170 Erythrocyte phagocytosis in IMHA One of the main mechanisms of anemia in IMHA is the removal of IgG- or C3coated erythrocytes by macrophages. An in vitro mononuclear phagocyte assay has been applied to demonstrate increased erythrophagocytosis in people with IMHA (Herron et al., 1986; Zupańska et al., 1987; Gallagher et al., 1983; Biondi et al., 2004; Biondi et al., 2001). A correlation was seen between the rate of phagocytosis and the degree of hemolysis in vivo (Gallagher et al., 1983) and the number of erythrocytebound IgG molecules (Zupańska et al., 1987). Most importantly, by use of a phagocytic assay, increased erythrophagocytosis was demonstrated in IMHA patients with both positive and negative direct antiglobulin test results (Biondi et al., 2001). These results suggest that a phagocytic assay could be used to assess either IgG/C3-negative or positive erythroid cells in dogs with suspected immune-mediated anemia. Erythroid cell phagocytosis in apoptotic conditions Receptors for phosphatidylserine on macrophages mediate phagocytosis of erythrocytes and erythroid precursors undergoing apoptosis. Beta-thalassemia is a condition known to cause oxidative stress, phosphatidylserine exposure, and apoptosis of erythroid cells in people. By applying an in vitro phagocytic assay, using either murine (Knyszynski et al., 1979) or human macrophages (Rachmilewitz et al., 1980), people with thalassemia have been shown to have circulating erythrocytes with increased susceptibility to phagocytosis when compared to healthy individuals. Treatment of erythrocytes of thalassemic patients with an antioxidative compound reduced their susceptibility to in vitro phagocytosis (Amer et al., 2008). In the same study, decrease in susceptibility to phagocytosis was suggested to be associated with decreased 171 erythrocyte oxidative stress, as demonstrated by reduction in reactive oxygen species (ROS) and increase in erythrocyte reduced glutathione (GSH) levels. Increased susceptibility to phagocytosis has also been demonstrated in erythroid precursors of human patients with beta-thalassemia major (Angelucci et al., 2002). Enhanced macrophagic attack was attributed to increased apoptosis, but also possibly to concurrent IgG-mediated phagocytosis. Rationale Phagocytosis of erythroid cells by macrophages is mediated mainly by IgG, C3, and phosphatidylserine. Selective and stage-selective rubriphagocytosis is common in PIMA, suggesting increased susceptibility of erythroid precursors to phagocytosis. Enhanced phagocytosis of erythrocytes and erythroid precursors has been demonstrated in people with immune- or apoptosis-mediated conditions by use of in vitro phagocytic assays. An in vitro phagocytic assay was developed based on previous studies, and applied to test our hypothesis that erythroid precursors from dogs with PIMA undergo increased phagocytosis compared to those from healthy dogs and from sick control dogs. Experimental design and methods Macrophages J774 murine macrophages (ATCC, Manassas, VA) were used as phagocytic cells. This cell line was chosen based on its expression of receptors for IgG (Sears et al., 1990) and C3 (Ralph et al., 1977), and on its ability to phagocytize IgG-coated cells (Weinshank et al., 1988; Schreiber et al., 1990) and apoptotic cells (McPhillips et al., 172 2009). Considering the phagocytic nature of these cells, phagocytosis mediated by alternative mechanisms might also be assessed. As an alternative, attempts were made to use canine monocyte/macrophage cell lines, anticipating that they would more accurately reflect cell interactions in dogs than a murine  cell  line.  We  tested  the  canine  cell  line  DH82,  known  to  express  Fcγ  receptors   (Sibley et al., 2011; Sibley et al., 2013), but we were not successful in promoting phagocytosis of IgG-coated erythrocytes when using these cells. We also attempted to use the 030-D canine macrophage cell line (Gebhard et al., 1995). However, these cell vials proved to be contaminated with fungus, hindering use of these cells in our studies. Phagocytic assay J774 cells were grown in complete medium (DMEM medium with 10% FBS and antimicrobials) and removed from culture dishes by scraping on the day of testing. A total of 200,000 macrophages were cultured in 1 mL of complete medium per well on Millicell EZ slides (4-well glass, sterile), at 37°C, with 5% CO2, for 3 hours. For each tested dog, 5 x 106 nucleated cells in bone marrow-derived Percoll fractions (top, middle, and bottom) were separately layered over J774-coated slides at a 25:1 ratio of nucleated bone marrow cells to phagocytic cells in 0.5 mL of complete medium (Biondi et al., 2004; Biondi et al., 2001; Brajovitch et al., 2009; Angelucci et al., 2002). After 3 hours, slides were washed with PBS, air-dried, stained with a Wright-stain, and examined under oil immersion. Phagocytosis was assessed by the number of nucleated cells phagocytized per 500 macrophages, but percentage of phagocytic macrophages was used in some preliminary experiments. Phagocytized nucleated cells were discriminated as intact erythroid cells, intact myeloid cells, or degraded cells (cell lineage 173 unrecognizable). Incubations were performed in duplicates when enough bone marrow nucleated cells were available, and average results were calculated. Initial experiments during assay characterization showed that murine J774 macrophages consistently and robustly phagocytized canine erythrocytes coated with canine IgG, which were made by incubating DEA1.1-positive erythrocytes with antiDEA1.1 typing serum (Figure 21B,C). Phagocytosis did not occur in negative control washed erythrocytes (not shown), or DEA1.1-negative erythrocytes incubated with antiDEA1.1 typing serum (Figure 21A). IgG positivity or negativity of control erythrocytes was confirmed by flow cytometry. Phagocytized erythrocytes looked distinct and countable, allowing quantification of the phagocytic activity. J774 macrophages did not phagocytize sucrose-treated canine erythrocytes (positive for canine C3 by flow cytometry, and presumed to be coated with C3b), suggesting failure to assess for canine erythroid cell complement opsonization. However, it is not clear which complement fragment might be present on canine cells in vivo (Appendix A), so ability of these cells to phagocytize naturally complement-opsonized canine erythrocytes could not be excluded. Figure 21: Representative images of phagocytic assay using murine J774 macrophages adherent to Millicell slides. Macrophages were washed after incubation with IgG174 Figure 21 (cont’d): negative erythrocytes (DEA 1-negative erythrocytes incubated with anti-DEA 1.1 typing serum) (A) or IgG-positive erythrocytes (DEA 1.1-positive erythrocytes incubated with anti-DEA 1.1 typing serum) (B, C). In B and C, most macrophages contain multiple, but distinct and countable phagocytized erythrocytes. Wright stain, 50× objective (A, B) and 100× objective (C). Assay sensitivity was assessed by incubating DEA1.1-positive erythrocytes with increasing dilutions of anti-DEA1.1 typing serum in order to coat erythroid cells with decreasing amounts of IgG. Positivity for IgG was determined by flow cytometry, and erythrocytes were then incubated with J774 macrophages. As anticipated, lower numbers of phagocytic macrophages were observed when using erythrocytes coated with lower amounts of IgG (Table 10), characterizing a dose-response of the assay with regard to IgG positivity on the target cells. The lowest amount of IgG, giving a MdFI of 196, equates to a relatively strong positive for RBC IgG in clinical samples. The relatively low amount of phagocytosis at these IgG levels suggested potential problems with assay sensitivity, but testing was begun to assess clinical samples. Table 10: Percentage of J774 macrophages engulfing DEA1.1-positive erythrocytes after incubation with different dilutions of anti-DEA1.1 typing serum to yield decreasing IgG positivity documented by flow cytometry. DEA1.1 typing serum dilution MdFI Phagocytic J774 macrophages (%) 1/32 1,472 96 1/64 828 96 1/128 470 77 1/512 196 16 MdFI = median fluorescence intensity of IgG-coated erythrocytes 175 Dogs and samples Bone marrow was collected from dogs with PIMA, healthy dogs, and unhealthy non-PIMA dogs. Mixtures of cells containing early-, mid-, and late-stage erythroid precursors were isolated using a Percoll gradient separation method, as described in Chapter 3. Expected outcomes Erythroid precursors of dogs with PIMA were expected to undergo increased phagocytosis compared to those of healthy and unhealthy non-PIMA dogs. Summary of experiment results and pitfalls encountered Preliminary results from four unhealthy dogs showed little phagocytosis, with few J774 cells containing degraded nuclei and cellular debris. Impaired identification of phagocytized cells prompted assessment of shorter time periods for cell incubation. IgG positive control erythrocytes were incubated with J774 macrophages for 15 and 30 minutes, in duplicates, and the average number of phagocytized erythrocytes per 200 macrophages was determined. There were averages of 247 and 439 phagocytized erythrocytes per 200 macrophages at 15 and 30 minutes, respectively, indicating that phagocytosis occurs as rapidly as 15 minutes after incubation, and that more phagocytized cells could be found at 30 minutes. To test if results of this timing experiment were reproducible with bone marrow nucleated cells, precursors from the top Percoll fraction and a mixture of erythroid precursors from the mid and bottom Percoll fractions (in order to have enough cells) from an unhealthy non-PIMA DEA1.1-positive dog were incubated with anti-DEA1.1 176 typing serum in order to artificially coat erythroid cells with IgG. After washing, cells were incubated with J774 macrophages, and the assay was terminated at 15, 30, 90, and 180 minutes. Additionally, J774 cells were incubated for two hours before adding bone marrow cells, instead of the three hours used previously. IgG coating of erythroid precursors was confirmed by flow cytometry. The number of phagocytized nucleated cells per 500 macrophages was assessed per well. The greatest amount of phagocytosis of any nucleated cell type, intact or degraded, occurred at 30 minutes for the top fraction and at 180 minutes for the mixture of cells from the mid and bottom fractions (Table 11). The greatest number of intact phagocytized erythroid precursors from the top fraction and from the mid and bottom fractions combined was at 30 minutes of incubation (Figure 22). The number of degraded cells within macrophages increased over time for cells from the mid and bottom fractions (Figure 22), but the same was not observed for the top fraction. Based on these findings, the assay was modified for incubation of bone marrow-derived nucleated cells with J774 macrophages for 30 minutes for prospective patient samples. Table 11: Phagocytosis of bone marrow-derived nucleated cells after incubation with J774 macrophages for different time periods. Phagocytized nucleated cells* per 500 macrophages Percoll Fraction 15 min 30 min 90 min 180 min Top 36 358 152 110 Mixture of mid and bottom 12 73** 99 137 *Intact or degraded, all types **Average of duplicates 177 Figure 22: Number of phagocytized bone marrow-derived nucleated cells from the mid and bottom Percoll fractions of a dog after IgG coating and incubation with J774 macrophages for different time intervals. The greatest number of intact erythroid precursors within macrophages occurred with 30 minutes of incubation. Degraded cells were present after just 15 minutes of incubation. Of the 14 dogs tested after optimizing the incubation period to 30 minutes, 2 PIMA, 1 unhealthy non-PIMA, and 5 healthy dogs were identified. The 6 remaining dogs had unclear diagnoses or were excluded because of hematologic neoplasia or high nonspecific binding of erythroid precursors on flow cytometric testing. No obvious differences were observed in phagocytic activity in different Percoll fractions among these dogs. The healthy dogs had the following mean (range) number of phagocytic cells per 500 macrophages: 14.1 (3.5-35.5), 10.9 (2.5-19), and 8.1 (4-18.5) for the top, mid, and bottom fractions, respectively, while the three patient samples yielded similar results of 1-26 and 7-18 for the top and mid fractions, respectively. Only one dog had enough cells to assess the bottom fraction, and that fraction had 9 phagocytized cells per 500 macrophages. The greatest degree of phagocytosis was in the top fraction of a 178 PIMA dog (26 per 500 macrophages), while the greatest degree of phagocytosis in a healthy dog was 35.5 per 500 macrophages, but most cells in the PIMA dog were not identifiable (22 of 26) and may not have been erythroid. At this time, the assay was reassessed. Unhealthy patients had sometimes markedly varying amounts of erythroid cells in their Percoll fractions, depending on their bone marrow abnormality and the myeloid-toerythroid ratios (M:E) found in their samples. For instance, late-PIMA dogs tended to have erythroid hyperplasia and low M:E, while dogs with early-PIMA or immunemediated neutropenia (IMN) had higher M:E, due to erythroid hypoplasia or myeloid hyperplasia, respectively. Based on that, additional steps were taken to evaluate the effect of M:E on phagocytosis of erythroid cells. It was hypothesized that dogs with high M:E could yield falsely low results for phagocytosis of erythroid precursors, because of low concentrations of erythroid cells in their Percoll fractions. Two experiments were done to address this question. The first one consisted of mixing IgG-coated erythrocytes from packed red cells with white blood cells (WBC) from buffy coat, both from blood, in different proportions in order to reach the following WBC:RBC ratios: 0.2 (mimicking the M:E in late-PIMA), 1.0, and 5.0 (mimicking the M:E in IMN), with an additional cell aliquot containing 100% erythrocytes. These cell aliquots were incubated with J774 cells for 30 minutes, with a fixed number of 5 x 10 6 nucleated cells (WBC) per well. The percentage of J774 cells containing phagocytized erythrocytes was determined by assessing 200 cells. There was a tendency for decreasing numbers of phagocytic macrophages as the WBC:RBC ratio increased (Table 12), supporting our suspicion that marrows with lower numbers of erythroid cells may yield fewer phagocytized erythroid precursors, even if erythroid cells are targeted for phagocytosis. 179 Table 12: Percentage of J774 macrophages engulfing IgG-coated erythrocytes after incubation with mixtures of erythrocytes and white blood cells of varying WBC:RBC ratio but constant total number. WBC:RBC ratio Phagocytic J774 macrophages (%)* All RBC 78 0.2 82 1.0 69 5.0 43 *average of duplicates based on assessment of 200 J774 cells The effect of normalizing the number of added erythrocytes with varied WBC:RBC ratios was then assessed by adding 5 x 106 erythrocytes per well with different number of WBCs to give the following WBC:RBC ratios: 0 (100% erythrocytes), 0.2, 1.0, and 5.0. Again, the percentage of J774 macrophages containing phagocytized erythrocytes was determined by assessing 200 macrophages. There was similar phagocytic activity in all wells except the one with the WBC:RBC ratio of 5.0 (Table 13). This suggested that a fixed number of erythroid cells per well would help normalize phagocytic results among patients with different M:E ratios, but high M:E ratios might still be problematic. The lesser phagocytosis with a WBC:RBC ratio of 5.0 might have been caused by an overwhelming total number of nucleated cells per well (30 x 10 6) leading to 1) physical blockage of macrophages from IgG-coated erythrocytes, 2) insufficient nutrients and oxygen that decreased J774 activity, or 3) removal of IgG-coated erythrocytes by Fcreceptor bearing WBCs present in the cell mixture. So, an Fc-receptor blocker (Fc receptor block, Innovex, Lincoln, RI) was tested, but it did not adequately block phagocytosis. Plans to reduce the total cell number and use only 1 x 10 6 erythrocytes per well were not completed. 180 Table 13: Percentage of J774 macrophages engulfing IgG-coated erythrocytes after incubation with mixtures of erythrocytes and white blood cells (WBC) of varying WBC:RBC ratio and fixed RBC number. WBC:RBC ratio Phagocytic J774 macrophages (%)* All RBC 97 0.2 96 1.0 96 5.0 73 *average of duplicates based on assessment of 200 J774 cells Outcome and future directions After pilot testing and recognition of a potential significant limitation of the assay because of varied numbers of erythroid precursors, this limitation was documented and partially addressed. However, further application was not pursued because of limited time, limited availability of bone marrow samples for testing, and recognition that there would be limited numbers of patients with enough cells to test for IgG, PS, and phagocytosis. Priority was placed on IgG and PS testing. Remaining concerns about the assay include its sensitivity to detect IgG-coated cells, as phagocytosis was not abundant when test cells were coated with amounts of IgG corresponding to strong clinical positivity for RBC IgG, and such levels were much higher than IgG positivity detected on erythroid precursors of any PIMA dogs. Moreover, phagocytosis of samples from healthy dogs was greater than expected, and recognition and differentiation of degrading nucleated cells proved difficult. Because bone marrow samples were tested for IgG and PS by flow cytometry on the day of collection, and these assays were time consuming, Percoll fractions were stored at 4°C overnight, and the phagocytic assay was performed on the day following collection. Cell storage might 181 have promoted degradation or expression of cell surface markers, increasing phagocytosis and the number of phagocytized degraded-appearing cells, even in samples from healthy dogs. Alternatively, these problems may have been a consequence of Percoll separation, which could have been damaging to the cell surface. Percoll separation was not assessed to see if it increased susceptibility to phagocytosis. Continued development and improvement of a phagocytic assay could include assessment of increasing assay sensitivity by stimulating IgG-mediated phagocytic activity of J774 macrophages with IFN-gamma (Weinshank et al., 1988), or by using a canine monocyte/macrophage cell line or fresh cells extracted from canine whole blood (Sampaio et al., 2007) or bone marrow (Tipold et al., 1998), which may have higher affinity for canine IgG. A potentially gentler method of processing bone marrow samples could be assessed, such as removal of nonerythroid cells by antibody coupled to magnetic beads as previously described (Angelucci et al., 2002). One could also assess using macrophages in suspension and assessing phagocytosis by flow cytometry (Bratosin et al., 1997). 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