5*! 'I I u I I III I I!“ I I II I I I — I l I I I :y l * I EVALUATION OF AN OPTICAL DEVICE FOR QUANTITATIVE STUDY OF ERYTHROCYTE AGGLUTINATION T5031: ‘09 Hm Degree of M. S. MICHIGAN STATE UNIVERSITY James John MacKenzie 1964 ISIS IIHIIIHIIIIIIIHIIIIllllllllllUllllIllilllllllllllllllll 31293 017640040 LIBRARY Michigan State University ABSTRACT EVALUATION OF AN OPTICAL DEVICE FOR QUANTITATIVE STUDY OF ERYTHROCYTE AGGLUTINATION by James John MacKenzie An optical device for measuring settling rates of agglutinating erythrocytes was studied. Its ability to differentiate between aggluti- nating and non-agglutinating settling blood suspensions was established. Experimental conditions of magnetic field were applied to determine their effect on agglutinating blood suspensions. No effect was found with the ABO blood system under the stated conditions. The D reaction in the Rh blood system was found not to be measurable by this device. A study was made of the quantitative aspects of the graphical record of agglutinating suspensions which proved inconclusive. EVALUATION OF AN OPTICAL DEVICE FOR QUANTITATIVE STUDY OF ERYTHROCYTE AGGLUTINATION BY James John Mac Kenzie A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physics and Astronomy 1964 ACKNOW LEDGMENTS I wish to thank Dr. Montgomery for all the encouragement and direction he has provided on this project. His patience and understanding have made this part of my studies a wholly enjoyable one. The contribution the American Red Cross made in providing blood samples is deeply appreciated. Special thanks go to Darrel Hart and Al Gladstone for their help in obtaining these samples. I want to thank also Josie Bennet and Gene Gardner for the work they performed in connection with this project. The work was sponsored by a National Institutes of Health research grant (No. 08967). *************** ii TABLE OF CONTENTS Page INTRODUCTION ........................ 1 Statement of Problem History of Investigation AGGLUTINATION ....................... 6 Antibodies and Antigens Erythrocytes Agglutination Reaction MATERIALS AND EXPERIMENTAL PROCEDURES . . . . . 14 P repa ration of Sample 3 Magnets P roc edure 3 RESULTS ............................ 21 CONCLUSIONS .............. . .......... 34 BIBLIOGRAPHY ........................ 35 APPENDIX ........................... 37 iii LIST OF FIGURES FIGURE Page 1. Device to measure optical density of blood sus- pension as a function of time ............. 3 2. A. Graphical record and physical appearance of non- agglutinating settling blood suspension in Beckman cell; B. Graphical record and physical appearance of agglutinating settling blood suspension in Beckman cell .......................... 4 3. Drawing of Red Blood Cell .............. 9 4. Test tube and microscopic observation of pre-zone, agglutination, and post-zone with bivalent antibody . 10 5. Two blood cells and antibody molecule in ionic solu- tion ..... . .................. . . 13 6. Magnet C1 with sample cell and horizontal field measurement from pole to pole at center of field . . 16 7. Field mapping in oersteds of magnet Cl (with pole face and tube) in the vertical plane through the center of the tube parallel to the pole face . . . . . 17 8. Magnet C2 with sample cell and horizontal field measurement from pole to pole at center of field . . 18 9. Field mapping in oersteds of magnet C2 (with pole face and tube)in the vertical plane through the center of the tube parallel to the pole face ..... 19 10. Plot of run V-15 showing (A) agglutinating and (B) non-agglutinating samples .............. 22 11. Regions of (A) non-agglutinating blood suspension, (B) agglutinating blood suspension .......... 23 iv LIST OF FIGURES - Continued FIGURE Page 12. Plot of two samples with temperature drop: A. Blood suspension with antibody; B. Blood sus- pension without antibody ............... 25 13. Incident light beam of A0 and intensity 10 on blood suspension . . . ................... 27 14. Plot of the log of the intensity of transmitted light against time for 8 different serum concentrations in V-187 . . ..... . ............... 31 15. Interpretation of settling curves which were ob- tained by plotting log of transmitted light intensity against time. A. Agglutination curve. B. Inter- face curve ....................... 32 16. Plot of the slope of [n( jnlm - In I) = In a - bt against concentration of serum for six runs . . . . 32 INTRODUCTION In this thesis we consider a problem in biophysics: the possible influence of magnetic fields upon the agglutination of human erythro- cytes in the antigen-antibody reaction. Many biological systems involve such a large number of variables that it is impossible to exer- cise control over them to the point where the laws of physics can be applied and meaningful conclusions drawn. We have chosen to investi- gate, in the specific area of bio-magnetism, human-erythrocyte agglutination in vitro as a relatively simple process. This agglutina- tion reaction has a high sensitivity that encourages its use in an experiment to detect small effects, as we know to be the case for magnetic fields. This reaction also exhibits a high degree of specificity with respect to its participants, and permits a quantitative determin- ation of the strength of those participants (1). In previous work in our laboratory, A. E. Smith found with the antibodies of the Rh system that erythrocyte agglutination was en- hanced by magnetic fields (2). This effect was observed visually by the method of Race and Sanger. The fields were homogeneous, with strengths from 20 to 5000 oersteds; and inhomogeneous, with gradients of 3, 000 oersted/cm. at a level of 4, 000 oersteds. With antibodies of the ABO blood system, no effect was found, but the data were not adequate to establish definitely a negative result. Independently of Smith, Foner at the Boston University School of Medicine confirmed enhancement of agglutination in a magnetic field, again by use of the Race-Sanger visual scoring system (3). The effect was noted for Anti-D and Anti-A, and also for a plant agglutinin. lectin, Both homogeneous and inhomogeneous fields were used at strengths between 1200 and 2600 oersteds. In these two cases, the resultsiwere obtained by methods that are to some extent subjective. Smith attempted to use the Coulter Counter as the basis for an objective measure of agglutination, but was unable to detect any response. He speculated that the electronic counter disrupted the aggregates and suggested use of a less disruptive method for evaluating agglutination, for example, sedimentation (2.). In our laboratory, H. P. Cole built a device to measure simultaneously the change of optical density with time in up to eight settling blood suspensions. A detailed account of the individual components and the mode of operation is given in his thesis (4). The device consists of a moving boom with light from the source at one end passing through an optical train to the other end. The light beam sweeps a series of tubes containing settling blood suspensions. The tubes, which are silicone-treated Pyrex rectangular spectrophotometer cells, are situated on an arc of a circle concentric with the circle traced out by the light source (see Figure 1). The light, after passing through the sample tubes, falls on the photocell whose output is fed directly into a Sargent recorder to provide a graphical record of transmitted light versus time. In a settling blood suspension that is agglutinating there will appear over. the depth of the liquid a more or less continuous optical- density gradient, which will alter as time goes on. In the absence of agglutination and with a homogenous distribution of erythrocyte sizes, the Optical density becomes a step function, constant below the inter- face of the descending erythrocytes, and very small above it (see Figure 2). Analyzing the graphs obtained under various concentrations of erythrocytes and antibody should enable us to determine in some 08TH. mo Gowuocsh .m mm coflmcoamzm poofim mo >fimCoD HwoSQO oudmmoz ow cogofl .H mmDDHh 0 iv > flog: «momoumm . x28. «ES: 3303?. mzmj mzm: 9 $63 . w . E , . . 9.. . a ado unused 200m SION LIGHT TRANSMIS TIME-" U) LIGHT TRANSMISSION TIME- FIGURE 2. A. Graphical record and physical appearance of non-agglutinating settling blood suspension in Beckman cell. B. Graphical record and physical appearance of agglutinating settling blood suspension in Beckman cell. quantitative manner the degree of agglutination. We must, of course, determine the reproducibility and the sensitivity of the instrument before any data for magnetic fields can be interpreted meaningfully. Let us examine the nature of agglutination to try to understand the meaning of the records obtained experimentally. AGGLUTINATION Human-erythrocyte agglutination is a long-studied reaction, involved in many standard procedures of hospitals, laboratories, and blood banks. Its understanding began in the early part of the twentieth century, when Landsteiner postulated different "types" of blood. He discovered the A and B types with the corresponding antigens on the blood cell, and an ‘0 type with neither a B nor A antigen. Next, - Von Decastello and Sturli found a fourth type of blood--AB, the red blood cell having both the A and B antigen (5). Many other blood groups have since beeniidiscovered and classified, including the A1 and A2 antigens into which A antigen can be broken down. One of the most important of these'groups is the Rh, discovered in 1939-40 by Landsteiner and Wiener (6). (We shall deal with only the ABC and Rh systems. In Table 1 these are tabulated. ‘ When an antigen entersia higher-order animal, one of the defense reactions enlisted in the organism is the production of antibodies that attack the invader and render it harmless. This reaction is called immunization. Upon subsequent invasion by the same antigen, the organism again produces antibodies, but much more rapidly. The manner of production of antibodies is imperfectly understood. 'There are two main theories that have evolved to explain the mechanism of antibody production in response to antigentic stimulus; neither can be reconciled wholly with the evidence. The first theory postulates that the antigenic molecule serves as a template around which the antibody molecule is formed. Pauling, in 1940, suggested that the lock-and-key arrangement of the antigen-antibody combination was due to the folding of the antibody polypeptide chain around the antigen (8). Under this 7 TABLE 1 (7, 8). ABC and Rh Blood Systems. Rh System of Blood Factors . . . . Reaction with Blood Gr0ups % Population Agglutmogen Aggluilmn . , Ann-I3 Rh+ ‘ 85% Rh none positive Rh’ 15% l none Anti-43F") negative ABO‘Syslem of Blood Factors Reaction with Blood Groupsi °/o POE-ulntion Agglutinogen l Agglutinin t’An'li—Seru'm Anti- A Anii- B O 45 °/o none . Anti-A Anta-Binegative negative . A * 4i ‘7. .. A .\ Anti—B positive. negative 8 l0% 8. > . Anti—A. Inegative positive AB 4%. A,B none, [positivelipositive ‘ theory, when the antigen has been removed from the organism the antibody production should stop; but it does not in some cases. The second theory states that the body contains within itself, irrespective of its immunological history, preformed antibody "receptors" of all types. When an antigen unites with one of these receptors, more free receptors (antibodies) are formed. The problem is how to explain the antibody production under the antigenic stimulus of artificial products of the laboratory. Foreknowledge of these artificial products on the part of the organism seems rather doubtful, as well as does a dual type of antibody formation to explain their production under these conditions (8). It might turn out that a combination of the two theories will be necessary. The antigenic molecules of interest to us are proteins having a molecular weight on the order of 15, 000. They reside on or in the surface of the human red blood cell. The antibody molecule is found in the gamma-globulin fraction of the blood serum and has a weight of about 150, 000 (9). As shown on the following page (FIGURE 3), the red blood cell is a bi-concave disk with average characteristics as follows (7): diameter, 7.5 u; thickness, 2. 3 u; surface area, 150 pa; volume, 87 (1.3. Born in the bone marrow, an erythrocyte has a life span of approximately 120 days as it passes through its various stages of development. Erythrocytes constitute 45% of the volume of whole blood, with plasma, platelets, and white cells making up the rest. The outer membrane of an erythrocyte consists of lipoprotein com- plex (10). Acidic lipids on the outer surface displace the isolectric point of the erythrocyte towards the acid side. In fact, the pH of red- cell ghosts is approximately 2 (8). A similar structure of protein, the stroma, exists within the surface structure and acts as a matrix to support the hemoglobin, which makes up most of the internal part of the erythrocyte. This structure of the erythrocyte makes it very sturdy and flexible. The hemoglobin accounts for the ability of the cell to transport oxygen _ from the lungs to the tissue of the body. 6‘<«“\'(‘1 ‘ 1 UTI' FIGURE 3. Drawing of Red Blood [Cell The phenomenon that our machine records is the sedimentation of agglutinating red blood cells. The two main theories of agglutination are the Bordet and the l_a_t£c_e_ theories. Under the Bordet theory, agglutination comes about because the antibody moleculeumerely makes the cells sensitive to the agglutinating action of the electrolyte in which they are suspended (11). This theory does not explain, of course, the specificity of the reaction observed upon mixing two types of erythro- . cytes and antibodies together. Instead of getting clumps made up of two different types of erythrocytes, one gets instead very specific aggregates made up of agglutinated erythrocytes of either one type or the other. The lattice theory, which in a simplified form says that an antibody molecule “holds hands" with two antigenic molecules, gives a satisfactory description of the observed specificity. But although the lattice theory explains the, specificity, it-does not explain completely 10 the action of erythrocytes in the presence of excess antibody. Boyd and Hooker exposed red blood cells to an excess of antibody, so that all the reactive sites were taken up by antibody molecules (12). Nevertheless, agglutination did take place. On the other hand, in many cases there is observed a maximum of agglutination with respect to serum concentration. The serum concentrations on either side of this maximum are called the Ere-zone in the case of low concentration and Est-zone in the case of high concentration. U in“ w (is/I U ‘4‘" v I‘Dre Zone Agglutination Post Zone OZAnhgen c:3:Annbody o O ' g . O foo - TOT [2% Pre Zone \ Agglutination Post Zone FIGURE 4. Test tube and mic rosc0pic observation of pre-zone, agglutination, and post-zone with bivalent antibody. 11 The number of combining sites where a B-antibody molecule may combine with a B antigen on a human red blood cell has been calculated by Filitti—Wormser to be 5 x 105. For D (Rho) sites, Boursnell, Coombs, and Rizk found approximately 5. 5 x 103 sites per cell (11). Supporting this finding are the results of Belkin and Wiener who prepared A, B, and Rho haptens from the stroma of human erythrocytes (13). Measuring the lowest titer of the A, B, Rho hapten serum that agglutinates the appropriate erythrocytes, they found that the Rho hapten serum titer was consistently lower. They concluded that the number of Rho haptens per red blood cell might be lower than in the case of A or B haptens. Agglutination can be thought of as taking place in two stages. The first is the simple combining, in some way, of the antibody with the antigen residing on the surface of the blood cell. The second stage is the combining of the antibody-antigen-cell complex with an antigen on another blood cell. The first stage of the reaction, that of joining the initial antigen with the free antibody molecule, involves three forces, singly or in combination: Coulomb, van der Waals, and hydrogen-bonding. The last two forces will be of short range, increasing markedly as the mole- cules approach each other. None of these forces in themselves are specific; a charged antigen, for example, cannot differentiate between equal charges on each of two different antibodies. The experimentally exhibited specificity of the reaction would then have to come about from some sort of complementary configuration between the antigen and anti- body molecule that would allow these forces to come into play at short distances. If Pauling's theory is correct, the “closeness” of fit of the antigen into the folded antibody would account for the specificity because of strong van der Waals forces, for instance, that bind them together. 12 Experiments have been done on changing the shape of an antigen com- bining group (11). A slight modification in shape decreased the strength of the antigen-antibody reaction, in support of the concept that comple- mentary shape is important. The Coulomb force does not enter into the first stage of the reaction because the blood factors (antigen and antibody) that we deal with do not have any positive or negative reactive groups in them. In the second stage, however, where the erythrocytes come together, agglu- tinating forces must clearly overcome the Coulomb force of repulsion due to the negative surface charge of the erythrocytes in suspension. We find that this repelling force associated with colloidal particles carrying negative charge is overcome by decreasing the pH of the solu- tion to the point where the particles absorbs H+ ions and are neutralized. Flocculation can then take place. This change of surface charge was first thought to play an important part in agglutination, with experiments on surface potential of bacteria lending support. Further experiments on reducing the surface charge of erythrocytes by charge-lowering agents, however, showed no increase in sensitivity to agglutination (11). This result indicates that the attachment of the antibody to the antigen of the blood cell does not alter the surface charge of the cell. When a negatively surface-charged blood cell is placed in an ionic solution, a positive cloud of ions surrounds it. In some way the antibody molecule bridges the gap between blood cells through this ion cloud, without allowing the Coulomb force of repulsion to develop. The antibody molecule is cigar shaped, with major and minor axes in our case of 263 X and 37.1 A respectively. This length of anti- body molecule could provide a link between the blood cells if it attached itself by the end points. The blood cells would then be sufficient distance apart that the Coulomb force would not be a problem. 13 (See Figure 5). It would follow from this model of end-point attachment that the antibody molecule is bivalent, as is generally agreed (l4). % -l- +- / / + + + + + + 1: . i + 5263A + M i + + + + + /+ + ;: i; ”/I/ ///+ + // FIGURE 5. Two Blood Cells and Antibody Molecule in Ionic Solution MATERIALS AND EXPERIMENTAL PROCEDURES Blood was obtained from the American Red Cross Blood Bank. The Red Cross draws 420 ml. of whole blood intravenously into a glass vessel containing 120 ml. of anticoagulant (A. C.D. mixture: disodium citrate 2.0-2. 5 g. , dextrose 3 g. , distilled water to 120 ml.). The blood is then refrigerated and stored at 40C. Blood so obtained is classified as outdated 21 days after date of donation. We obtain blood 2-4 days after it has become outdated. Two 5-c. c. portions of whole blood plus A. C. D. mixture were removed from the Red Cross storage bottle by a syringe and transferred to two vacuum containers (evacuated rubber-stoppered test tubes). These containers were stored at 4°C for up to a week, 1. O-ml. portions being removed from them as needed. In preparing blood suspensions for the experiment, a 1. O-ml. portion of blood was removed from the vacuum container and placed in a 10 x 75-mm. test tube which was then filled to the top with saline solution. The tube was centrifuged at 3000 rpm for 5 minutes; the supernatant liquid was then removed and fresh saline added. This pro- cess was repeated three times so that the A. C. D. mixture and the plasma constitutents of the whole blood were thoroughly washed out. A suitable volume of packed red blood cells was then removed from the tube with a micropipette. These packed erythrocytes were added to saline in order to make the desired initial suspensions. Depending on the number of samples being measured in the machine, equal amounts of the initial blood suspension were added to equal dilut- ing amounts of saline, or of saline and serum. This procedure resulted in separate blood suspensions with varying amounts of serum but with the same concentration of erythrocytes. l4 15 The serum was supplied by Ortho: Anti-A B, Anti-A, Anti-B, Anti-D; and by Dade: Anti-A, Anti-B. The serum was kept refrigerated at 40C. The Anti—D runs were made at incubation temperatures of approximately 370C, and the other runs at room temperature. Two permanent magnets provided fields over the length of the tube. Magnet Cl had a gap of 0.69 inch between identical flat circular pole faces 0. 76 inch in diameter. The field at the center of the gap was 4100 oersteds as measured by a Rawson-Lush rotating-coil gaussmeter (Type 723). The sample cells, with outside dimensions 12 x 12 x 48 mm., provided a lO-mm. light path through the liquid. During the run the cell was placed between the pole faces at equal distances from each face, with the center of the volume of the cell at approximately the center of the field as shown in Figure 6. The field strength in the vertical plane through the center of the tube parallel to the pole face is shown in Figure 7. The other permanent magnet, C2, had one circular pole face 1. 5 inches in diameter, and one conical pole piece of base diameter 1. 5 inches and height . 922 inch. Its apex was.625 inch from the circular pole face. The cell was placed next to the apex as shown in Figure 8. The field mapping in the vertical plane through the center of the tube parallel to the pole face is shown in Figure 9 for magnet C2. In a typical run the experimental procedure was as follows. The light source was turned on at least 1/2 hour before the start of a run so as to reach a steady state. A heating element was switched on when the samples required incubation above room temperature. In such cases a continuous record of the temperature was kept before and during the run. The blood suspensions were prepared in individual 20-ml. glass vials. Each of these vials contained 10-ml. of blood suspension, suf- ficient to fill two sample cells. The sample cells were washed thoroughly l6 .45 a ‘ KlLOOERSTED N A b IN CDv - FIGURE 6. Magnet Cl with sample cell and horizontal field measure- ment from pole to pole at center of field. after each run, and then dried with cotton swabs and absorbent lens paper. The tubes were wiped inside and outside after drying in order to remove any film deposits. The cells were then placed in the machine, care being taken to keep them perpendicular, since inclina- tion of the tube changes the rate of sedimentation (14). The windows were adjusted so that approximately the same amount of light energy for each cell would fall on the photocell as indicated by the graphical record. When adjustments were completed, the proper amounts of antibody were added to the separate vials. These vials 17 12‘42i 625 680 760 810 900 9:55 9.15 880 815 715 615 10 755 80 980 110 1200 12:55 12715 1150 105 875 750 8 890 1050 1250 1475 1640 1700 16% 1495 1280 1070 875 ' 6 1050 1260 1600 2000 2340 2475 2350 19175 1600 1275 1025 4 1210 1530 2000 2660 3240 .. 27:5 2610 1900 1500 1160 // NI 1300 1590 2365 3250 [3 )0 40210 3825 150 2215 1650 1256 lI)=/in.9 I 7 6 5 4/3 1 1) 1 3 4 6 8 12”». 1340 1760 2500 3425 40100 410 3950 330 2400 1720 1315 2 1/1 1330 1720 2340 3155 3180 39:15 3719/ 3060 2255 1680 1330 N /r 12 0 1550 200 2560 3070 32550 300 2500 1910 1550 1250 6 1160 1430 1660 1970 2240 23:15 225 1950 1600 1400 1160 8 100 1245 1400 1550 16EO 17:15 160 1500 1410 12150 1050 10 9pc 11(0 1200 1295 1350 1375 1360 1305 12:15 110 975 12:125.. 880 960 1030 1100 1145 11550 115 11:10 1050 10f0 880 FIGURE 7. Field mapping in oersteds of magnet Cl (with pole face and tube) in the vertical plane through the center of the tube parallel to the pole face. 18 .43 O .. KILOOERSTED N .01 O O 6 O I’ 8 DIN FIGURE 8. Magnet C2 with sample cell and horizontal field measure- ment from pole to pole at center of field. were shaken well to ensure complete mixing of suspension and antibody, and the contents were pipetted into the sample cells. The height of the bottom of the meniscus above the window was identical for each cell in order to-assure that the lightlpassedithrough the same portion of settling suspension in each sample.‘ Thelwindows were placed as near the bottom of the cell as possible, to provide a long period of observation before the interface fell across the window. Inasmuch as the concentration gradient becomes spread out in agglutinating . . l9 1 oclfn. 990 1175 1370 15‘I0 1710 18115 180 I750 ITOO 1300 1230 IOFZO era 7410 97) 1160 l 0 1710 2000 2210 2005 2430 23JI) 2020 1710 1415 11160 9"(5 80 103013215 1610 2150 25.15 2800K' 005 307:0 28 40 205 1640 1315 1070 890 O \ IZPO 150) 1950 25(0 % 3300 35,45 3500 3 2900 244’0\ 180 145 1130 925 12751625 2150 2740/20) 3640 4050 4000 3 3275 2615 015 15601225 9215 U % . , a 1 11 1 .145. , - 1315 1675 2225 zsiokzw 3700 4050 4500 39 338p 2793 1410 1610 124 990 1300 1635 2170 2750 K15 36q 4 0 40I510 3 320 2700/20 1510 120 900 14"5 1155 9410 IF g—I ~O ‘5 121251510 1990 25:10 2905 aclo 3%0 3515 33 29 112151363 17715 as 2593 2875 3cm 300 28 2551) 2190 1675 131.5 100 815 IOIFO 119 D 14*) l7IIO .20d 5 2325 21:0 24C 0 Z ZOTO 17*) 14115 11"0 9<)0 8 25 agllh. - 9 5 1015 12 0 1400 1600 17 0 1800 18 0 1700 1600 1400 1200 10115 300 750 FIGURE 9. Field mapping in oersteds of magnet C2(with pole face and tube)in the vertical plane through the center of the tube parallel to the pole face. 20 suspensions as time progresses, observation at the lower portion of the tube provides more extensive data. The runs lasted from four to eight hours and were controlled by an automatic timer. RESULTS A total of 187 runs were made under various conditions of erythrocyte concentration and type, antibody concentration and type, temperature, and magnetic field. The first fifteen runs were per- formed to investigate and develop the effectiveness of the machine as an instrument for detecting the difference between agglutinating and non-agglutinating systems. A problem encountered initially was con- vection currents. These were reduced by placing heat absorbers in the light beam in the form of a 3 x 3 x 3 inch clear plastic tank con- taining water and a 2 inch square piece of heat-absorbing glass. Under the pr0per conditions of erythrocyte and antibody concen- tration, the graphical record for the agglutinating system was found to be measureably different from that of the non-agglutinating system (see Figure 10). The separate regions of the graphs are interpreted as follows: (see Figure 11). Agglutinating sample 1. Suspension remains nearly homogeneous in field while agglutination sets in. 2. Agglutinated particles settle out of field. 3. Interface falls across window. 4. All material has fallen past window. Non-agglutinating sample 1. Suspension remains at constant Optical density in field. 2. Interface falls across window. 3. All material has fallen past window. 21 22 .moagmm mcfimcfidfimmmicdc Amy can monumcfigmmm :3 @339? mT> a?“ mo uofinm .oH HMDOHW mmDOI «no N O q d N m NOISSIWSNVHI 1H9” 23 ’ l l 2 l 11.2 . O ' | U) I W I E ’ ' U) | Z l q " I c: | /./ I- I / p. ' l I l 0 I.\ .1 .. I TlME I I l : 2 . 3 , I B. _ I : / 2 : , , 9 l l U) I I 22 ‘ I . 2 I I U) _ | Z I <1 I K I I— ~ ' l-- I I I 0 - I .J I L l 1 l J__ l l J I 5 TIME FIGURE 11. Regions of (A) non-agglutinating blood suspension, (B) agglutinating blood suspension. 24 The next 61 runs were made with the samples kept in an inhomo- geneous magnetic field, to see if any gross effects appeared. Forty- one runs on A, B, and D antibody were made with magnet Cl. With the ABO blood group, no difference in the graphs was discern- ible between samples agglutinating in the field and those outside it. Various combinations of concentrations and temperature were tried. The runs made with the D antibody showed no evidence of aggluti- nation during settling. Concentrations of blood cells and antibody were the same as those used in previous work with the Coulter Counter in which agglutination was detected. Examination of the settled blood on the bottom of the tube showed that the cells had agglutinated. Therefore, although the cells did not agglutinate while settling, they did agglutinate once they had reached the bottom. Such delayed agglutination would indicate an important difference in the conditions necessary for aggluti- nation between the D and A-B antibodies. One explanation suggested for this behavior is that the antibody and antigen molecules in the D re- action need a long time of contact to effect a bond. Therefore, although the cells do not agglutinate when settling, the D reaction does take place during the prolonged contact at the bottom of the sample tube. This dif- ference in behavior between ABC and D systems under comparable erythrocyte concentration could be explained under the complementary- configuration theory of antigen-antibody reaction by saying that the configuration for the D system is more complicated than that for the ABO system. The D participants consequently, require a longer period of time to "fit" together to the point where the short-ranged forces discussed previously could become effective. Very possibly‘the dif- ference in the number of reactive sites where the D and ABC reactions can take place on the blood cell is a contributing factor to the behavior. In any case, it was found that the D reaction was unsuitable for investi- gation by this machine. 25 The next series of runs was made with magnet C2, again on the ABO system. Twenty runs were performed without any discernible differences in the graphical results. One observation made at this time, which may eventually give some insight into the nature Of the agglutination reaction, was that Of the effect Of a time-varying tempera- ture on settling blood suspensions. With a rate of change of temperature of the order of 2-30C drop per hour, a non-agglutinating settling blood suspension formed no interface, and its graphical record was identical in form to that Of a agglutinating sample without the time-varying temperature. The graphical characteristics of agglutination at a steady tempera- ture appeared when a run was performed with a temperature drOp whether the samples contained antibody or not (see Figure 12). The magnitude Of the effect on the agglutinating sample was greater because of the combined action of antibody plus temperature drop. Further investigation into the phenomenon might lead tO more understanding Of the forces involved in agglutination. U! 0) .b / \e LIGHT TRANSMISSION N l L l l O I 2 3 4 HOURS FIGURE 12. Plot Of two samples with temperature drop: A. Blood suspension with antibody; B. Blood suspension without antibody. 26 The next series Of runs was designed to investigate the repro- ducibility and the sensitivity Of the machine. With runs after V-77, magnetic fields were not applied to the samples. The test specimens were four pairs of two samples each, all eight having the same blood concentration, but each pair having a different antibody concentration. Various factors influenced the reproducibility from run tO run. The blood samples obtained from the Red Cross were not from the same donor each time. Since variation in sensitivity to antibody con- centration is common among individuals, it is not surprising to find a variation in response with such blood. Aging of the blood is another factor causing variation. Deterioration takes place even though the blood is refrigerated. Variations in ambient temperature about the machine would also affect the agglutination rates. Although each of these factors in themselves might produce only a slight difference, in combination they might account for the run-tO-run variability noted in our data. Before we investigate the sensitivity of the instrument it would be advisable to consider the mathematical treatment of light trans- mission as described by a modified form Of the Lambert-Beer law (15). A parallel beam of light with cross sectional area A falls on a sus- pension Of path length Almade up of spherical particles Of diameters di dispersed in a fluid where i indicates the different diameters. The re are mi of these particles in a gram Of particle material corresponding to each di' The projected area per gram of material is: z ni k <11z , (1) where k is a comprehensive constant. The total projected area Of these particles per cm3 Of suspension is: 27 2 where C is the concentration Of the suspension. The projected area of particles in light path is Aaflcznikdf. (3) 1 Expression 3 is equal tO the change in effective cross sectional area (-A A) Of the beam going through the suspension. —AA = AAIC Enikdkz (4) 9J5 -13 7T 0 i D 5 FIGURE 13. Incident light beam of A0 and intensity 10 on blood suspension. I go I 2 l0 A! c 7;: nikdiz (5) A 2 lnA-f- =jC?Jnikdi (6) 28 Some of the light is blocked by the particles when a beam of light falls on a suspension. The initial beam of area A0 with intensity Id becomes, after passing through the suspension, a beam of area A; with same intensity. A photocell will record the light passing through the suspension as being of area A0 but of lower intensity 1;. The two energies must be the same. 10 Al : A0 11 (7) in = in. A1 1 So: [n '11-9- 3: IC Znikdiz . (8) I 1 Letting V = C 2 ni k di2 1 In?L = 1 vm (9) l The factor V (t) is the blocking areahof material per unit volume ‘ in the light beam path. This factor is a function of time because of ‘ the change in distribution‘of particle diameters resulting from aggluti- nation. The major assumptions in the derivation are: (a) multiple scattering is absent; (b) the projected area ofthe particles determines the amount of light that is blocked; (c) k is a. constant not dependent on particle ’size. 'The concentrations. of blood suspensions were sufficiently dilute that mutual interference between particles, such as multiple scattering or hindered settling, was not important. The assumption that this projected area is equal to the light blocked and k is constant is made on.the« basis that the erythrocyte dimensions are large compared with the wave length of the light. 29 We have just seen how knowledge of the size distribution of agglomerates would permit calculation of the optical density in a particular stratum of an agglutinating suspension at a particular instant. Analysis of the sedimentation process, along the lines . described in Cole's thesis, would in principle permit the determina- tion of the time dependence of the particle distribution at a particular stratum in terms of the initial distribution throughout the suspension. Moreover, we should be able to invert this time dependence to solve the converse problem of inferring the course of the reaction from the observed settling curves. In practice the theory is too complicated to carry out either process. Hence we look for empirical expressions which will let us use the method phenomenologically, and which hope- fully will ultimately point to a theoretical scheme for agglutination. Hence we search for parameters to characterize the observed curves, and study their dependence on relevant variables. Schematically, it is easy to see from Figure 2 how agglutination affects the course of the settling curve. We have seen that in the absence of agglutination the transmission remains low until the inter- face from a relatively homogeneous distribution of erythrocytes passes before the window. As the concentration of antibody is increased, Figure 14 shows that stronger agglutination produces a nonuniform time-varying distribution which results in an increase in transmission at very early times. Furthermore, the point at which the transmission shoots up rapidly (corresponding to the passage of the interface in the absence of antibody) moves to earlier times with increasing concentration, as does the place where the transmission levels off at Im- It is not clear what parameters will be most useful, and much of our effort has been expended in searching for those. 30 From Figure 14 it can be seen that each plot of log of the trans- mitted light intensity against time can be represented by a combination of two different curves as indicated in Figure 15. The first curve (A) indicates the agglutination process and intersects the second curve (B) which represents the interface falling. across the path of the light beam. Several different types of quantitative analysis were applied to the experimental graphs without leading to consistent results. Among these analyses were the slopes of various portions, height and distance along the time axis at various percentages of final value, and intersection of agglutinating and interface curves. The first part of the total curve was finally chosen as the portion best indicating the difference in antibody Concentration between samples and the one being most reliable for analysis. This portion would corres- pond to region 2 of the agglutinating sample in Figure 11. The equation for this portion is given by: in I = In 10, - ae”bt a, b, fin Ia: are constants This curve goes to in I = in In, - a at t = 0 and approaches the line In I = in lo, as t —->' (D In 10) - finl= ae”bt ln(£nlm- £nl)= Ina-ht Choosing Inlm and plotting I n ( Zulu, - In I) against time, we can obtain b and a. The value of b so obtained was plotted against concentration of antibody in the sample. In Figure 16 we have the slope of In ( lnlm - $11) = [n a - bt plotted against serum concentration for 6 different runs. There is indicated some sort of general relationship between b and concentration which seems to hold for lower concentrations of antibody 31 .FwAIKV Cd.” mCOMwNHwCQUCOU ESHMm adonomfip w HOW meat. omcfimmm Em: poufigmcmnu mo >fimc8§ 9.3 mo mob oat mo uofinm 3: mmDOHh IIEL ON 1 3 x‘ \\ v. 92:. v a N. O. 1 d «O no N a 0d 1901 32 noo_o_ C‘J' TIME FIGURE 15. Interpretation of settling curves which were obtained by plotting log of transmitted light intensity against time. A. Agglutination curve. B. Interface curve. 25.. 0 =168 x #69 . / O 3'74 // 2.0b 0 3,76 ’50) A =l77 x’/ . =l79 ’ o is, . // /Q’ A LU // Q ,/ x g :.o_ , ,3 U) 0 I, ’ ’ml/ 05 _ I I I o x’ . . _. . O 0.! 0.2 0.3 0.4 CONC ENTRATION OF SERUM FIGURE 16. Plot of the slope of IMZnIm - In I) = [n a - bt against concentration of serum for six runs. 33 and which breaks down as the concentration increases. Further analysis showed, that although this equation somewhat predicted the behavior of a family of curves with concentration of serum, as the parameter there were other variables not under] complete control which caused this analysis to break down. CONCLUSIONS 1. For certain immune systems, antibody concentrations may be assessed by observing the sedimentation rate of agglutinating erythrocytes. The sedimentation rate is determined by observing the time dependence of the optical density at a given stratum of agglutinat- ing erythrocytes suspended in a plasma-saline medium. A. The sedimentation rate in the ABO agglutination process may be used to assess antibody concentration quantitatively, though not very precisely. B. The Anti-D reaction in the Rh system was found unsuitable for study by the sedimentation process with the apparatus described herein. 2. No effect on ABC system agglutination was detected by the sedimentation process as observed by this machine under the stated condition of magnetic field. 3. A search for useful parameters characterizing the strength of the agglutination reaction as determined by the sedimentation curves was undertaken. The earliest portion of the settling curve was established as holding the most promise for quantative under- standing of the agglutination process. The analysis remains unsatis- factory, probably because of imperfect control of all variables in the experiment. 34 BIBLIOGRAPHY 35» 10. 11.. 12. 13. 14. 15. BIBLIOGRAPHY . Kabat, Elvin A. Experimental Irnmunochemistry. Bannerstone House , Illinois , 1961 . Smith, A. E. Ph. D. Thesis, Michigan State University, 1963. . Foner, B. G. "Human Erythrocyte Agglutination in Magnetic Fields, " Boston University School of Medicine. . Cole, H. P. M. S. Thesis, Michigan State University, 1963. . Blood Group Antigfns and Antibodies. Ortho Pharmaceutical Corporation, New Jersey, 1960. The Rh Factor. Ortho Pharmaceutical Corporation, New Jersey, 1955. . Albritton, E. C. Standard Values in Blood. W. B. Saunders Co., Philadelphia , 1952 . . MacFarlane, R. G. and Robb-Smith, A. H. T. Functions of the Blood. Academic Press, New York, 1961. . Pollard, E. C. and Setlow, R. B. Molecular Biophysics. Massa- chusetts: Addison-Wesley Publishing Co. , Inc. , 1962. Behrendt, H. Chemistry of Erythrocytes. Bannerstone House, Illinois, 1957. Boyd, William C. Fundamentals of Immunology. New York: Inter- state Publishers, 1956. Boyd, W. C. and Hooker, S. B., Proceedings of the Society of Biological Medicine, 19, 491 (1938). Belkin, R. B. and Wiener, A. S. Proceedings of Society of Experi- mental Biological Medicine, _5_6_, 214 (1944). Cushing, J. E. and Campbell, D. H. Principles of Immunology. New York: McGraw-Hill, 1957. Rose, H. E. and Lloyd, H. B. Journal of the Society of Chemical Industry, Transactions and Communications, 65, 52 (March, 1946). 36 APPENDIX DA TA 37 APPENDIX Blood Type and DA TA No. Samp. No. Samp. Conc. Run No. Conc. in Mag Out Mag Temp. Serum Type v-1 0+ (150 1/100 ml) 0 46“ room 0 - V-Z 0+ (150 A/lOO ml) 0 4c room 0 - V—3 0+ (150 A/IOO ml) 0 4c room 0 - V-4 0+ (150 X/IOO ml) 0 4c room 0 - (2 with V-5 0+ (150 X/IOO ml) 0 4oz withodaom 0 — (2 with V-6 0+ (150 A/IOO ml) 0 4c2 veaalséfiaom 0 - V-7 A (50 A/33. 3 ml) 0 4c *1: room 0 - V-8 A (50 A/18 cc) 0 2c, Zs room 0.036 A V-9 A (50 A/18 cc) 0 2c, Zs room 0.0071 A V-10 A (50 A/18 cc) 0 2c, 2s room 0.021 A V-ll A (50 A/18 cc) 0 2c, 2s room 0.036 A v-12 A (50 V18 cc) 0 2c, 23 room 0.036 A v-13 A (50 >./18 cc) 0 2c, 23 room 0.036 A V-14 B (50 A/15 cc) 0 2c, 2s room 0.036 B v-14 B (50 >./15 cc) 0 2c, 2s room 0.071 B v-15 B (50 1/15 cc) 0 2c, 2s room 0.036 B V-l6 B (50 A/15 cc) "ls 1c, 2s room 0.036 B V-17 B (50 A/15 cc) ls 1c, 23 room 0.036 B V-18 B (50 A/15 cc) ls lc, 2s room 0.036 B v-19 B (50 1/15 cc) ls 1c, 28 room 0.036 B V-20 0+ (50 A/15 cc) ls 1c, 2s room 0.036 D V-Zl A (50 A/15 cc) Is 23 room 0.036 A v-22 A (20 1/15 cc) ls 2s room 0.022 A V—23 A (20 A/15 cc) ls Zs room 0.021 A v-24 A (20 1/15 cc) ls 2s room 0.043 D v-25 0+ (20 1/15 cc) ls 23 37° 0.043 D V-26 0+ (50 >./15 cc) ls 2s 39° 0.125 D v-27 0+ (50 21/15 cc) ls 25 38° 0.125 D V-28 0+ (50 1/10 cc) Is 23 37° 0.125 D = without antibody with antibody 38 Continued- - 39 Run N Blood Type and No. Samp. No. Samp. Conc o. Conc. 1n M_ag Out Mag Temp. Serum Type v-29 A+ (50 x/ls cc) 1 ° v-30 A+ (50 1/15 cc) 1: 1:1: 37° 0.125 D V-31 0+ (BOA/10 cc) 1c 1c,ls 37o 0.25 D V-32 0+ (100 A/IO cc) 1c 1c,ls :70 0.25 D V-33 0+ (50A/10 cc) 1c 1c,ls 7o 0.25 D V-34 0+ (50 k/IS cc) 1c 1c, 18 37o 0.25 D v-35 0+ (50 1/15 cc) 1c 1c,1 37° 0.25 D V-36 0+ (50 k/IS CC) 1c 1c, 1: 37o 0.25 D V-37 0+ (50 A/15 cc) 1c 2c, 37o 0.25 D V-38 0+ (50 A/lS cc) 1c 2c 370 O - V-39 AB+ (50 A/15 cc) ls 25 37o O - V-40 AB+ (50 k/15 cc) ls 23 37 0.036 B V-41 AB+ (50 x/15 cc) 1s 2s mgm 0.036 B v-42 AB+ (50 1/15 cc) ls 25 33 fail!) 0.036 B v-44 AB+ (50 1/15 cc) ls Zs mgm 0.036 B V-45 AB+ (50 A/15 cc) ls 23 33ofai£lé 0.036 B V-46 AB+ (50 A/15 cc) Is 23 320 0.036 B V-47 AB+ (50 A/15 CC) Is 28 300 0.036 B V-48 AB+ 1(50 A/15 cc) ls 2 O ’38 00.036 B V-49 AB+ (50 A/15 cc) ls ZS 33of¢11F° 33 0.036 B v-so AB+ (50 1/15 cc) 0 38 35° mung 0.036 B (1 in durimy) 35 falling 0.036 B V-51 AB+ (50 1/15 cc) (:1 . 33 35° falling 0.036 B v-52 AB+ (50 1/15 cc) ls m dim” ° v-53 AB+ (50 1/15 cc) Is 2s 3 ° 35 0.036 B 5 fell and 0.036 B V-54 AB+ (50 x 15 ° then rose v-55 AB+ (50 les 2:) 1: :8 37° faufng 0.036 B V-56 AB+ (50 A/15 cc) ls ZS 390 fallfng 0.036 B * v-57 AB+ (50 )./15 cc) ls 28 37° falung 0.036 B new mag.V—58 AB+ (50 A/15 cc) ls ZS 38 falling O. 036 A V-59 AB+ (50 A/15 cc) ls 28 50°31 0.036 B V-60 AB+ (50 A/15 cc) 1s ZS 3465 fapmg 0.036 B V-61 AB+ (50 x/15 cc) ls l8 1 37° falling 0.036 B V-62 AB+ (50 A/15 CC) 13 1C, IS 37 falling 0.018 B V-63 AB+ (50 x/15 cc) Is 2:, S 3655 0.036 B V-64 AB+ (50 X/15 CC) Is 2 36 0.036 A v-65 A+ (50 x/15 CC) 18 2s rogm 0.036 A v-66 A+ (100 x/15 CC) 13 25 27 room 0.036 A V-67 A+ (100 A/lS cc) ls 28 room 0.036 A V-68 A+ (100 A/lS cc) ls ZS room 0.036 A 5 room 0.014 A Continued- - Blood Type and No. Samp. No. Samp. Conc. Run No. Gone. in Mag Out Mag Temp. Serum Type V-69 Ark (100 X/IS cc) ls 25 room 0.0071 A V-70 A+ (100 A/IS cc) ls 25 room 0.0036 A v-71 A+ (100 x/15 cc) 15 25 room 0.0043 A v-72 A+ (100 1/15 cc) 15 25 room 0.071 A v-73 A+ (100 1/15 cc) 15 25 room 0.071 A v-74 A+ (100 1/15 cc) 15 2s 37°-35° 0.036 D v-75 A+ (50 1/15 cc) 15 2s 37°-40° 0.071 D V—76 A+ (50 1/15 cc) 15 23 37° 0.071 D v-77 A+ ( 300 1/15 cc) 15 25 room 0.044 D V-78 A+ (200 1/30 cc) 0 85 room 0.005- A 0.020 V-79 A+ (200 A/30 cc) 0 85 room 0.006- A 0.009 V-80 A+ (200 X/30 cc) 0 85 room 0.011-0. 014 A V-81 A+ (200 A/30 cc) 0 85 room 0.005-0.021 A V-82 A+ (200 1/30 cc) 0 85 room 0. 0110014 A V-83 A+ (200 1/30 cc) 0 85 room 0.010-0.016 A V-84 A+ (200 1/30 cc) 0 85 room 0.0lO-0.016 AB V-85 A+ (300 1/30 cc) 0 85 room 0.0lO-0.016 AB V-86 A+ (200 1/30 cc) 0 85 room 0. GIG-0.016 AB V-87 A+ (200 X/30 cc) 0 85 room 0. 012-0.018 AB V—88 A+ (200 1/30 cc) 0 85 room 0.012-0.018 AB V-89 A+ (200 1/30 cc) 0 85 room 0,012-0. 018 A V-90 A+ (200 A/30 cc) 0 85 room 0. 005-0. 020 A v-91 A+ (200 >./30 cc) 0 85 room 0.005-0.020 A v-92 A+ (200 x/30 cc) 0 85 room 0.005-0.020 A v-93 A+ (200 1/30 cc) 0 85 room 0.005-0.020 A v-94 A+ (200 1/30 cc) 0 85 room 0.005-0. 020 A V-95 B+ (2.00 A/30 cc) 0 85 room 0.010-0.04 B V-96 B+ (200 A/30 cc) 0 85 room 0.00-0.030 B v-97 B+ (200 1/30 cc) 0 85 room 0.00-0.030 B V-98 B+ (200 A/30 cc) 0 85 room 0.0-0.020 B v-99 3+ (200 >./30 cc) 0 85 room 0.0-0.0120 AB v-100 . . 3+ (200 >./30 cc) 0 85 room 0.0-0.012 AB v-101 B+ (200 1/30 cc) 0 85 room 0.004-0.01 AB v-102 B+ (200 >./30 cc) 0 85 room 0.0-0.012 AB v-103 B+ (200 1/30 cc) 0 85 room 0.0-0.015 AB v-104 B+ (200 1/30 cc) 0 85 room 0.02-0.05 AB v-105 B- (200 1/30 cc) 0 85 room 0.01-0.025 AB V-106 B- (200 A/30 cc) 0 85 room 0.0-0.015 AB v-107 B- (200 1/30 cc) 0 85 room 0.0-0.012 AB V-108 B+ (200 1/30 cc) 0 85 room 0.0-0.012 AB v-109 AB+ (200 1/30 cc) 0 85 room 0.012 AB Continued-- 41‘ Blood Type and . Samp. No. Samp. Conc Run No. Conc. Mag Out Mag Temp. Serum Type v-110 B+ (200 V30 cc) 0 85 room 0.0-0.012 AB v-111 B+ (150 V30 cc) 0 85 room 0.0-0.012 AB V-112 B+ (150 A/30 cc) 0 85 room 0.0-0.012 AB v-113 B+ (200 V30 cc) 0 85 room o.0-0.012 AB V-ll4 B+ (200 A/30 cc) 0 45 room 0.0-0.012 AB V-llS AB+ (150 X/30 cc) 0 85 room 0 -0.012 AB V-116 AB+ (200 V30 cc) 0 83 room 0 -0.016 AB v-117 AB+ (150 V30 cc) 0 85 room 00-0 016' AB V-118 AB+ (200 A/30 cc) 0 85 room 00-0.016 AB V—ll9 AB+ (150 A/30 cc) 0 85 room 00-0.03 AB V-lZO AB+ (150 A/30 cc) 0 85 room 00-0.03 AB V-lZl AB+ (150 A/30 cc) 0 85 room 00-0.03 AB V-lZZ AB+ (200 A/30 cc) 0 85 room 00-0.03 AB V-123 AB+ (200 A/30 cc) 0 85 room 00-0.03 AB V-124 AB+ (200 A/30 cc) 0 88 room 00-0.03 AB V-lZS AB+ (200 X/30 cc) 0 85 room 00-0.03 AB V-126 AB+ (200 A/30 cc) 0 88 room 00-0.03 AB V-lZ7 AB+ (200 A/30 cc) 0 85 room 00-0.03 AB V-128 AB+ (200 A/30 cc) 0 85 room 00-0.03 AB v-129 AB+ (200 V30 cc) 0 85 room 00-0.03 AB v-130 AB+ (200 V30 cc) 0 85 room 0.005-0.02 AB v-131 AB+ (200 V30 cc) 0 85 room 0.005-0. 02 AB v-132 AB+ (200 V30 cc) 0 85 room 0.005-0.02 AB v-133 AB+ (200 V30 cc) 0 83 room 0.005-0.02 AB v-134 AB]- ,(200 V30 cc) 0 85 room 0,005-0.02 AB v—135 AB+ (200 V30 cc) 0 85 room 0.005-0.02 AB V-l36 AB+ (200 V30 cc) 0 85 room 0.005-0.02 AB v-137 AB+ (200 V30 cc) 0 85 room 0.005-0.02 AB V-l38 AB+ (200 A/30 cc) 0 85 room 0.005-0.02 AB v-139 AB+ (200 V30 cc) 0 8 5 room 0.005-0. 02 AB v-140 AB+ (200 V30 cc) 0 8 5 room 0. 005-0. 02 AB V-l41 AB+ (200 A/30 cc) 0 8 5 room 0.005-0. 02 AB V-l42 AB+ (200 A/30 cc) 0 8 5 room 0. 005-0. 02 AB v-143 AB+ (200 V30 cc) 0 8 5 room 0.005-0.02 AB V-144 AB+ (200 V30 cc) 0 8 5 room 0.005-0.02 AB v-145 AB+ (200 V30 cc) 0 8 5 room 0.005-0.02 AB V-l46 AB+ (200 V30 cc) 0 8 5 room 0.005-0.02 AB v-147 AB+ (200 V30 cc) 0 85 room 0. 005-0.02 AB V-148 AB+ (200 V30 cc) 0 8 5 room 0.005-0.02 AB v-149 AB+ (200 V30 cc) 0 8 5 room 0.005-0.02 AB v-150 AB+ (200 V30 cc) 0 8 5 room 0.01-0.04 AB v-151 AB+ (200 V30 cc) 0 85 room 0.01-0.04 AB V-152 AB- (200 A/30 cc) 0 85 room 0.005-0. 02 AB C ontinued- - 42 Blood Type and No. Samp. No. Samp. Conc. Run No. Gone. in Mag: Out Mag Temp. Serum ije V-153 AB- (200 A/30 cc) 0 85 room 0.005-0.02 AB V-154 AB- (200 A/30 cc) 0 85 room 0.005-0.02 AB V-lSS AB- (200 A/30 cc) 0 85 room 0.005-0.02 AB V-156 AB- (200 A/30 cc) 0 85 room 0.01-0.04 AB V-157 AB+ (200 A/30 cc) 0 85 room 0.015-0.03 AB V-158 AB+ (200 V30 cc) 0 85 room 0.005-0.02 AB V-159 AB+ (200 A/30 cc) 0 85 room 0.015-0.03 AB V-160 AB+ (200 V30 cc) 0 85 room 0.015-0.03 AB V-161 AB+ (300 V30 cc) 0 88 room 0.000-0.02 AB V-162 AB+ (400 A/30 cc) 0 85 room 0.007-0.025 AB V-163 AB+ (400 V30 cc) 0 8s room 0. 007-0. 026 AB V-164 AB+ (500 A/30 cc) 0 85 room 0.007-0.028 AB V-l65 AB+ (400 A/30 cc) 0 85 room 0.015-0.039 AB V-166 AB+ (500 A/30 cc) 0 85 room 0. 02-0. 05 AB V-167 AB+ (500 A/30 cc) 0 85 room 0. 02-0. 05 AB V-168 AB+ (475 71/30 cc) 0 85 room 0.01-0.04 AB V—169 AB+ (400 V30 cc) 0 85 room 0015-0. 04 AB V-170 AB+ (500 A/30 cc) 0 85 room 0.01-0. 04 AB v-171 AB+ (500 V30 cc) 0 85 room 0.02-0.05 AB v-172 AB+ (500 V30 cc) 0 85 room 0.01-0-04 AB V-l73 B+ (400 A/30 cc) 0 85 room 0.01-0.04 AB v-174 B+ (400 V30 cc) 0 85 room 0.015-0.045 AB V-175 B+ (400 A/30 cc) 0 85 room 0.015-0.045 AB V-176 B+ (400 A/30 cc) 0 85 room 0.015-0.045 AB V-l77 B+ (400 A/30 cc) 0 85 room 0.015-0.045 AB V—178 B+ (400 A/30 cc) 0 85 room 0.035 AB V-l79 B+ (400 A/30 cc) 0 85 room 0.015-0.045 AB V-180 B+ (400 A/30 cc) 0 85 room 0.011-0.045 V-181 B+ (400 V30 cc) 0 85 room 0.010-0.045 V-182 B+ (400 A/30 cc) 0 85 room 0.01-0.045 AB V-183 B+ (400 A/30 cc) 0 85 room 0.005-0.090 AB v-184 AB+ (400 V30 cc) 0 88 room 0.005-0.040 AB v-185 AB+ (400 V30 cc) 0 85 room 0.005-0. 040 AB V-186 AB+ (400 V30 cc) 0 85 room 0.005-0.040 AB V-187 AB+ (400 V30 cc) 0 85 room 0.005-0. 040 AB “111111111 1111))“