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I . . . l 3 IV In. or: . .III I. I « I .IIII. I. ID I A I . a . OII . I II 2 I II . rflf I '- KAHN PRECIPITATION TEdT WITH SPECIAL REFERENCE TO THE HEATING OF SERUH. THESIS Submitted to the Faculty of the Kichigan state College in partial fulfillment of the requirements for the degree or’Master of Science. \ B 3k. 3% Jr J2- Info Be“,Kurtz, Do V. I... June 1928. THESlS C ON +3 h‘ V Y .- ; *3 U) A I INTRODUCTION. KAHN'S STUDIES ON THE PRENONSNA OF PRECIPITATION. THE KAHN TEST. PREPARATION AND STANDARDIZATION OF ANTIJEN. PREPARATION OP SLRUA. ROUTINU DIAaNOSTIC T ST. QUANTITATIVE PROCEDURE. REVIEW OF PREVIOUS RXPERINLNTS ON finnTINd OP Smfidm. RXPSRIULNTAL. OUTLINE OF PROCEDURS. EXPERIMENTS.1 - VIII CONCLUSIONS. ACKNOWLEDGMENT. , BI BLI OGRAPHY . 102150 KIRK PRECIPITATION TEdT WITH SPECIAL REFEREKCE TO TqE HEATING OF SERUM.‘ In the Kahn and Wessermann tests it is customary to heat the serum for 30 minutes at 56°C. before carrying out the test. In the case of the Wassermann test the heating treatment is term- ed "inactivation" since it is the means of destroying the native complenent. In the Kahn reaction, on the other hand, in which conplement does not play a role, heating is resorted to because it actually increases the sensitiveness of the serum. Therefore the term inactivation is not properly used in this test. Regard- ing this increase in sensitiveness Kahn has shown that serum heat- ed at 56°C. is more sensitive in his test than unheated serum. No adequate explanation for this increase in sensitiveness has yet been prOposed. In the present paper, Kahn's studies on the phenomena of precipitation are discussed, the essentials of Hahn procedures with serum are reviewed, a summary of previous eXperiments on the effect of heating serum is given, and some new experiments on the heating Of serum are presented. These last mentioned experiments represent an attempt to deterrine whether the re- sults obtained by heating the serum above 56°C. for a relative: 1y short period of tire might not parallel the results obtained in the regular procedurgfxwhich the serum is heated at 56°C. *In presenting the theoretical and technical aspects of the Kahn test, reference is constantly made to the two books listed as (l) and (2) in the bibliography. 2. for 30 minutes. Any reduction in the duration of the heating period would be of practical importance, espec- ially in blood transfusions or emergency surgical cases. KAHN'S STUDIES ON THE PHENONENA OF PRECIPITATION. In the Kahn test for syphilis, serum is mixed with antigen suspension in one or more prOportions. The mix? ture is shaken under standard Conditions, physiological salt solution added, and the results read on the basis of the presence (positive serum) or absence (negative serum) of a precipitate. The antigen used is a specially prepared al- coholic extract of dried heart muscle which is mixed with physiological salt solution to form the antigen-saline sus- pension. Practically all alcoholic extracts of animal tissue, when properly mixed with synhilitic serum, will produce pre— cipitates after a certain amount of incubation. Therefore it would appear that to evolve a precipitation test for syphil- is would be an easy procedure. However no precipitation test had met with any great degree of success until Yahn, studying the factors affecting precipitation with syphilitic serum, worked out a test embodying Optimum conditionsibr precipitation. The conditiors established are as follows: 1. Optimum concentration. 2. Instability of antiven-saline susnension. 3. Serum antigen suspension prOportion 4. Agitation. The rate and degree of precipitation is markedly affected by concentration of the ingredients. Excess- ive dilution of the serum with salt solution, or of the antigen with alcohol or salt solution will so delay the precipitation reaction that incubation at higher tem- peratures is necessary. On the other hand, excessive concentration, especially of the beef heart extractives in the antigen may actually inhibit the precipitation reaction. If the ingredients are employed at certain optimum concentrations the precipitate is formed almost immediately without incubation and thus the contamination that so oiten accompanies incubation is eliminated. When the antigenasaline mixture consists of very fine highly dispersed particles (an opaleeeent mixture) the precipitation reaction is not as marked as when the antigen-saline mixture contains relatively large particles. If, on the other hand, the particles in suspension are not readily dispersed when brought in contact with saline or serum, the suspension cannot be used in a diagnostic test since particles would as found in the serum-antigen suspension mixture with bOth negative and positive serum. In other words a coarse readily dispersable antigen-saline suspension is demanded by the condition 0! this test. This 4.. necessitates the use of a standard controlled method of preparinr the antigen-saline suspension. Experiments indicate that for complete precipitation in any given serum-antigen suspension, there must exist a prOper quantitative relationship between the reacting substances of the serum and the reacting substances of the antigen. A given volume of a strongly potent serum will react best with a relatively large amount of antigen sus- pension (as 1 part serum to 1/3 part! of antigen suspen- sion), whereas the same volume of a weakly positive serum will react best with a smaller volume oifantigen suspension {as 1 part of serum to 1/12 part of antigen suSpension). Therefore it appeared that in order to be successful a precipitation test should include varying proportions of serum and antigen. This principle is used by Fahn in his standard procedure which consists of a E-tube test, the technique of which will be described later. It was discovered by Fahn in his early work that agitation definitely increased the precipitates in tne weakly positive serums. Although shaking is not necessary in strongly potent serumsit does render them more easily read and is always employed in the regular procedure. fihe effect of shaking was studied by Kahn, and Optimum condi- tions for the rate of shaking and for the length of time were worked out. 5. THE KARI“! TEST Preparation and standardization of antigen. Kahn antigen is an alcoholic extract of dehydrated powdered beef heart, the preparation of which is, in brief, as follows: Fifty grams of powdered beef heart are extracted with 200 cc. of ether for ten min- utes and filtered. Three subsequent ether washings are made with 150 cc. of ether reapectively, using the same extraction period of 16 minutes. After the last filtration, the beef heart is spread out on paper and dried until every trace of ether odor is removed. The beef heart is now weighed (it will be found that approx- imately 5 grams of beef heart will be lost during the process of extraction with ether) and 5 cc. of 953 alco- hol is then added to each pram o: the dry beef heart, and allowed to extract in the dark at room temperature for three days. At the end of the three day extraction period the beef heart is separated from the alcoholic ex— tract by filtration and discarded. The extract is now ready for cholesterolization. 0.63 of pure cholesterol is added to the extract. The dissolving o: the Cholester- 01 is facilitated by gently rotating tie flask containing the mixture in warm water. After the Cholesterol is thor- oughly dissolved the antigen is filtered to render it free from foreign particulate matter. The anti an is then ready to be titrated. The purpose of the titration is to determine the smallest amount of physiological salt solution waich will produce a suspension capable of redissolving upon the further addition of saline or neg- ative serum.‘ It is desirable (but not necessary) to Ob- tain an antigen which has a titer of 1 cc. antigen plus 1.1 cc. of physiolOgical salt solution. Antigensprepared from dried beef heart by the stand- ard nethod will frequently vary in sensitiveness due to variations in the biological product employed. A certain degree of antigen sensitiveness was selected as standard on the basis of clinical and serological studies carried out jointly by the Iichigan Department Of Wealth and the Department of Dermatology and Syphiloloyy at the University of Zichigan fospital. Standardization. If an antigen prepared according to the required method is not identical in sensitiveness with standard antigen as shown by its reactions with synhilitic serum, the antiaen must be modified in sen- sitiveness in such a way that it wil; correspond with the standard. The correction method involves an in- crease or decrease of the concentration of beef heart ‘ Solution of the suspension particles as a preliminary step also takes place in the case of positive serum. It is, however, almost imrediately succeeded by reprecipitation, which does not occur in the ease of negative serum or of salt solution. 7. extractives of the antigen, or the addition to the antigen of an extract known as sensitizing reagent, especially prepared from ether extractives of beef heart. Preparation of serum. Serum used in the Kahn test must be clear and free from particles. Therefore the s ecimen of blood must be either centrifugalized or allowed to stand for some tire in order that the clear serum may be separated from the clot and cells. The serum after being heated for 30 minutes at 56°C. in a water bath, is ready to be tested. If serum is to be kept several hours after heating before being tested , a subsequent heating period of 10 minutes at 56°C. should be applied. Serums when allowed to stand for sore time at ice box temperatures tend to become cloudy. This cloudiness usually disappears When the serum is reheated for 10 minutes. Routine diagnostic test. The routine Fahn test is a 3- tube procedure in which three different proportions of serum CA) and antigen suSpension are mixed. Into tubes 1, 2 and supported in a metal rack are measured respectively, 0.05 cc., 0.025 cc. and 0.0125 cc. antigen suspension. To each of the three tubes is added 0.15 cc. of previously heated serum. Che rack of tubes containing the antigen suspension and serum is shaken for three minutes at about 280 oscillations per minute. To the first tube containing 0.05 cc. antieen suspension is added 0.1 cc. saline. To the second and third tubes containing 0.025 cc. and 0.0125 cc. of antiven suspension is added 0.5 cc. saline 0 Reading and interpretation of results. The negative reac- tions appear Opalescent but free from visible precipitate. The positive reactions are marked by the formation of precipitates which may be very fine and barely visible to the unaided eye (in doubtful reactions) or may show increasingly marked precip- itates up to a well defined precipitate suspended in a clear medium (++++, four plus reactions). The intermediate precipit- ates are graded +, ++ and +++, reSpectively. The following scheme summarizes the steps of the procedure: Tube 1 Tube 2 Tube 3 berum:Antiyen Suspension 3:1 6:1 12:1 Antigen suspension, cc. 0.05 0.025 0.0125 Serum, cc. 0.15 0.15 0.15 Saline, cc. 1.0 0.5 0.5 Quantitative procedure. In the quantitative procedure, antigen suspension is mixed in C16 proportion of 1 to 15 with increasing dilutions of serum. The serum dilutions are nade according to the following scheme. Dilution No. Dilution Ratio. (1) 1 a undiluted serum (2) 5 a 0.2 cc. undiluted serum plus 0.8 cc. nornal saline. (3) 10 = 0.7 cc. of (2) plus 0.7 cc. normal saline. (4) 20 z 0.2 cc. of (3) plus 0.2 cc. normal saline. (5) 30 a 0.2 cc. of (3) plus 0.4 cc. normal saline. (6) 40 = 0.1 cc. of (3) plus 0.3 cc. normal saline. (7) 50 a 0.1 cc. of (3) plus 0.4 cc. normal saline. (8) 60 a 0.3 cc. of (3) plus 0.5 cc. normal saline. Eight tubes are placed in a rack and 0.01 cc. antigen suspen- sion is pipetted into each tube. The serum dilutions in 0.15 cc. amounts are then added to the tubes containing the antigen suspen- sion, pipettirg fron the highest to lowest dilution. The rack of tubes is shaken 3 minutes, 0.5 cc. salt solution is added to each tube and the results read. The routiui E-tube test previous- ly described does not differentiate the potency of two, 4-plus serums, yet one serur may contain a much greater number of reacting units than the other. The quantitative test was designed to measure relatively the potency of a serum Which is strongly positive. The relative potency expressed in Kahn reacting units may be computed according to the formula S u 4 D, 8 representing serum in terms of reacting units, and D representing the highest serum dilution ratio in which there is a definite precipitate. Thus, if 1:5‘ represents the highest dilution of serim capable of producing a precipitate, 4 x 5 or 20 reacting units are contained in this serum. _Likewise if 1 : 40 represents the highest dilution in wuich a serum gives definite precipitation the serum contains 4 x 40 or 160 units. In the quantitative test a definite precipitate (++++, +++ or ++) is considered as positive, and a wear precipitate (+ or g) as negative. Additional procedures include quantitative and qualitative tests with soinal fluids, tests with fluids from syphilitic lesions and micro tents to be used only When minute amounts of serum or spinal fluid are available. ‘ Serum dilution ratio is taken as the ratio of the volume of diluted serum at a given dilution to the volume of the serum before dilution. Thus if a serum is diluted 1 to 5, the serum dilution ratio is 5. 10. REVIEW OF PREVIOUS STUDIES ON THE HEATING OF SERGE; As has already been mentioned the heating of serum within certain limits markedly increases its sensitiveness. Relatively few quantitative studies have been carried out on this important phase of the Kahn procedure. A brief summary of the work that has been done follows. In Hahn's original studies he found that serum heated for 50 minutes at 56°C. gave much stronger reactions than did a portion of the same serum unheated. In a series of 177 tests all of which pave either a +++ or ++++ reaction when the serums were heated for 30 minutes at 56°C., only 48 gave equally strong reactions when the unheated serums were used. An experiment to determine the length of the heating period of serum at 56°C. which would give the best results were carried out as follows. Unheated serums were divided into 6 parts, 1 part of each serum was allowed to remain un- heated and the remaining parts were heated at 560C. for 5, 10, 20, 30 and 60 minutes, respectively, after which they were tested by means of the regular procedure. It was shown that the more potent the serum the suorter tie heating period required. Strong serums did not require any heating while the somewhat less potent ones required only five min- utes. In Fensral there was no difference in potency between serums heated for 60 minutes and those heated for 50 minutes 11. except for a sliynt increase in sensitiveness in the ones heated for 60 minutes. It was found that there was no drop in sensitiveness of the SSTJMS after 80 minutes heat- ing and but a slight drop in sensitiveness after 5 hours. Other experiments were carried out at temperatures higher than 56°C. and longer than 30 minutes. A number of serums were heated 30 minutes at 56°C., samples with- drawn the remaining portion of each serum heated at 62°C., samzles being withdrawn and tested after 30, 60 and 120 minutes, respectively. The additional herting at 62°C. caused only a slight fall after 3-0 minutes, but became quite awpreciable after 60 minutes and very pronounced after 120 \ minutes. Kendrick’made some interesting observations on the relation between the heating of serum and sensitiveness of the reaction in Wassermann and Kahn tests. A number of pos- itive serums were eiamined by both tests before being heated and also exarined after being heated at 56°C. for varying perioos of time. In the case of the ”assernann test the most sensitive results rare, in general, obtained with the unheated serums. Jerims heated for 30 minutes were about equal in sensitiveness to the unheated serum. A sidden continuous drOp in sensitiveness was observed after 56 min- utes heating. In the Xahn test, on tie o;her hand, the sensitiveness was least with the inheated seruns and rose steadily as t.e heating period was increased. “he sensitive- ness of the bahn test was less than taet of tie ”essermann *See reference (5) in the Bibliopraphy. 12. test when the unheated serums were used. Wassermann and Hahn tests gave similar results when the heating period was 30 minutes, and the Fahn test has more sensitive for longer heating periods. In sore unpublished studies from Fahn's laboratory, Eagle and To Dermott carried out three series of experi- ments on the heating of serums, in which thev compared the effect of heating serum for various periods of time at 60°C., 58°C., and 45°C., with that of standard heating treat- ment for 30 minutes at 56°C., the results of which follow. In the first series of tests 15 serums were divided into 4 portions which were heated as follows. The first portion heated at 56°C. for 30 minutes served as a con- trol. The second nortion was heated at 60°C. for 10 min- utes, the third and fourth portions at the same temperature, bit for 15 and 20 minutes, respectively. The sensitiveness of the serums were comparable to that of tue control when the heating at 60°C. lasted 10 minutes; it was less than that of the control when the heating lastad5 and 20 minutes. In the seCUnd series seruns were heated rt 58°C. for 10, 15 and 20 minutes and:it was found that they were less sensitive than that of the control. In the third series, serums were heated at 45°C. for 15 and 30 minutes and have mucx less sensitive readtions than the control. 13. Present Experiments on the Heating of Serums. The present studies on the heating of serum were prompted largely by the growing interest in, and the prac- ca tkbility of, the iahn test. In these studies both the 3- tube and quantitative procedures, previously described, were used in a somewhat muiified form. Instead of the regular 3-tube test, a 1-tube test was emnloyed using a serum-anti— gen suSpension rztio of 12, which is the sane as that used in the back tube of the regular test. Since only small amounts of individual serums were available, it was nec- essary to reduce the amounts of serum and antigen suSpen- sion to one-half of that which is ordinarily used in the 12:1 serum-antigen suspension proportion. Thus, .006 cc. of antigen suspension was pipetted to the bottom of Kahn tubes by means of a 0.2 cc. pipette graduated in .001 cc. To the antigen suspension was added .075 cc. of serum also with a 0.2 cc. pipette. rthe teats were shaken in the standard way, 0.05 cc. of saline added and the results read. The quantitative procedure used was according to stand rd requirements except that one-half the usual amount of serum, namely, 0.1 cc. was used in the preparation of the serum di- lutions, and die to the small amount of serum available, di— lution ratio 1 was not employed. The serum dilutions were prepared according to tee following scheme. 14. Dilution Dilution Io. Ratio. 1 5 0.1 cc. undiluted serum + 0.s cc. salt solution. 2 10 0.3 cc. of dilution 1 + 0.5 cc. salt solution. 3 20 0.2 cc. of dilution 2 + 0.2 cc. salt solution. 4 30 0.1 cc. of dilution 2 + 0.2 cc. salt solution. 5 40 0.1 cc. of dilution 2 + 0.3 on. salt solution. 6 50 0.2 cc. of dilution 3 + 0.3 cc. salt solution. 7 60 0.2 cc. of dilution 5 + 0.1 cc. salt solution. Standard antigen was used throuahout these studies. 5he seruns used in these experiments were carefully removed by means of a bulb pinette from the clots after sufficient amounts had been withdrawn for the routine diatnostic test for syphilis in tie laboratories of the Eichiyan Department of Vealth. The numbers given the Specimens in the laboratory were used in this exoeriment in order to facilitate checking our technique. rihe serums were then examined for cells or foreign particles. Those which were badly hemolized or showed material which could not be removed were considered undersirable and discarded. Each serum was divided into 2 portions. One portion was heated for 30 minutes at 56°C. and served as a control. The other portion was heated at a higher temperature for varying periods 0: time. The seruns were kept stonpered during the meeting period in order to prevent evaporation. Experiment Lo. 1. Fine hundred fifty individual serums were used in this exneriment. The portion of the serur serv- ing as a dontrnl was heated for 30 minutes at 56°C., while the other portion was heated for 10 minutes at 60°C. A qual- itative l—tube procedure was employed in making the tests, and 15. the results recorded on a 4-p1us basis in Table I. Identical results were obtained in 915 (96.5%) of the serum: tested. The results obtained from the other 35 serums showed variations ranging from + to +++. The data revealed that in only 11 (1.1%) serums, was there a difference of more than +. fitn 6 of these 11 serums the control showed more sensitive reactions than those heated for 10 minutes at 60°C. while the remaining 5 gave more sensitive reactions when heated for 10 min. utes at 60°C. It appears from this exneriment with serum tested by meanscf the qualitative l-tube procedure, that a heating period of 10 minutes at 60°C. is equivalent to the regular heating period of 30 minutes at 56°C. Experiment No. II. The same heating treatment, 10 minutes at 60°C., was used with 65 Serums in a second series of experiments in which the quantitative procedure was used. The results obtained are listed in Table 2. There was agreement in 59 (91%) of the serums. In the other 6 (95) serums the variations never exceeded the difference between the number of Kahn units given by one dilution, and the number that would be given by the next hiémer or next lower dilution. Tiis experiment thus confirms the results of the previous experiment. 16. Experiment Ko. III. Prompted by the satisfactory results obtained with the serums heated for 10 minutes at 60°C. a series of tests was? undertaken using a 5 minute heating period and a temperature of 62°C. The l-tube qualitative method was employed. The results as obtained from 260 Serums are presented in Table III. In 245 (94.23) of the serums, identical results were obtained. The remainina 15 (5.83) gave variations ranging from + to +++. Twelve of the ssrums which showed variations gave more sensitive reaction in the control, while tie remaining 3 rave more sensitive reaction when heated for 5 minutes at 62°C. Although most of the serim in this experiment gave the same results wnen the heat treatment was 5 minutes at 6200., yet we find that the rajority of tie ones showing variations pave more sen- sitive results when heated ascording to the standaru nethod. This sugyeSts that 62°C. may be higher than the Optimum heat- ing temperature for serum. however, had other time neriods at 62°C. been tried, better results might h:ve been obtained. Experiment 30. IV. In this experiment one portion of vach of 35 serums were heated for 3 minutes at 65°C. and the relaining nortion of each serum heated according to the stand- ard method. The results observed are listed in Table 1V. Comparable reellts were observed in only 27 (77%) of the serurs. Less sensitive results were shown in 7 (20%) of the serums. In only one serim was the sensitiveness greater when heated for 3 minutes at 650C. than When heated according to the standard method. 17. Experiment K0. V. A few serums were heated for 5 min- utes at 68°C. and comparison made as usual with the control. None of the Serums were coagulated when subjected to this heating treatment. The results are recorded in Table V. From experiments III, IV and V, it appears that a tem- perature of 68°C. is not sufficiently high to destroy all tie serum reacting substances and this prevent precipitation with antigen but, on the other hand, it appears that most satisfactory results are obtained when the heating temper- ature is below 62°C. Experiment 10. VI. An experiment was carried out to determine the relative sensitiveness of a serum heated at 56°C. for varying tire periods. Several four plus seruns were pooled for this study. The pooled serum was poured into individual vials whicn were placed in a water bath at 56°C. At various intervals a vial was removed and its contents tested immediately with the quantitative proced- ure. The shortest heating period was 10 minutes, and the longest was 300 minutes. The results are plotted in Tig- ure 1, in waica the ordinates represent serim sensitiveness in Hahn units, and the abscissae represents duration of the heating period in minutes. Its sensitiveness was increased by the 10-minute heating period and became consistently great— er as the herting period of 40 minutes was reached. Ionper heating periods decreased the sensitiveness, but even the maximum heating period tried, 300 minutes, did not reduce 18. the sensitiveness to that of the unheated serum. Experiment No. VII. A number of vials containing portions of a given lot of pooled serums were placed in a water bath at 60°C. At stated times a vial was withdrawn and the serum tested with the quantitative procedure. The results are represented graphically in Tigure 2. It appears from this experiment that Serums when heated at 60°C. reach their maximum sensitiveness in a very short time, the opti- mum heating period in this case being between 2 and 14 rin- utes. If the heating period is prolonged beyond 14 minutes a fall in sensitiveness is observed, and when the heating period is prolonged beyond 80 minutes for the serums tested the sensitiveness is less than that of tie unheated serum. Experiment No. VIII. In order to determine the per- sistence of the increase of sensitiveness brought about by heating, the following experiment was carried out. Equal por- tions of a given lot of pooled serum were poured into a number of vials and placed at 60°C. At given intervals 3 vials were removed; the contents of one of these vials was used for an immediate test (quantitative procedure) and the contents of the other 2 vials were similarlz tested after having stood at ice box temperature for 5 minutes and at room temperature for 1 hour, respectively. The results are represented graphics ly in Tigure 3. This experiment would indicate that the increase in sensitiveness due to heating is destroyed when the heated serum 19. is alloyed to stand at room teperature for an hour before being tested, and that t'e increase is reduced when the serum is placed at 7°C. before beiny tested. It would thus appear that a heated serix gives its maximum sensi- tiveness when tested within a short time after receiving the heat treatment. It must be remembered that this ex- perimfnt was carried out with bit a single lot of pooled serim and tiat hence tie results may not aoply in all cases. Since the presistence of the heatinfl effect on thw sensitiveness of serum is of practical importance in the laboratory, further experiments are planned to test the general applicability of this observation. C NCLUJIOES 1. It appears from thisstudy that a heating treatment of serum for 10 minutes at 60°C. gives practically the same results as the regular heating period of 30 minutes at 56°C., irreSpective of whether the serums are tested with the qual— itative l-tube procedure or with the quantitative procedure. 2. heating temperatures of 62°C. and over are found to be less satisfactory in the Fahn procedure with serums than heating temperatures of from 56°C. to 60°C. 20. 3. Serums heated at 60°C. appear to reach their maximum sensitiveness in from about 2 to 10 minutes while serums heated at 56°C. require a much longer per- iod. 4. derums remaining at room temperature for on hour or at 7°C. for 5 minutes after being heated 10 min- utes at 60°C., give less sensitive reactionsthan when tested immediately. ELBIE I EFIIG! OF DIFFERENT HEATING PERIODS OF SERUM UN SENSITIYENESS OFIKAHN REACTIONS. No. of Serum: 144 746 Reaction after heating for 30 minutes at 56°C. H” +++-O ++++ +++ +++ ++ ++ ++ ++ ++ [-9 H- 12-2-27V Reaction after heating for 10 minutes at 60°C. +4-44- +++ + ++++ +++ ++ I+ +++ ++ H- H' |+ ++ l+ 2‘31! 11 ‘IIIIOI 0F DIFFEREN! HEATING PERIODS GP BERUYS on ssusirrvsnnss or QUINTIIATIVE EARN REACTIONS. No. of Serums No. of Kahn units after No. of Kahn.units after heating serum for 50 heating serum for 10 minutes at 55°C. minutes at 60°C. 1 280 280 2 240 240 1 200 200 1 160 160 3 120 120 1 .120 so 1 ‘80 4O 8 4O 40 s ‘20 ' ’ 4o 1 '20 ' 4 13 20 20 30 4 4 ’ variations. 12-2-27V 12-2-27V mu III arm: 0? DIM! HEATING PERIODS OI SERUM 0N SENSITIVENESS OF KAHN REACTIONS. No. of scrumm ‘Roastion after heating Reaction after heating for 30 minutes at 56°C. for 5 minutes at 6290. 23 ++++ ++++ 1 ++++ +++ 1 ++++ ++ 1 ++++ + i 1 +++ +++... 2 +++ +++ 4 +++ ++ 1 +++ 1.". 1 ++ ++++ 2 ++ ++ 1 ++ + 1 ++ 1: g...- 'l' + 1 1; .1: 1 i. - 1 u + 216 - ' - TJBDI IV EFFECT OF DIFFERENT HEJTING PERIODS OF SERUM ON SENSITIVENESS OF KEEN REACTIONS. No. of Serum: Reaction after heating Reaction after heating for 30 minutes at 5500. for 5 minutes at 65°C. 11 ++++ ++++ 1 ++++ .+++ 1 ++++ :_ 2 +++ +++ 3 +++ +4» 4 ++ ++ 1 ++ .t .1 + ++ 2 + + 3 + ;: 1 + - 1 a. a 4 - - 12-2-27V .hUXb ..\.\\< 8m. OWN. 09w 9Q 8\ 8 0% QR 8 D“. 0% 0m. QN Q\ Q f + I / 9,; Sun (ll/0)] \ < ... .h\00\k66\ metxx \ECKMCCCD kOK 0.9%.».0 mobxbunx EBKUQ K0 $03.0on \ 6Q hUsSstV ed 98 6me Newsboeewmsmsvsesfiwsmwememh S m a. .hD0.\\MQ\ NRA? xtbkbkkxxu {0.x 0.0% *6 bu$bus\ EDKOO \O kuutuxonux m. ..mxk 0 . an Lit/0% O {'2’} 0m. 0‘ km. ..00.\.\66\ htxxbhhx Next. met?! o\\o.\ .Dahtfix EOOK *6 L30..\ 6C0 MECUQQ$W KOXXO Dwain-fix EOKUW. ..U llllll .DQNK‘Qx mnx\\0~s\ tax m£.§o\\o\ UK was taken. .35.“ c swim but}. Eaton .muulll. .303...an mtxxbhfi .kQXKO %\0.\0\\0NR\R\.\ \thhfit Exukbh. .VIIII IDQ0%U.V h0$3tx§ SWQQ\\\W\.«.\§‘M.\N\ \\ Q\ um. hm. .thkahK “RAD. *tbk6\\.\\nv \0.\ .UoOW «n.0, ~00$66Q EBKQH \0 Motuxonx m. st 9% sat/an m/a/y'” ACMIO‘TILEDC‘EZ'ENT I wish to express my appreciation of the warm encouragement given me by Dr. R. L. Kahn, at whose suggestion this study was undertaken. I am also indebted to Dr. Grace Lubin and "iss Elizabeth Kc Dermott for helpful surgestions and cooperation while carrying out these experiments. 21. BIBLIOGRAPHY (1) Kahn, R. L.: Serum Diagnosis of syphilis by firecipitation. 1924. (2) The Kahn Test — A Practical Guide. 1928. (in manuscript} (3) Kendrick, P. L.: Comparative Heat Resis- tance of Serum in the Kahn and W253- ermann Tests. Reprint series, Kich- igan Department of Health, December, 1925. ROOM USE ONLY i.s.~...t.-.... D r. ..aob. ova ..‘vo: .a | 2' 3 i8 21 .3 Q .5 fl!IIHIHIHUIIIHIHHHIIIHMUIHIHIHIHII"Hill!NIH 293 031 1451424