USA > Ohio > Hancock County > Findlay > Twentieth Century History of Findlay and Hancock County, Ohio, and Representative Citizens > Part 129
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* Where the expression "0° C." is used in this paper in de- scribing my own experiments it is to be understood that the tubes were placed in a vessel of ice water ranging in temperature from 0° C. to about 2° C.
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groups. Similarly, if a member of Group III contains an iso- hemolysin it can act only against the corpuscles of Groups I and II. The iso-hemolysin of Group IV is effective only against corpuscles of Groups I, II and III. The corpuscles of Group IV are not subject to the action of iso-hemolysin and from what has just been said, the corpuscles of members of the other three groups are never hemolysed by the serum of a member of the corresponding group.
The group to which an individual belongs can be readily determined by testing the agglutinating action of his serum against the corpuscles of the four different groups, or by test- ing the action of serum from the four different groups against the corpuscles of the individual to be classified. This will be made clearer by taking an example.
To determine the group to which an individual, X, belongs :
First Method.
I.
II.
III.
IV.
1
Serum X 0.25 cc. + Corp. Gr. 1 0.25 cc.
0
2
Serum X 0.25 cc. + Corp. Gr. II 0.25 cc.
8
Serum X 0.25 cc. + Corp. Gr. III 0.25 cc.
4
Serum X 0.25 cc. + Corp. Gr. IV 0.25 cc.
0
ofot
0 0 oo++
o+++
Column I shows the resulting agglutination if X is a mem- ber of Group I and similarly, Columns II, III, and IV show the resulting agglutination if X is a member of Group II, III or IV, respectively.
Second Method.
I.
II.
III.
IV.
1
Corp. X 0.25 cc. + Serum Gr. 1 0.25 cc.
2
Corp. X 0.25 cc. + Serum Gr. II 0.25 cc.
8
Corp. X 0.25 cc. + Serum Gr. III 0.25 cc.
+++0
++00
0
0
4
Corp. X 0.25 cc. + Serum Gr. IV 0.25 cc.
+
0
Columns I, II, III and IV show the resulting agglutination if X is a member of Group I, II, III or IV, respectively.
An analysis of the above tables shows that in order to classify an individual it is not necessary to test his blood against blood from all four groups. It is sufficient if his serum is tested against the corpuscles of Groups II and III, or if his corpuscles are tested against the serum of Groups II and III.
Thus if the serum of the individual to be classified aggluti- nates corpuscles of neither Group II nor III, it must belong to Group I. If it agglutinates corpuscles of Group III, and not those of Group II, it must belong to Group II. If it agglutinates corpuscles of Group II and not those of Group III, it must belong to Group III, while if it agglutinates cor- puscles of both Groups II and III, it must belong to Group IV.
It is just as easy to determine the group if one tests the corpuscles of the individual in question against the serum of Groups II and III.
By the methods just described it was determined to which groups the cases here reported belonged and the presence or absence of normal iso-hemolysin repeatedly observed. It will be sufficient to give in detail the results of a single such de- termination.
Serum and corpuscles were obtained, as previously de- scribed, from four normal individuals representing Groups
I, II, III and IV, also from Case I, G. C., whose group was to be determined. Tests were carried out acording to the fol- lowing protocol :
PROTOCOL I .- Case I, G. C.
Tube No.
Serum 0.25 cc.
Corps. (5% susp.) 0.25 cc.
Aggl.
Hem.
Aggl. Hea
Gr. 1.
Gr.
I.
Gr. II.
Gr. III.
0
0
0
:
Gr. IV.
0
0
..
G. C.
0
0
6
Gr. II.
Gr. I.
+
0
7
8
9
Gr. IV.
0
10
G. C.
Gr. III.
Gr. I.
87ºC. 2 hrs.
0 0
11 12 13
14 15
16
Gr. IV.
Gr. I.
17
Gr. II.
18
:
Gr. III.
Gr. IV.
..
G. C.
21 29
G. C. ..
Gr. II.
23
Gr. III.
24
Gr. IV.
25
G. C.
This experiment shows that the corpuscles of the patier :. G. C., are neither agglutinated nor hemolysed by the seruz of Groups I, II, III or IV, therefore the patient is a men- ber of Group IV. This is confirmed by the fact that he: serum agglutinates the corpuseles of Groups I, II and III. I: is further evident that the patient's serum contains normal is- hemolysin, since it is able to hemolyse the corpuscles : Groups I, II and III at 37° C. without the mixtures havin: been previously exposed to the cold.
In addition to the normal iso-agglutinin and iso-hemolrs: contained in the patient's serum, which is effective agains corpuscles of Groups I, II and III without the action of cel: (Tubes 21, 22 and 23) there is another hemolysin preser: peculiar to paroxysmal hemoglobinuria, which only becomes evident after the mixture of serum and corpuscles is subject: to a low temperature followed by a high temperature (Tube 24 and 25).
Attention is here directed to another point of difference ie- tween the action of the hemolysin peculiar to paroxysmi hemoglobinuria and normal iso-hemolysin. I have pointed out elsewhere that normal iso-hemolysis is always accompanin. or preceded by iso-agglutination. The above experimir: shows that the hemolysis caused by the hemolysin peculiar F paroxysmal hemoglobinuria may occur entirely independent: of agglutination (Tubes 24 and 25).
The hemolysin which characterizes the disease with which we are dealing is often referred to as an auto-hemolysin, sin: the patient's serum is able to bring about the solution of bi- own corpuscles, but as previous investigators have shown a: I have repeatedly confirmed, the patient's serum is able : hemolyse not only his own corpuscles but those of other bet .. globinuric patients and normal individuals, therefore it has : iso-hemolytic, as well as an auto-hemolytic action.
This iso-hemolytic action of the hemolysin peculiar paroxysmal hemoglobinuria, which becomes evident on! :
Digitized by
0
0
0
0
5
Gr. II.
Gr. III.
0
87ºC. 2 hrs.
0 0 0 1 0 00+++++++000++00+0+00
0 0 oo+++000++0000000
0°C. 12 hr.
19 20
Gr. II.
Gr. III.
..
Gr. IV.
G. C.
87º℃ 2 hrs.
0
0
87ºC. 2 hrs.
0
0
+
0
.
Gr. I.
0
erum of hemoglobinuric patients, as well as normal indi- iduals and which does not require previous chilling for its lemonstration.
Bearing in mind the fact that it does not fully define the emolytic power of the serum, I shall use the term auto- emolysin to distinguish that property which characterizes the erum of hemoglobinuric patients from the normal iso-hemo- vtic property which may be possessed by the serum of both ormal and hemoglobinuric individuals.
Similar tests carried out on the other two cases showed that Case II, F. L., is also a member of Group IV, while Case III, . S., is a member of Group III and that the serum of both ases contained, in addition to their auto-hemolysin, normal jo-hemolysin.
It now became of interest to determine if the auto-hemo- "sin of hemoglobinuric patients could bring about the solu- ion of the corpuscles of all individuals; for example, cor- uscles for which the serum in question contained a normal 30-hemolysin.
Before this question can be discussed, certain facts must be iven which resulted from the experiments undertaken to olve the next point in the investigation, namely :
3. Determination of the relation of temperature to the nion of (a) amboceptor and corpuscle, (b) complement and mboceptor-corpuscle.
Divergent views are held on this subject. As previously mentioned, Donath and Landsteiner hold that the union of uto-amboceptor to the corpuscle takes place only at a low emperature, that the union of complement occurs subse- uently, and only at a higher temperature. Meyer and Em- erich concur in this view, while Hoover and Stone maintain at a low temperature is necessary, not only for the union of ne amboceptor and corpuscle but also for the union of the omplement, a higher temperature being necessary, however, or the lytic action of the complement.
It seemed easy to approach this problem by means of ordi- ary reactivation experiments ; accordingly, some of the hemo- obinuric serum was inactivated by heating to 56° C. for 20 inutes and the following test performed : 0.5 cc. Pt. serum a. + 0.25 cc. Pt. corps. (5% susp.) 0° C. ¿ hr. 37° C. 5 in. + 0.25 cc. complement (non-lytic serum of normal in- vidual) 37° C. 2 hrs. Result: no hemolysis. The mixture is then held at 0° C. for } hour, followed by 37° C. 2 hours. esult: hemolysis.
The above experiment would seem to indicate that a low mperature was necessary for the union of complement as ill as amboceptor; on the other hand, it might be argued at the amboceptor united with the corpuscle during the first posure to a low temperature, but that dissociation took ice when the temperature was elevated before the comple- ent was added, hence the failure of hemolysis at the end of : first exposure of 2 hours at 37º C. The positive result emolysis) occurring after the exposure to 0° C., followed a second exposure of 2 hours at 37° C., might be explained
-
plement now being present united as soon as the temperature reached a suitable degree and before dissociation could take place between the amboceptor and corpuscle.
That this explanation is correct seems improbable. Disso- ciation is said to be hastened by temperatures approaching that of the body and by mechanical measures, such as shaking. In the above experiment mechanical factors were carefully avoided and the exposure to 37° C. was very brief, only five minutes being allowed for the temperature of the mixture to rise from 0℃. to 37° C.
In the next experiment the possibility of dissociation under the influence of temperature was avoided, but mechanical factors (washing the corpuscles) were introduced.
0.5 cc. Pt. serum ina. + 0.25 cc. Pt. corps (5% susp.) 0° C. ¿ hour. The corpuscles were then removed by centrifugaliza- tion at a low temperature (0° C. to 5° C.) and washed twice with salt solution at 0° C., resuspended in 0.5 cc. salt solution and 0.25 cc. of complement at 0° C. (non-lytic serum of a nor- mal individual) added, while the suspension of corpuscles was still at 0° C. This mixture was then held at 37° C. for 2 hours, and as no hemolysis resulted the temperature was low- ered to 0° C. for $ hour and then again held at 37º C. for 2 hours. Result : no hemolysis.
The failure of hemolysis in this experiment might possibly be ascribed to dissociation of the amboceptor and corpuscle under the influence of the mechanical disturbance of washing.
The following experiment indicates that the explantions thus far offered are not correct. 1 cc. Pt. serum + 0.1 cc. Pt. corps., containing as little salt solution as possible, were sub- jected to a temperature of 0° C. for } hour, then centrifu- galized at a low temperature (0° -5° C.), the supernatent fluid removed and saved for further tests, and the corpuscles washed three times with salt solution at 0° C. to remove all the serum from them. They were then suspended in 0.75 cc. salt solution and kept at 37° C. for 2 hours. Result: no hemolysis. Complement was now added (0.25 cc. of normal non-lytic serum) and the temperature maintained at 37° C. Result : prompt hemolysis.
The following seems to be the only explanation which will harmonize the results of all the foregoing experiments : Union between the auto-amboceptor and the corpuscle takes place only at a low temperature in the presence of comple- ment; the complement does not enter into the combination, or at least not permanently, at this temperature. Complement does unite with the corpuscle-amboceptor combination at 37º C. with resulting solution of the cell.
The last experiment further shows that the union between amboceptor and corpuscle is a fairly stable one, since repeated washing at a low temperature and subsequent standing for 2 hours at 37 C. does not cause dissociation.
If the above explanation is correct, then the serum in the last experiment, which was removed after standing in contact with the corpuscles at a low temperature, should have been deprived of its auto-amboceptor, but not of its complement
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(provided the proper quantitative relations were observed). The following tests were made to determine these points :
0.25 cc. serum from previous test + Pt. corps. 0.25 cc. (5% susp.) 0° C. § hr. 37° C. 2 hrs. = no hemolysis.
0.25 cc. serum from previous test + 0.25 cc. complement (normal non-lytic serum) + Pt. corps. 0.25 cc. (5% susp.) 0° C. ¿ hr. 37° C. 2 hrs. = 0 hemolysis.
0.25 cc. serum from above + 0.25 cc. Pt. serum ina. + 0.25 cc. Pt. corps. 0℃. ¿ hr. 37° C. 2 hours = + hemo- lysis.
These three tests show that all of the auto-amboceptor had been removed from the patient's serum by contact with pa- tient's corpuscles at a low temperature, but that complement remained, thus corroborating the findings when the corpuscles were examined and found to have anchored amboceptor, but not complement, at the low temperature.
If this interpretation of the results is correct, it furnishes an example of a hitherto undescribed function of complement ; namely, the ability to effect a combination between two other bodies (amboceptor and corpuscle) without itself entering permanently into the combination.
We are now in a position to consider the question pre- viously mentioned as to the ability of the auto-hemolysin con- tained in the serum of a hemoglobinuric patient to bring about the solution of corpuscles for which the same serum contains an iso-hemolysin.
4. Separation of the auto- and iso-hemolysin contained in the same serum in order to determine if the auto-hemolysin is capable of dissolving those individuals' corpuscles for which the serum in question contains an iso-hemolysin.
Reference to Protocol I, of experiments carried out on the serum of Case I, G. C., shows that the serum of this patient contained an iso-hemolysin for the corpuscles of a normal in- dividual belonging to Group I, for one belonging to Group II and for one belonging to Group III (Tubes 21, 22 and 23) in addition to the auto-hemolysin for an individual belonging to Group IV and for her own corpuscles.
In order to determine if the auto-hemolysin is capable of dissolving the corpuscles of the individuals belonging to Groups I, II and III it is first necessary to remove the iso- hemolysin. To this end, serum and corpuscles were obtained as follows:
No. 3 normal individual belonging to Group III.
No. 4 normal individual belonging to Group IV.
No. 5, Case I, G. C., hemoglobinuria patient, Group IV. A portion of serum 5 (Case I, G. C.) was inactivated by heating at 56° C. for 20 minutes and the experiments were performed as shown in Protocol II.
Tubes 1 to 9 inclusive, in Protocol II are controls and show as follows:
Tube 1 shows that serum 4, which was used as complement contained no iso-hemolysin for corpuscles 3.
Tube 2 shows that serum 5 contained an iso-hemolysin for corpuscles 3 (reading at end of first two hours at 37º C.).
Tubes 3 and 4 show that serum 5 contains no iso-hemolysin for corpuscles 4 and 5 (reading at end of first two hours at
37° C.), but that it does contain an auto-hemolysin fe: these corpuscles (reading after } hour at 0° C. + 2 hours e: 37° C.).
Tube 5 shows that the iso-hemolysin in serum 5 was it- activated for corpuscles 3 by heating to 56° C. for 20 minutes.
Tube 6 shows that the auto-hemolysin (if effective againe. corpuscles 3) had been inactivated by heating to 56° C. f .: 20 minutes.
Tube 7 shows that the auto-hemolysin contained in serum 5 for corpuscles 5 had been inactivated by heating to 56° C. for 20 minutes.
Tube 8 shows that serum 4 was capable of activating the iso-amboceptor contained in serum 5 for corpuscles 3.
Tube 9 shows that serum 4 was capable of activating the auto-amboceptor contained in serum 5 for corpuscles 5.
PROTOCOL II.
To absorb the iso-hemolysin from hemoglobinuric serum leaving the 1.> hemolysin and determine if the latter is capable of dissolving an individualit puscles (No. 3) for which the hemoglobinuric serum contains an iso-hr-molysi
1. Ser. 4, X cc. + Corps. 8, X cc. 87ºC. 2 hrs .= 0hz c
2. Ser. 5, X cc. + Corps. 3, X cc. 37ºC.2 hrs. - + bem. 0ºC. 12 hr. SrºC. 9 hrs. = +b: 8. Ser. 5, & cc. + Corps. 4. X cc. 37ºC. 2 hrs. = 0 hem. 0℃. 14 hr. 87ºC.9 hrs. = +b: 4. Ser. 5, 14 cc. + Corps. 5, X cc. 87ºC. 2 hrs. == 0 hem. 0℃. 14 hr. 37°℃.9 hrs. =- k:
5. Ser. 5 ina., 14 cc. + Corps. 8, 14 cc. 37°C. 2 hrs = 0hz
6. Ser. 5 ina., 14 cc. + Corps. 8, 14 cc. 0℃. 19 hr. 87℃. 2 hrs = 1ke:
7. Ser. 5 ina., 14 cc. + Corps. 5, 14 cc. 0°C. 19 hr.37ºC.2 br& = Ik:
8. Ser. 5 ina., 1 cc. + Corps. 8, 1/ cc. + Comp. (Ser. 4) 14 cc. 87ºC. 2 hrs. = +k: 9. Ser. 5 ina., 14 cc. + Corps. 5, 14 cc. + Comp. (Ser. 4) 4 cc. 0℃. 12 hr. 87ºC. 2 hrs. = + Mes
10. Ser. 5 ina., 11/2 cc. + Corps. 8. ris cc. 87ºC. 2 hrs. Centrifugalize, save serum, wash corps.
11. Ser. tube 10, 14 cc. + Corps. 8, 1/ cc. + Comp. (Ser, 4) 14 cc. 12. Ser. tube 10, 14 cc. + Corps. 5. 14 cc. + Comp. (Ser. 4) 14 cc. 0℃. 15 hr. 87ºC. 2 hrs. = + ka
18. Ser. tube 10, 14 oc. + Corps. 8, 14 cc. + Comp. (Ser. 4) 14 cc. 0℃. 15 hr. 87ºC. 9 hrs. = + bez 14. Washed corps. tube 10+ NaCl % cc. + Comp. (Ser. 4) 14 cc. 87ºC.2 hrs .= +kz 1
15. Ser. 5 ina., 11/2 cc. + Corps. 8, Ils cc. 0ºC. 2 hrs. Centrifugalize, save serum, wash corps. 87ºC. 9 hrs. = 0kr
16. Ser. tube 15, 14 co. + Corps. 8, 1/ cc. + Comp. (Ser. 4) 14 cc. 17. Ser. tube 15, 14 cc. + Corps. 5, 14 cc. + Comp. (Ser. 4) 1 cc. 0℃. 15 hr. 87ºC. 9 hrs. = +ts
18. Ser. tube 15, 14 cc. + Corps. 8, 14 cc. + Comp. (Ser. 4) 14 cc. 0℃. 1/2 hr. 87ºC. 2 hrs = the 19. Washed corps. tube 15+ NaCl &co. + Comp. (Ser. 4) 14 cc. 87º.C2hra .= +bez
Wherever inactive serum was used in the above experiment it is indicated !! the abbreviation " ina." Elsewhere active serum was used. The corpuscles were used in 5% suspension except in tubes 10 and 15, where al *** supernatant salt solution after centrifugalization was removed from the corpos.# before they were used.
Tube 10 was to absorb the iso-amboceptor from serum 5 in- active at 37° C. by means of corpuscles 3.
Tube 11 shows that all of the iso-amboceptor for corpuseles 3 had been absorbed from serum 5 inactive in tube 10. Tube 12 shows that the auto-amboceptor had not been re moved from serum 5 inactive in tube 10.
Tube 13 shows that the auto-amboceptor contained in serum 5 inactive was capable of dissolving an individual's corpuseles (corpuscles 3) for which the hemoglobinuric serum contained an iso-hemolysin, Q. E. D.
Tube 14 shows that the corpuscles in tube 10 had anchored an iso-amboceptor from serum 5 inactive, which was not sepa- rated from the cells by washing twice at room temperature with 10 cc. salt solution at each washing.
Tube 15 was to absorb the iso-amboceptor from serum 5 inactive at 0° C. by means of corpuscles 3. (I have previously
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1
-
87ºC.2 hrs = 0ks
ig tubes confirmed this statement).
Tubes 16, 17, 18, 19 gave results identical with those given y tubes 11, 12, 13, 14, again proving that the auto-hemolysin f hemoglobinuric serum is capable of dissolving an indi- idual's corpuscles for which the serum contains an iso- emolysin.
Having absorbed the iso-hemolysin from hemoglobinuric erum and left the autohemolysin, it seemed desirable, in rder to complete this part of the study, to absorb the auto- emolysin from hemoglobinuric serum and leave the iso- emolysin. This was readily accomplished in the following manner :
Blood was obtained from-
No. 1. A normal individual belonging to Group I.
PROTOCOL III.
absorb the auto-amboceptor from hemoglobinuric serum leaving the iso-ambo- ır.
r. 5, 1/ cc. + Corps. 1, 1/ co. 87ºC. 2 hrs. = + hem. 0℃. 12 hr. 87ºC. 2 hrs .= + hem. r. 5, 14 cc. + Corps. 4, 14 cc. 87ºC. 2 hrs. = 0 hem. 0°℃. 14 hr. 37ºC. 2 hrs .= + hem. r. 5, 4 cc. + Corps. 5, 14 cc. 87℃. 2 hrs. = 0 hem. 0℃. 14 hr. 87℃. 2 hrs .= + hem. r. 4. 14 cc. + Corps. 5. 14 co. 87ºC.2 hrs. = 0 hem. 0℃. 1/2 hr. 87ºC. 2 hrs .= 0 hem. r. 5 ina., 1 co. + Corps. 1, 14 cc.
87ºC. 2 hrs. - 0 hem. 0ºC. 15 hr. 87ºC. 2 hrs .= 0 hem.
r. 5 ina., 14 cc. + Corps. 5, 1/ co. 0°C. 1% hr. 37°C. 2 hrs .== 0 hem. r. 5 ina., 14 co. + Corps. 1, 14 cc. + Comp. (ser. 4) 1/10 co. 87ºC. 2 hrs .= + hem. r. 5 ina., 1/ co. + Corps. 5, 14 cc. + Comp. (ser. 4) 1/10 cc. r. 5, 1 cc. + Corps. 5, 1/20 cc. 0ºC. 14 br. Centri- 0°℃. 14 hr. 87℃. 2 hrs .= + hem. ugalize at 0℃. same serum and wash corpuscles hree times at 0ºC.
ie-half the washed corps. from tube 9+ NaCl 0.45 cc. 87°C. 2 hrs .= 0 hem. le-half the washed corps. from tube 9+ NaCl 0.45 co.
- Comp. (ser. 4) 1/10 cc. 87ºC. 2 hrs .= + hem.
r. tube 9, 14 cc. + Corps. 5, 1/ cc. 0°℃. 1/g hr. 87℃. 2 hrs. = 0 hem.
ir. tube 9, 1/ cc. + Corps. 5, 14 co. + Comp. (ser. 4) 1/10 cc.
0°C. 1/2 hr. 87ºC. 2 hrs .= 0 hem. r. tube 9, 14 cc. + Corps. 5, 1/ cc.+Ser. 5 ina., 14 co. 0℃. 1/3 hr. 87℃. 2 hrs .= + hem. r. tube 9, 1 cc. + Corps. 1, 14 cc. 87ºC. 2 brs .- + hem.
›rever inactive serum was used in the above experiments it is indicated by the viation "ina." Elsewhere active serum was used.
corpuscles were used in 5% suspension except in tube 9, where all the super- t salt solution after centrifugalization was removed from the corpuscles ethey were used.
No. 4. A normal individual belonging to Group IV.
No. 5. Case I, G. C., hemoglobinuric patient, Group IV.
Tubes 1 to 8 inclusive are controls and show as follows :
Tube 1 shows that serum 5 contains an iso-hemolysin for rpuscles 1 (reading at the end of first 2 hours at 37º C.). Tubes 2 and 3 show that serum 5 contains an auto-hemo- sin for corpuscles 4 and 5 (reading after } hour at 0º C. d 2 hours at 37° C.).
Tube 4 shows that serum 4 contains neither iso- nor auto- molysin for corpuscles 5, therefore it can be used for com- ment, provided it is able to reactivate serum 5 inactive.
Tube 5 shows that both the iso- and auto-hemolysin con- ned in serum 5 for corpuscles 1 were inactivated by heating 56° C. for 20 minutes.
Tube 6 shows that the auto-hemolysin contained in serum or corpuscles 4 was inactivated by heating to 56° C. for 20 nutes.
Tubes 7 and 8 show that serum 4 (complement) is capable
serum o Inactive.
Tube 9 was to absorb the auto-amboceptor from serum 5 by means of corpuscles 5 at 0° C. Hemolysis was prevented by keeping the mixture at a low temperature until after the serum had been removed from the corpuscles and the latter thoroughly washed with salt solution.
Tube 10 shows that the corpuscles did not anchor, at least not permanently, both auto-amboceptor and complement at 0° C.
Tube 11 shows that the corpuscles did anchor the auto- amboceptor at 0° C., that dissociation did not occur as a re- sult of thorough washing of the corpuscles and that comple- ment united at 37° C. with consequent solution of the cor- puscles. This and tubes 12, 13, 14 are a repetition and con- firmation of experiments done many times under subhead 3; namely: Determination of the relation of temperature to the union of (a) amboceptor and corpuscle (b) complement and amboceptor-corpuscle.
Tube 12 shows that serum 5 after absorbtion with cor- puscles 5 in tube 9 at 0° C. was unable to dissolve cor- puscles 5.
Tube 13 shows that the failure of hemolysin in tube 12 was not due to lack of complement.
Tube 14 shows that the failure of hemolysis in tube 12 was due to lack of auto-amboceptor only. -
Tube 15 shows that the auto-amboceptor was absorbed from serum 5 in tube 9, leaving iso-amboceptor and complement,. Q. E. D.
Hoover and Stone were able to absorb the auto-amboceptor from hemoglobinuric serum in the absence of complement, as indicated by the following quotation, to which reference has previously been made: "Thus we have shown that if in two stages, 10 drops of a 10% suspension of red cells be added to (1 cc.) the serum and exposed to cold for one hour each time, the amboceptor can be exhausted from the inactivated hemo- globinuric serum." It seems probable that this result is to be explained on the basis of a mechanical removal due to the relatively large amount of corpuscles used. Even so they found it necessary to absorb in two stages.
In my efforts to separate the auto- and iso-amboceptor in hemoglobinuric serum I found it necessary to use the mini- mum quantity of corpuscles which would completely remove the one in order that the other be left. In case an excess of corpuscles was employed both auto- and iso-amboceptor would be removed.
5. Resistance of the red blood corpuscles.
Certain investigators have observed that the red blood cells of hemoglobinuric patients studied by them were less resistant to hypotonic salt solution than normal, while other investi- gators have found no difference between normal corpuscles and the corpuscles of their patients in relation to hypotonic salt solution. Dr. Helen Watson kindly tested this point for me in my three cases. The corpuscles of Cases I and II were tested on two occasions, an interval of about two months elapsing between tests. The corpuscles of Case III were only
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JOHNS HOPKINS HOSPITAL BULLETIN.
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tested once. The corpuscles of all three patients showed definitely increased resistance to hypotonic salt solution. The only exception to this rule was Case II, whose corpuscles, at the time of the second test, showed the same resistance as those of several normal individuals, tested at the same time, but on the occasion of the first test they showed a markedly greater resistance than normal corpuscles.
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