Vox Sang. 37: 21-29 (1979)
Posttransfusional lrnmunologic Thrombocytopenia A Case Report jean-Pierre Soulier, Claude Patereau, Nicole Gobert, Pierre Achach and Jean- Yves Muller Centre national de Transfusion sanguine, Paris
Abstract. A case of posttransfusional immunologic thrombocytopenia is reported in a 75-year-old PP-negative woman. This was the second episode of postoperative and posttransfusion thrombocytopenia in the same patient who had had only one pregnancy. Both thrombocytopenic episodes were subclinical and discovered by systematical hematologic study. A potent anti-PP antibody (titer 1/128) was demonstrated by the platelet-indirect radioactive Coombs test which appeared more sensitive than other platelet immunological assays used. The very long duration of the thrombocytopenia is discussed as well as the therapeutic possibilities in such cases. Posttransfusional thrombocytopenic purpura (PTTP) was first described simultaneously in 1959 by Zucker et al. [21] and by van Loghem et al. [18]. Shulman et al. [ 131 made a detailed clinical and serological study of this very rare syndrome, observed in PI*'-negative women about a week after transfusion. No more than 20 cases have been published in nearly 20 years (only one in France IS]), thus any additional serologically well-studied case of PTTP is worth reporting.
Case Report Mrs. M A U . . ., 75 years old, was admitted to the Clinique Labrouste in November 1977, for a total hip replacement. At the time of her admission, on November 26, the platelet count was 190,OOO/mm3.
Mrs. MAU.. . was born from a first cousin's marriage, which may play a role in the fact that her platelets were found to be PW-negative (only 2.5% of the normal French population). Her blood group is 0 Rh-positive. When 20 years old, she had a son, who died in his first year of life, and about whom there is no further information. In March 1974, she underwent orthopedic surgery for a fracture of the neck of the femur. One unit of blood was transfused preoperatively, and one postoperatively on day 5 , which was accompanied by chills and fever. A clear thrombocytopenia was present 2 weeks after the transfusion, and was only revealed by the platelet count: April 11, 1974: 30,000 platelets; April 24, 1974: 50,000 platelets; April 27, 1974: 39,000 platelets. No hemorrhagic complications were observed, and this thrombocytopenia appears to have been subclinical, and was therefore forgotten, explaining why 3 years later no special precautions were taken prior to transfusing the patient during and after a second surgical procedure. In 1977, the new thrombocytopenia which developed was discovered by a systematic hemato-
22
Soulier/Patereau/Gobert/Achach/Muller
Table I. Successive hematological data Date
Hematocrit
Platelet counts
Serology
PIRC
1977 November 26 November 28
December 5 December 10 December 11 December 12 December 14 December 15
38
190,000
36 26 37 38 37
2 units of blood (chills) I unit of blood (chills) 35,000 20,000
TEG (r+k=14 min; am = 44 mm) Megakaryocytes rare
10,000
10,000 10,Ooo < 10,000
December 20 December 29
38 38
< 10,000 < 10,Ooo
1978 January 5 January 11 January 16 January 20 February 7
38 39 37 37 42
< 10,Ooo 10,000 20,000
Megakaryocytes numerous t+t
11128
TEG (r+k=12 min; am =40 mm)
1/128 11128 return home
1/128
45,000
45,000 105,000 138,000
logical study. On November 28, the day of surgery, 5 units of blood were transfused. The hematocrit
was 41%. No hemorrhagic syndrome was observed, and more especially, no local hematoma at the site of surgery. However, a decrease in the hematocrit from 36% on December 5 to 26% on December 10 was noticed, and two additional units of blood were transfused. This transfusion was not well tolerated chills, cephalalgia and feyer being observed. The day after, a new blood transfusion
-
direct Coombs (cf. table 1V)
5 units of blood hip replacement
December 17 December 18 December 19
February 23 March 23 April 17
Remarks
1/64 (dubious 1/128) 1/64
dubious
1/64 1/64
produced similar reactions. It was at this time (December 12, 1977) that the thrombocytopenia was discovered: 35,000 platelets/mmy. No petechiae, no ecchymosis or other hemorrhages in spite of a systematic anticoagulant treatment. The oral anticoagulant (Sintrom) was immediately stopped and platelet counts were made every day (table 1). The platelets decreased from 35.000 to 10,000 and they remained at this very low level for 1month. Thromboelastograms were made twice at an interval of
Posttransfusional Immunologic Thrombocytopenia
2 weeks (December 15 and 29). They showed the usual defects found in thromhocytopenia (r + k = 14 min; maximal amplitude 40 mm). In spite of the risks involved in this elderly patient, the surgeon chose not to use corticosteroids for fear of infection and failure of the total hip arthroplasty. It was considered to transfuse P W negative blood but this was kept in reserve (see Discussion), since the PW-negative platelets of the recipient were affected by the exogenous P1A conflict. This conservative attitude proved to be justified since no hemorrhagic complications were oherved. Two bone marrow specimens were examined. On December 16, the marrow was 'somewhat diluted' and the megakaryocytes considered as 'rare'; on December 22 it was of normal density and the rnegakaryocytes were increased. On January 20, 1978, 6 weeks after surgery, the hematocrit was well stabilized (between 37 and 39%), with a platelet count of around 20,000. This thrombocytopenia was especially slow to correct spontaneously, and on April 17, the platelet count was 138,000.
Serological Study Platelet Indirect Radioactive Coombs (PIRC) This method has already been described [ 141. Briefly: 50 pl of platelet suspension (108) are mixed for 1 h at 37 "C with 50 pl of serum. The platelets are washed 3 times with saline. 125I-labelled goat IgG, active against human IgG, is added and kept at 37 "C for 30 min. After centrifugation, the supernatant ( S * ) is kept and the platelet sediment (PS") is washed (W*). The test is done in triplicate. The fixation percentage of goat IgG on platelets is: PS* PS* + s*+ w*
Controls are done using P P t and PP-negative platelets, as well as AB normal serum.
Fixation higher than 10% is considered as a positive test. Fixation lower than 6% denotes a negative test. HLA antibodies (in the presence of platelets having the corresponding antigen) may give a fixation of around 10%. PIA*antibodies may give a fixation as high as 2 0 4 0 % . Platelet Direct Radioactive Coombs The same test may be used as a direct test using the patient's platelets if their number is high enough (over 50,00O/mm3). Other Tests Platelet aggregation of normal PIA'+ platelet rich plasma. This was done according to Deykin and Hellerstein [4]using an aggregometer. Platelet serotonin release was done according to Dechavanne et al. [3]. Complement fixation on platelets was done by Mrs. Jullien using standard methods [13]. Thromboagglutination was done according to Van der Weerdt [17]. Platelet factor 3 assay was done according to the Hirschman and Gralnick technique [S], but dextran sulfate was used instead of kaolin to activate the contact factors. Lymphocytotoxicity antibodies and HLA typing were done by L. Hullt using standard techniques [16].
Results of the Serological Study The platelet factor 3 availability test and the platelet serotonin release were both negative. The thromboagglutination and the complement fixation test were both weakly positive. The undiluted patient serum produced an aggregation curve using normal PIA1+PRP, in contrast with normal AB serum.
Soulier/Patereau/Gobert/Achach/Muller
L4
Table 11. PIA1 typing of Mrs. M A U . . . platelets. Percent of radioactivity fixed by the patient's platelets in the presence of anti-PIA1serum and of AB normal serum, in comparison with normal PWpositive platelets and PIA1-negative Serum
Platelets PI"'+
~
~~
~
Anti-PIA1serum (LAB ...) AB serum
~~~
PlAl-
MAU ...
~
16.2%
4.5%
5.1%
3.5%
3.5%
4.1%
Lymphocytotoxicity was positive using a lymphocyte panel of known phenotype: antiHLA-9 and anti-B12 antibodies were found in the patient's serum.
antibody was titrated by serial dilutions (table IV). This strong anti-PIA1 antibody was regularly found at each of the subsequent PIRC assays, the titer remaining at least 1 to 64 (dubious results with the 1 to 128 dilution). This high titer persisted for 5 months after the discovery of the thrombocytopenia (table I). Direct Platelet Radioactive Coombs Due to the very low platelet count, the results of this test were not considered significant before February 23 (table V).
Discussion
This case represents a further example of the rare syndrome of posttransfusion imPlatelet Indirect Radioactive Coombs Clear-cut results were obtained only with munologic thrombocytopenia and it is the this test, for PIA typing as well as for the second recorded in France, the first being demonstration of strong anti-PIA1 antibodies reported by Dubarry et al. [ 5 ] . This patient in the serum of the patient. Results of the was a multitransfused female, with one pregP1*1 typing are summarized in table 11. nancy in her past history; her platelets were Mrs. MAU . . . platelets are PIAi-negative PIA1-negative and her serum contained an (PlA2PP). No anti-PlA* antiserum is avail- anti-PP antibody of high titer. In spite of her age (75) and the severe able to test such platelets. thrombocytopenia (less than 10,000 for several weeks), the patient had no apparent Assay for Anti-PIA1Antibodies by the hemorrhagic symptoms. However, a sudden PIRC Test A typical assay for anti-PP antibodies is fall in the hematocrit (36-26% between the shown in table 111. It is seen that PIA1-posi- 7th and the 12th postoperative day) was obtive platelets gave a uniformly high fixation served and 3 units of blood were transfused. of radioactivity (mean 30%) where PIA1-neg- A low platelet count was discovered, and ative platelets fixed less than 10%. Some thus a second episode of subclinical postfixation on PP-negative platelets was due to operative thrornbocytopenia was diagnosed. the presence of the HLA antigen on these It is not possible to decide today if the reacplatelets which reacted with the anti-HLA tions observed after a 3-year interval were antibody of Mrs. MAU . . . Besides anti-HLA due only to anti PI*' antibodies, since we antibodies, a strong specific anti-PIA1 anti- know that the patient's serum also contained body is thus present in the patient's serum anti-HLA antibodies which were able to reexplaining the posttransfusion reactions. This act with the transfused leukocytes.
Posttransfusional Immunologic Thrombocytopenia
25
Table 111. Detection of anti-PIA1antibody in the patient's serum by the PIRC test
Tested serums
Mrs. MAU.. . serum Dec. 1977 (anti-HLA-9 +++ ND anti-HLA-12 ++ ND) PIRC: 1
AB control serum
2
mean,%
1
2
mean, %
PlA'-positive plateletA
ABO-Rh group - name HLA group O + ; COR ... A l , 28; 88, 18
31.14
23,46
27,3
5.22
5.22
5.2
O + ; GAU ... A2, 3; 88, 14
37.13
35.91
36.5
3.96
3.31
3.6
O + ; DAV ... A3, 10; B14, W38
37.26
32.98
35.1
O + ; FOU ... A3, 29; B12, 18
33.24
30.00
31.6
3.19
3.62
3.4
O + ; HOR ... A3, W31: B18, 7
33.39
32.36
32.9
O + ; C H I ... A2, 9; 81, 12
34.38
31.44
32.9
O + ; GRA ... A l , W32; B12, W21
31.98
29.58
30.8
A + : MER ... A2, - ; BI, I2
7.87
8.90
8.4
5.18
5.86
5.5
O f ; DE F... A2, 9: B18, -
10.04
10.50
10.3
5.10
5.31
5.2
O + ; L E G ... W19, W31; B5, 8
6.60
6.29
6.5
5.00
5.12
5.1
A - ; MOR ... AI, W31; B8, 8
6.94
6.26
6.6
5.64
6.01
5.9
0 + :RE1 ... A2, 1 I ; B5, W35
4.41
8.80
6.6
5.00
5.29
5. I
O + ; BOR ... A l l , W19; B18,-
5.26
6.34
5.8
3.78
4.09
3.9
PI A'-negative platelets
Soulier/Patereau/Gobert/Achach/Muller
26
Table IV. Titration of Mrs. MAU.. . anti-PIA1antibody: percent of radioactivity bound on PIA1-PIA2platelets Sera
Dilution (saline) ~~
ND
1 /2
1/4
1 /8
1/16
1/32
1/64
1/128
1/256
29.25 3.40 4
31.84 1.26 4
31.67 3.08 3
27.99 1.74 4
22.61 2.34 3
17.20 2.25 8
11.59 0.52 4
6.57 0.90 5
4.32 0.44 6
2.40 0.14 2
2.16 0.01
2.24 0.33 2
2.11 0.08 2
MAU... Dec. 77
Yo SD n S. AB % SD n
3.41 0.30 2
2
Mean number of assays, SD = standard deviation; n = number of observations.
Table V. Direct radioactive Coombs test using the patient’s platelets Date
Patient platelet count
Percent of radioactivity found patient platelets
Dec. 77 to Feb. 78 Feb. 7,78
1st platelets
2nd platelets
1.37 1.29 1.3
2.72 2.64 2.7
6.95 7.28 mean, % 7.1
2.74 2.64 2.7
2.57 2.45 2.5
1 2
2.80 2.69 2.75
2.36 2.35 2.36
not enough platelets
45,000
1
4.53 4.47 mean, % 4.5 2
Feb. 23,78
45,000
1
2
April 17,78
control platelets
138,OOO
2.81 2.83 mean, % 2.8
Posttransfusional Immunologic Thrombocytopenia
The platelet indirect Coombs test (PIRC) appears to be the most sensitive of the tests used for the detection of anti-PIA1 antibodies and the typing of platelets in the P P system. The significance of these observations lies in the fact that they concern a second episode of subclinical posttransfusion thrombocytopenia, and also in the magnitude and duration of the thrombocytopenia. The platelet increase was very slow. Below or near 10,000 for 1 month, it doubled each successive month, reaching 138,000 5 months after the onset. This very slow recovery, compared to other published cases, may be explained by the numerous stimuli. Such a repeated stimulation by the large dose of PIA’antigen produced a strong and sustained anamnestic response. However, it is not yet clear why severe thrombocytopenia develop in sensitized individuals. Shulman et al. [13] have shown that the administration of her own anti-PlA’-positive antibody to a patient after recovery from posttransfusion purpura did not cause thrombocytopenia, whereas infusion of a smaller quantity of the serum into a PP’-positive individual resulted in thrombocytopenia. It is conceivable that attachment of antibody plus PIA1 antigen from transfused platelets (forming an immune complex) on the recipient’s PIA1-negative platelets may result in thrombocytopenia, just as antigen-antibody complexes are adsorbed on platelets which are ‘innocent bystanders’ in drug purpura. However, the persistence of thrombocytopenia for several months is difficult to understand, such immune complexes being usually rapidly cleared once the drug is stopped. To detect antibodies on the surface of our patient’s platelets, we applied a direct radioactive Coombs test. The results are shown in table V. Only one of the three tests was posi-
27
tive (when platelets were 45,000). Such a positive Coombs test only means that an abnormal amount of immune globulins was present on the recipient’s platelets 3 months after the onset, but it does not indicate whether they are autoantibodies or alloantibodies (or alloimmune complexes). Alloantibodies with cross-reactivity were suggested by Morrison and Mollison [ 101, postulating that the anti-PIA1antibody could have some common specificity for PIA1+and PIA1-negative platelets. Experimental evidence for this hypothesis is lacking and it does not explain why the patient’s platelets are left unharmed before the transfusion. To explain the duration of the thrombocytopenia, in addition to the hyperstimulation, there is also the possibility that the megakaryocytes were severely injured by the high titer antibody. A first sternal puncture done 2 weeks after surgery showed ‘rare’ megakaryocytes but this sample was considered as overdiluted. A second marrow specimen 2 weeks later showed an increased number of megakaryocytes. Another factor which may have some bearing on the persistence of the thrombocytopenia may be the lack of treatment. Most of the other reported cases (about 20) have received prednisone which may shorten the immunologic reaction. In a case of posttransfusion purpura published by Vaughan-Neil et al. [19], which was tested only for HLA antibodies and not for PIA’ antigen or antibody, it is said that: ‘corticosteroids in conventional doses have been used in a number of cases but there is no evidence that this treatment influenced the course of the disease, since several weeks elapsed before the platelet count returned to normal. Our patient treated with massive doses (2 g daily) of intravenous methylprednisone-sodium-succinate for the first 3
28
days improved rapidly . . . We therefore consider short courses of high dose corticosteroids worthy of further trial in the treatment of posttransfusion purpura.' In our case, the surgeon chose not to use corticosteroid, fearing severe infection. This conservative attitude proved to be justified in spite of the risk of intracranial complications, since no bleeding occurred. PIA1-negative blood donors were available for compatible transfusions in case the need would arise. Some patients have been treated by exchange transfusion using n x ma1 blood (PIA1+).We wonder if such treatment is to be recommended for patients with a high antibody titer. In fact, the exchange of more than one blood volume is necessary to decrease the antibody titer significantly, and the administration of new PIA1+ antigen may be a source of restimulation. In a case published by Cimo and Aster [ 2 ] , there was an increase of antibody titer and a transient fall in blood platelets after a 60% exchange of the blood volume, before the definitive correction of the thrombocytopenia, which took 2 months. This case of posttransfusional thrombocytopenic is, as is the rule, found in a PP-negative female. Only two exceptions among the welldocumented cases were published [20]. Our finding, that no bleeding was observed, suggests that PTTP may pass unnoticed and that it may be less exceptional than appears from the few published cases. Moreover, even if serological investigations are done, the antibodies may not always be detected. The PIRC test that we described appears much more sensitive than other conventional tests for detecting platelet ant ibodies. The serum of all patients with acute postoperative thrombocytopenia should be exam-
Soulier!Patereau/Gobert/Achach/Muller
ined for the presence of platelet alloantibodies, especially when dealing with pretransfused multiparous patients. When anti-PIA1 antibodies are found, subsequent blood transfusions should be considered with great care. We have selected a dozen PIA1-negative donors who are willing to give blood in case of neonatal thrombocytopenia due to anti-PW. Their blood could also be used to transfuse patients with anti-PIA1 to prevent posttransfusional thrombocytopenia. Once this has developed after a transfusion, it is questionable if transfusions with PI*'-negative blood are useful, since the PIA1-negative donor platelets may also be destroyed. Gerstner et al. [6] found that their patient continued to bleed, with fatal result, when they tried to treat PTTP by transfusion of PIA1-negative platelets, but the number of transfused platelets may have been too small: it is well known that platelets from at least 4-6 units of blood are necessary to achieve a clinical effect in ordinary cases of acute hemorrhagic thrombocytopenia. It may be helpful in the future to store frozen PI*'-negative platelets from several donors to treat severe cases of PTTP. References 1 Abramson, N.; Eisenberg, P . D . , and Aster, R. H.: Post-transfusion purpura: immunologic aspects and therapy. New Engl. J . Med. 291: 1163 (1974). 2 Cimo, P. L. and Aster, R. H.: Post-transfusion purpura. Successful treatment by exchange transfusion. New Engl. J . Med. 287: 290 (1972). 3 Dechavanne, M.; Lagarde, M.; Arnaud, P.; Thouverez, J. P.; Creyssel, R. et Viala, J . J.: Mise en tvidence in vitro d'anticorps antiplaquettaires au cours du purpura thromboptnique idiopathique par la mesure de I'excrttion plaquettaire de 1GsCrotonine. Path. Biol., Paris 22: 17 (1974).
Posttransfusional Immunologic Thrombocytopenia
4 Deykin, D. and Hellerstein, L.. J.: The assessment of drug-dependent and isoimmune antiplatelet antibodies by the use of platelet aggregometry J. clin. Invest. 51: 3142 (1972). 5 Dubarry, J. J.; Moulinier, J.; Quinton, A.; Labat, J. P. et Merle, M. c . : Un cas de purpura thrombop6nique post-transfusionnel. Bord. mid. 4: 911 (1970). 6 Gerstner, J.B.; Smith, M. 1.; Davis, K.D.; Aster, R. H., and Cimo, P.: Post-transfusion purpura. Therapeutic failure of PIA1-negative platelet transfusion. Abstr. 17th Annu. Meet. Am. SOC.of Hematology, 1974. 7 Gockerman, J. P. and Shulman, N. R.: Isoantibody specificity in posttransfusion purpura. Blood 41: 817 (1973). 8 Hirschman, R. J. and Gralnick, H. R.: A simplified platelet factor 3 (PF-3) assay for the rapid detection of platelet isoantibodies and an antiplatelet factor in ATP and SLE. J. Lab. clin. Med. 84: 292 (1974). 9 Howard, J. E.; Glassberg, A. B., and Perkins, H. A.: Post-transfusion thrombocytopenic purpura: a case report. Am. J. Hemat. l: 339 (1976). 10 Morrison, F. S. and Mollison, P. L.: Post-transfusion purpura. New Engl. J. Med. 275: 243 (1966). 11 Morse, E. E.: Topics in clinical medicine. Posttransfusion thrombocytopenic purpura. Johns Hopkins med. J. 121: 365 (1968). 12 Nichols,R. J.;Davies,P., and Kenwright, M. G.: Thrombocytopenic purpura after blood transfusion. Br. med. J. ii: 582 (1970). 13 Shulman, N. R.; Aster, R. H.; Leitner, A., and Hiller, M. C.: Immunoreactions involving platelets. V. Post-transfusion purpura due to a complement fixing antibody against a genetically controlled platelet antigen. A proposed mechanism for thrombocytopenia and its relevance in ‘autoimmunity’. I. clin. Invest. 40: 1597 (1961).
29
14 Soulier, J. P.; Patereau, C., and Drouet, I.: Platelet indirect radioactive Coombs test. Its utilization for PlAl grouping. Vox Sang. 29: 253 (1975). 15 Svejgaard, A.; Pederson, M. F.; Hensen, 0. H., and Kissmeyer-Nielsen, F.: Post-transfusion purpura caused by a specific platelet isoantibody. Dan. med. Bull. 14: 41 (1967). 16 Terasaki, P. I. and McClelland, J. D.: Microdoplet assay of human cytotoxins. Nature, Lond. 204: 998 (1964). 17 Weerdt, C. M. Van der; Veenhoven-Von Riesz, L. E.; Nijenhuis, L. E., and Loghem, J. J . Van: The Zw blood group system in platelets. Vox Sang. 8: 513 (1963). 18 Loghem, J . J. Van, jr.; Dorfmeijer, H., and Hart, M. Van der: Serological and genetic studies on a platelet antigen (Zw). Vox Sang. 4: 161 (1959). 19 Vaughan-Neil, E.F.; Ardeman, S.; Bevan, G.; Blakeman, A. C., and Jenkins, W. 3.: Posttransfusion purpura associated with unusual platelet antibody (anti-PIB1). Br. med. J. i: 436 (1975). 20 Zeigler, Z.; Murphy, S., and Gardner, F. H.: Post-transfusion purpura: a heterogeneous syndrome. Blood 45: 529 (1975). 21 Zucker, M. B.; Ley, A. B.; Borrelli, J.; Mayer. K., and Firmat, 3.: Thrombocytopenia with a circulating platelet agglutinin, platelet lysin and clot retraction inhibitor. Blood 14: 148 (1959).
Received: October 5, 1978 Accepted: January 15, 1979 Jean-Pierre Soulier, Centre national de Transfusion sanguine, 6, rue Alexandre Cabanel, F-75015 Paris (France)
Posttransfusional lrnmunologic Thrombocytopenia A Case Report jean-Pierre Soulier, Claude Patereau, Nicole Gobert, Pierre Achach and Jean- Yves Muller Centre national de Transfusion sanguine, Paris
Abstract. A case of posttransfusional immunologic thrombocytopenia is reported in a 75-year-old PP-negative woman. This was the second episode of postoperative and posttransfusion thrombocytopenia in the same patient who had had only one pregnancy. Both thrombocytopenic episodes were subclinical and discovered by systematical hematologic study. A potent anti-PP antibody (titer 1/128) was demonstrated by the platelet-indirect radioactive Coombs test which appeared more sensitive than other platelet immunological assays used. The very long duration of the thrombocytopenia is discussed as well as the therapeutic possibilities in such cases. Posttransfusional thrombocytopenic purpura (PTTP) was first described simultaneously in 1959 by Zucker et al. [21] and by van Loghem et al. [18]. Shulman et al. [ 131 made a detailed clinical and serological study of this very rare syndrome, observed in PI*'-negative women about a week after transfusion. No more than 20 cases have been published in nearly 20 years (only one in France IS]), thus any additional serologically well-studied case of PTTP is worth reporting.
Case Report Mrs. M A U . . ., 75 years old, was admitted to the Clinique Labrouste in November 1977, for a total hip replacement. At the time of her admission, on November 26, the platelet count was 190,OOO/mm3.
Mrs. MAU.. . was born from a first cousin's marriage, which may play a role in the fact that her platelets were found to be PW-negative (only 2.5% of the normal French population). Her blood group is 0 Rh-positive. When 20 years old, she had a son, who died in his first year of life, and about whom there is no further information. In March 1974, she underwent orthopedic surgery for a fracture of the neck of the femur. One unit of blood was transfused preoperatively, and one postoperatively on day 5 , which was accompanied by chills and fever. A clear thrombocytopenia was present 2 weeks after the transfusion, and was only revealed by the platelet count: April 11, 1974: 30,000 platelets; April 24, 1974: 50,000 platelets; April 27, 1974: 39,000 platelets. No hemorrhagic complications were observed, and this thrombocytopenia appears to have been subclinical, and was therefore forgotten, explaining why 3 years later no special precautions were taken prior to transfusing the patient during and after a second surgical procedure. In 1977, the new thrombocytopenia which developed was discovered by a systematic hemato-
22
Soulier/Patereau/Gobert/Achach/Muller
Table I. Successive hematological data Date
Hematocrit
Platelet counts
Serology
PIRC
1977 November 26 November 28
December 5 December 10 December 11 December 12 December 14 December 15
38
190,000
36 26 37 38 37
2 units of blood (chills) I unit of blood (chills) 35,000 20,000
TEG (r+k=14 min; am = 44 mm) Megakaryocytes rare
10,000
10,000 10,Ooo < 10,000
December 20 December 29
38 38
< 10,000 < 10,Ooo
1978 January 5 January 11 January 16 January 20 February 7
38 39 37 37 42
< 10,Ooo 10,000 20,000
Megakaryocytes numerous t+t
11128
TEG (r+k=12 min; am =40 mm)
1/128 11128 return home
1/128
45,000
45,000 105,000 138,000
logical study. On November 28, the day of surgery, 5 units of blood were transfused. The hematocrit
was 41%. No hemorrhagic syndrome was observed, and more especially, no local hematoma at the site of surgery. However, a decrease in the hematocrit from 36% on December 5 to 26% on December 10 was noticed, and two additional units of blood were transfused. This transfusion was not well tolerated chills, cephalalgia and feyer being observed. The day after, a new blood transfusion
-
direct Coombs (cf. table 1V)
5 units of blood hip replacement
December 17 December 18 December 19
February 23 March 23 April 17
Remarks
1/64 (dubious 1/128) 1/64
dubious
1/64 1/64
produced similar reactions. It was at this time (December 12, 1977) that the thrombocytopenia was discovered: 35,000 platelets/mmy. No petechiae, no ecchymosis or other hemorrhages in spite of a systematic anticoagulant treatment. The oral anticoagulant (Sintrom) was immediately stopped and platelet counts were made every day (table 1). The platelets decreased from 35.000 to 10,000 and they remained at this very low level for 1month. Thromboelastograms were made twice at an interval of
Posttransfusional Immunologic Thrombocytopenia
2 weeks (December 15 and 29). They showed the usual defects found in thromhocytopenia (r + k = 14 min; maximal amplitude 40 mm). In spite of the risks involved in this elderly patient, the surgeon chose not to use corticosteroids for fear of infection and failure of the total hip arthroplasty. It was considered to transfuse P W negative blood but this was kept in reserve (see Discussion), since the PW-negative platelets of the recipient were affected by the exogenous P1A conflict. This conservative attitude proved to be justified since no hemorrhagic complications were oherved. Two bone marrow specimens were examined. On December 16, the marrow was 'somewhat diluted' and the megakaryocytes considered as 'rare'; on December 22 it was of normal density and the rnegakaryocytes were increased. On January 20, 1978, 6 weeks after surgery, the hematocrit was well stabilized (between 37 and 39%), with a platelet count of around 20,000. This thrombocytopenia was especially slow to correct spontaneously, and on April 17, the platelet count was 138,000.
Serological Study Platelet Indirect Radioactive Coombs (PIRC) This method has already been described [ 141. Briefly: 50 pl of platelet suspension (108) are mixed for 1 h at 37 "C with 50 pl of serum. The platelets are washed 3 times with saline. 125I-labelled goat IgG, active against human IgG, is added and kept at 37 "C for 30 min. After centrifugation, the supernatant ( S * ) is kept and the platelet sediment (PS") is washed (W*). The test is done in triplicate. The fixation percentage of goat IgG on platelets is: PS* PS* + s*+ w*
Controls are done using P P t and PP-negative platelets, as well as AB normal serum.
Fixation higher than 10% is considered as a positive test. Fixation lower than 6% denotes a negative test. HLA antibodies (in the presence of platelets having the corresponding antigen) may give a fixation of around 10%. PIA*antibodies may give a fixation as high as 2 0 4 0 % . Platelet Direct Radioactive Coombs The same test may be used as a direct test using the patient's platelets if their number is high enough (over 50,00O/mm3). Other Tests Platelet aggregation of normal PIA'+ platelet rich plasma. This was done according to Deykin and Hellerstein [4]using an aggregometer. Platelet serotonin release was done according to Dechavanne et al. [3]. Complement fixation on platelets was done by Mrs. Jullien using standard methods [13]. Thromboagglutination was done according to Van der Weerdt [17]. Platelet factor 3 assay was done according to the Hirschman and Gralnick technique [S], but dextran sulfate was used instead of kaolin to activate the contact factors. Lymphocytotoxicity antibodies and HLA typing were done by L. Hullt using standard techniques [16].
Results of the Serological Study The platelet factor 3 availability test and the platelet serotonin release were both negative. The thromboagglutination and the complement fixation test were both weakly positive. The undiluted patient serum produced an aggregation curve using normal PIA1+PRP, in contrast with normal AB serum.
Soulier/Patereau/Gobert/Achach/Muller
L4
Table 11. PIA1 typing of Mrs. M A U . . . platelets. Percent of radioactivity fixed by the patient's platelets in the presence of anti-PIA1serum and of AB normal serum, in comparison with normal PWpositive platelets and PIA1-negative Serum
Platelets PI"'+
~
~~
~
Anti-PIA1serum (LAB ...) AB serum
~~~
PlAl-
MAU ...
~
16.2%
4.5%
5.1%
3.5%
3.5%
4.1%
Lymphocytotoxicity was positive using a lymphocyte panel of known phenotype: antiHLA-9 and anti-B12 antibodies were found in the patient's serum.
antibody was titrated by serial dilutions (table IV). This strong anti-PIA1 antibody was regularly found at each of the subsequent PIRC assays, the titer remaining at least 1 to 64 (dubious results with the 1 to 128 dilution). This high titer persisted for 5 months after the discovery of the thrombocytopenia (table I). Direct Platelet Radioactive Coombs Due to the very low platelet count, the results of this test were not considered significant before February 23 (table V).
Discussion
This case represents a further example of the rare syndrome of posttransfusion imPlatelet Indirect Radioactive Coombs Clear-cut results were obtained only with munologic thrombocytopenia and it is the this test, for PIA typing as well as for the second recorded in France, the first being demonstration of strong anti-PIA1 antibodies reported by Dubarry et al. [ 5 ] . This patient in the serum of the patient. Results of the was a multitransfused female, with one pregP1*1 typing are summarized in table 11. nancy in her past history; her platelets were Mrs. MAU . . . platelets are PIAi-negative PIA1-negative and her serum contained an (PlA2PP). No anti-PlA* antiserum is avail- anti-PP antibody of high titer. In spite of her age (75) and the severe able to test such platelets. thrombocytopenia (less than 10,000 for several weeks), the patient had no apparent Assay for Anti-PIA1Antibodies by the hemorrhagic symptoms. However, a sudden PIRC Test A typical assay for anti-PP antibodies is fall in the hematocrit (36-26% between the shown in table 111. It is seen that PIA1-posi- 7th and the 12th postoperative day) was obtive platelets gave a uniformly high fixation served and 3 units of blood were transfused. of radioactivity (mean 30%) where PIA1-neg- A low platelet count was discovered, and ative platelets fixed less than 10%. Some thus a second episode of subclinical postfixation on PP-negative platelets was due to operative thrornbocytopenia was diagnosed. the presence of the HLA antigen on these It is not possible to decide today if the reacplatelets which reacted with the anti-HLA tions observed after a 3-year interval were antibody of Mrs. MAU . . . Besides anti-HLA due only to anti PI*' antibodies, since we antibodies, a strong specific anti-PIA1 anti- know that the patient's serum also contained body is thus present in the patient's serum anti-HLA antibodies which were able to reexplaining the posttransfusion reactions. This act with the transfused leukocytes.
Posttransfusional Immunologic Thrombocytopenia
25
Table 111. Detection of anti-PIA1antibody in the patient's serum by the PIRC test
Tested serums
Mrs. MAU.. . serum Dec. 1977 (anti-HLA-9 +++ ND anti-HLA-12 ++ ND) PIRC: 1
AB control serum
2
mean,%
1
2
mean, %
PlA'-positive plateletA
ABO-Rh group - name HLA group O + ; COR ... A l , 28; 88, 18
31.14
23,46
27,3
5.22
5.22
5.2
O + ; GAU ... A2, 3; 88, 14
37.13
35.91
36.5
3.96
3.31
3.6
O + ; DAV ... A3, 10; B14, W38
37.26
32.98
35.1
O + ; FOU ... A3, 29; B12, 18
33.24
30.00
31.6
3.19
3.62
3.4
O + ; HOR ... A3, W31: B18, 7
33.39
32.36
32.9
O + ; C H I ... A2, 9; 81, 12
34.38
31.44
32.9
O + ; GRA ... A l , W32; B12, W21
31.98
29.58
30.8
A + : MER ... A2, - ; BI, I2
7.87
8.90
8.4
5.18
5.86
5.5
O f ; DE F... A2, 9: B18, -
10.04
10.50
10.3
5.10
5.31
5.2
O + ; L E G ... W19, W31; B5, 8
6.60
6.29
6.5
5.00
5.12
5.1
A - ; MOR ... AI, W31; B8, 8
6.94
6.26
6.6
5.64
6.01
5.9
0 + :RE1 ... A2, 1 I ; B5, W35
4.41
8.80
6.6
5.00
5.29
5. I
O + ; BOR ... A l l , W19; B18,-
5.26
6.34
5.8
3.78
4.09
3.9
PI A'-negative platelets
Soulier/Patereau/Gobert/Achach/Muller
26
Table IV. Titration of Mrs. MAU.. . anti-PIA1antibody: percent of radioactivity bound on PIA1-PIA2platelets Sera
Dilution (saline) ~~
ND
1 /2
1/4
1 /8
1/16
1/32
1/64
1/128
1/256
29.25 3.40 4
31.84 1.26 4
31.67 3.08 3
27.99 1.74 4
22.61 2.34 3
17.20 2.25 8
11.59 0.52 4
6.57 0.90 5
4.32 0.44 6
2.40 0.14 2
2.16 0.01
2.24 0.33 2
2.11 0.08 2
MAU... Dec. 77
Yo SD n S. AB % SD n
3.41 0.30 2
2
Mean number of assays, SD = standard deviation; n = number of observations.
Table V. Direct radioactive Coombs test using the patient’s platelets Date
Patient platelet count
Percent of radioactivity found patient platelets
Dec. 77 to Feb. 78 Feb. 7,78
1st platelets
2nd platelets
1.37 1.29 1.3
2.72 2.64 2.7
6.95 7.28 mean, % 7.1
2.74 2.64 2.7
2.57 2.45 2.5
1 2
2.80 2.69 2.75
2.36 2.35 2.36
not enough platelets
45,000
1
4.53 4.47 mean, % 4.5 2
Feb. 23,78
45,000
1
2
April 17,78
control platelets
138,OOO
2.81 2.83 mean, % 2.8
Posttransfusional Immunologic Thrombocytopenia
The platelet indirect Coombs test (PIRC) appears to be the most sensitive of the tests used for the detection of anti-PIA1 antibodies and the typing of platelets in the P P system. The significance of these observations lies in the fact that they concern a second episode of subclinical posttransfusion thrombocytopenia, and also in the magnitude and duration of the thrombocytopenia. The platelet increase was very slow. Below or near 10,000 for 1 month, it doubled each successive month, reaching 138,000 5 months after the onset. This very slow recovery, compared to other published cases, may be explained by the numerous stimuli. Such a repeated stimulation by the large dose of PIA’antigen produced a strong and sustained anamnestic response. However, it is not yet clear why severe thrombocytopenia develop in sensitized individuals. Shulman et al. [13] have shown that the administration of her own anti-PlA’-positive antibody to a patient after recovery from posttransfusion purpura did not cause thrombocytopenia, whereas infusion of a smaller quantity of the serum into a PP’-positive individual resulted in thrombocytopenia. It is conceivable that attachment of antibody plus PIA1 antigen from transfused platelets (forming an immune complex) on the recipient’s PIA1-negative platelets may result in thrombocytopenia, just as antigen-antibody complexes are adsorbed on platelets which are ‘innocent bystanders’ in drug purpura. However, the persistence of thrombocytopenia for several months is difficult to understand, such immune complexes being usually rapidly cleared once the drug is stopped. To detect antibodies on the surface of our patient’s platelets, we applied a direct radioactive Coombs test. The results are shown in table V. Only one of the three tests was posi-
27
tive (when platelets were 45,000). Such a positive Coombs test only means that an abnormal amount of immune globulins was present on the recipient’s platelets 3 months after the onset, but it does not indicate whether they are autoantibodies or alloantibodies (or alloimmune complexes). Alloantibodies with cross-reactivity were suggested by Morrison and Mollison [ 101, postulating that the anti-PIA1antibody could have some common specificity for PIA1+and PIA1-negative platelets. Experimental evidence for this hypothesis is lacking and it does not explain why the patient’s platelets are left unharmed before the transfusion. To explain the duration of the thrombocytopenia, in addition to the hyperstimulation, there is also the possibility that the megakaryocytes were severely injured by the high titer antibody. A first sternal puncture done 2 weeks after surgery showed ‘rare’ megakaryocytes but this sample was considered as overdiluted. A second marrow specimen 2 weeks later showed an increased number of megakaryocytes. Another factor which may have some bearing on the persistence of the thrombocytopenia may be the lack of treatment. Most of the other reported cases (about 20) have received prednisone which may shorten the immunologic reaction. In a case of posttransfusion purpura published by Vaughan-Neil et al. [19], which was tested only for HLA antibodies and not for PIA’ antigen or antibody, it is said that: ‘corticosteroids in conventional doses have been used in a number of cases but there is no evidence that this treatment influenced the course of the disease, since several weeks elapsed before the platelet count returned to normal. Our patient treated with massive doses (2 g daily) of intravenous methylprednisone-sodium-succinate for the first 3
28
days improved rapidly . . . We therefore consider short courses of high dose corticosteroids worthy of further trial in the treatment of posttransfusion purpura.' In our case, the surgeon chose not to use corticosteroid, fearing severe infection. This conservative attitude proved to be justified in spite of the risk of intracranial complications, since no bleeding occurred. PIA1-negative blood donors were available for compatible transfusions in case the need would arise. Some patients have been treated by exchange transfusion using n x ma1 blood (PIA1+).We wonder if such treatment is to be recommended for patients with a high antibody titer. In fact, the exchange of more than one blood volume is necessary to decrease the antibody titer significantly, and the administration of new PIA1+ antigen may be a source of restimulation. In a case published by Cimo and Aster [ 2 ] , there was an increase of antibody titer and a transient fall in blood platelets after a 60% exchange of the blood volume, before the definitive correction of the thrombocytopenia, which took 2 months. This case of posttransfusional thrombocytopenic is, as is the rule, found in a PP-negative female. Only two exceptions among the welldocumented cases were published [20]. Our finding, that no bleeding was observed, suggests that PTTP may pass unnoticed and that it may be less exceptional than appears from the few published cases. Moreover, even if serological investigations are done, the antibodies may not always be detected. The PIRC test that we described appears much more sensitive than other conventional tests for detecting platelet ant ibodies. The serum of all patients with acute postoperative thrombocytopenia should be exam-
Soulier!Patereau/Gobert/Achach/Muller
ined for the presence of platelet alloantibodies, especially when dealing with pretransfused multiparous patients. When anti-PIA1 antibodies are found, subsequent blood transfusions should be considered with great care. We have selected a dozen PIA1-negative donors who are willing to give blood in case of neonatal thrombocytopenia due to anti-PW. Their blood could also be used to transfuse patients with anti-PIA1 to prevent posttransfusional thrombocytopenia. Once this has developed after a transfusion, it is questionable if transfusions with PI*'-negative blood are useful, since the PIA1-negative donor platelets may also be destroyed. Gerstner et al. [6] found that their patient continued to bleed, with fatal result, when they tried to treat PTTP by transfusion of PIA1-negative platelets, but the number of transfused platelets may have been too small: it is well known that platelets from at least 4-6 units of blood are necessary to achieve a clinical effect in ordinary cases of acute hemorrhagic thrombocytopenia. It may be helpful in the future to store frozen PI*'-negative platelets from several donors to treat severe cases of PTTP. References 1 Abramson, N.; Eisenberg, P . D . , and Aster, R. H.: Post-transfusion purpura: immunologic aspects and therapy. New Engl. J . Med. 291: 1163 (1974). 2 Cimo, P. L. and Aster, R. H.: Post-transfusion purpura. Successful treatment by exchange transfusion. New Engl. J . Med. 287: 290 (1972). 3 Dechavanne, M.; Lagarde, M.; Arnaud, P.; Thouverez, J. P.; Creyssel, R. et Viala, J . J.: Mise en tvidence in vitro d'anticorps antiplaquettaires au cours du purpura thromboptnique idiopathique par la mesure de I'excrttion plaquettaire de 1GsCrotonine. Path. Biol., Paris 22: 17 (1974).
Posttransfusional Immunologic Thrombocytopenia
4 Deykin, D. and Hellerstein, L.. J.: The assessment of drug-dependent and isoimmune antiplatelet antibodies by the use of platelet aggregometry J. clin. Invest. 51: 3142 (1972). 5 Dubarry, J. J.; Moulinier, J.; Quinton, A.; Labat, J. P. et Merle, M. c . : Un cas de purpura thrombop6nique post-transfusionnel. Bord. mid. 4: 911 (1970). 6 Gerstner, J.B.; Smith, M. 1.; Davis, K.D.; Aster, R. H., and Cimo, P.: Post-transfusion purpura. Therapeutic failure of PIA1-negative platelet transfusion. Abstr. 17th Annu. Meet. Am. SOC.of Hematology, 1974. 7 Gockerman, J. P. and Shulman, N. R.: Isoantibody specificity in posttransfusion purpura. Blood 41: 817 (1973). 8 Hirschman, R. J. and Gralnick, H. R.: A simplified platelet factor 3 (PF-3) assay for the rapid detection of platelet isoantibodies and an antiplatelet factor in ATP and SLE. J. Lab. clin. Med. 84: 292 (1974). 9 Howard, J. E.; Glassberg, A. B., and Perkins, H. A.: Post-transfusion thrombocytopenic purpura: a case report. Am. J. Hemat. l: 339 (1976). 10 Morrison, F. S. and Mollison, P. L.: Post-transfusion purpura. New Engl. J. Med. 275: 243 (1966). 11 Morse, E. E.: Topics in clinical medicine. Posttransfusion thrombocytopenic purpura. Johns Hopkins med. J. 121: 365 (1968). 12 Nichols,R. J.;Davies,P., and Kenwright, M. G.: Thrombocytopenic purpura after blood transfusion. Br. med. J. ii: 582 (1970). 13 Shulman, N. R.; Aster, R. H.; Leitner, A., and Hiller, M. C.: Immunoreactions involving platelets. V. Post-transfusion purpura due to a complement fixing antibody against a genetically controlled platelet antigen. A proposed mechanism for thrombocytopenia and its relevance in ‘autoimmunity’. I. clin. Invest. 40: 1597 (1961).
29
14 Soulier, J. P.; Patereau, C., and Drouet, I.: Platelet indirect radioactive Coombs test. Its utilization for PlAl grouping. Vox Sang. 29: 253 (1975). 15 Svejgaard, A.; Pederson, M. F.; Hensen, 0. H., and Kissmeyer-Nielsen, F.: Post-transfusion purpura caused by a specific platelet isoantibody. Dan. med. Bull. 14: 41 (1967). 16 Terasaki, P. I. and McClelland, J. D.: Microdoplet assay of human cytotoxins. Nature, Lond. 204: 998 (1964). 17 Weerdt, C. M. Van der; Veenhoven-Von Riesz, L. E.; Nijenhuis, L. E., and Loghem, J. J . Van: The Zw blood group system in platelets. Vox Sang. 8: 513 (1963). 18 Loghem, J . J. Van, jr.; Dorfmeijer, H., and Hart, M. Van der: Serological and genetic studies on a platelet antigen (Zw). Vox Sang. 4: 161 (1959). 19 Vaughan-Neil, E.F.; Ardeman, S.; Bevan, G.; Blakeman, A. C., and Jenkins, W. 3.: Posttransfusion purpura associated with unusual platelet antibody (anti-PIB1). Br. med. J. i: 436 (1975). 20 Zeigler, Z.; Murphy, S., and Gardner, F. H.: Post-transfusion purpura: a heterogeneous syndrome. Blood 45: 529 (1975). 21 Zucker, M. B.; Ley, A. B.; Borrelli, J.; Mayer. K., and Firmat, 3.: Thrombocytopenia with a circulating platelet agglutinin, platelet lysin and clot retraction inhibitor. Blood 14: 148 (1959).
Received: October 5, 1978 Accepted: January 15, 1979 Jean-Pierre Soulier, Centre national de Transfusion sanguine, 6, rue Alexandre Cabanel, F-75015 Paris (France)
