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Obstetrics & Gynecology 1999;93:169-173
© 1999 by The American College of Obstetricians and Gynecologists
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ORIGINAL RESEARCH

Maternal Antiplatelet Antibodies in Predicting Risk of Neonatal Thrombocytopenia

FRANÇOISE BOEHLEN, MD, PATRICK HOHLFELD, MD, PHILIPPE EXTERMANN, MD and PHILIPPE DE MOERLOOSE, MD

From the Departments of Internal Medicine and Gynecology and Obstetrics, University Hospital, Geneva; and the Department of Gynecology and Obstetrics, University Hospital, Lausanne, Switzerland.

Address reprint requests to: Philippe de Moerloose, MD Hemostasis Unit University Hospital 1211 Geneva 14 Switzerland E-mail: phdm{at}diogenes.hcuge.ch


   Abstract
Top
Abstract
Patients and Methods
 Results
 Discussion
 References
 
Objective: To determine the incidence of maternal antiplatelet antibodies in cases of thrombocytopenia during pregnancy, using the monoclonal antibody–specific immobilization of platelet antigens assay; and to assess the usefulness of this assay for predicting risk of neonatal thrombocytopenia.

Methods: A total of 6770 pregnant women were included in the study, and the monoclonal antibody–specific immobilization of platelet antigens assay was done when platelet counts were less than 150 x 109/L. Platelet counts were determined in 6103 newborns.

Results: The incidence of maternal thrombocytopenia was 11.6% (95% confidence interval [CI] 10.8, 12.4). Among newborns, 1.3% (95% CI 0.5, 2.7) born to thrombocytopenic mothers were thrombocytopenic, compared with 0.4% (95% CI 0.2, 0.6) born to nonthrombocytopenic women. Antiplatelet antibodies were detected in 37 (8.6%) of 430 thrombocytopenic women; autoantibodies were detected in 28 cases (circulating or bound to platelets), alloantibodies in eight cases, and an association of alloantibodies and autoantibodies in one case. The positive and negative likelihood ratios for predicting neonatal thrombocytopenia were 4.6 and 0.7, respectively.

Conclusion: The monoclonal antibody–specific immobilization of platelet antigens assay did not predict the risk of neonatal thrombocytopenia in an unselected population of thrombocytopenic pregnant women.

Thrombocytopenia in pregnant women is relatively common,1 the majority of cases classified as gestational or incidental. It is important to differentiate gestational from autoimmune thrombocytopenia, particularly because the risk of neonatal thrombocytopenia is different in the two groups.2–5

There is no specific laboratory test to distinguish immune thrombocytopenic purpura (ITP) from gestational thrombocytopenia; both diagnoses are based mainly on exclusion criteria.6 Because no laboratory tests based on maternal platelet counts2,3,7 or detection of antiplatelet antibodies4,7–11 predict the occurrence of neonatal thrombocytopenia, the only reliable method of identifying fetal thrombocytopenia is fetal blood sampling. This method is controversial, risky, and not widely available.11–14

New antiplatelet antibody assays, using monoclonal antibodies, such as the monoclonal antibody–specific immobilization of platelet antigens assay,15 are accepted as sensitive and specific assays for ITP,16 but their usefulness for predicting risk of neonatal thrombocytopenia has never been tested in a large unselected population of pregnant thrombocytopenic women. We report the findings of an 18-month prospective study in which platelet counts were measured in 6770 mothers and 6103 of their newborns. The aims of our study were to determine the incidence of antiplatelet antibodies in cases of maternal thrombocytopenia, using a monoclonal antibody–specific immobilization of platelet antigens assay, and to correlate the results with the occurrence of thrombocytopenia in newborns.


   Patients and Methods
Top
Abstract
 Patients and Methods
Results
 Discussion
 References
 
This study was performed in two university hospitals, in Lausanne and Geneva, Switzerland, and received the approval of both hospitals’ ethics committees. There were no exclusion criteria. A whole blood count including a platelet count was done on admission to the labor ward or during the last month of pregnancy. In cases in which platelet counts were less than 150 x 109/L, informed consent was obtained and women were enrolled in the study. Then, another platelet count on ethylenediaminetetra-acetic acid (EDTA) and citrated blood was done and if thrombocytopenia was confirmed, antiplatelet antibodies (both circulating and bound to the platelets) were assessed by a monoclonal antibody–specific immobilization of platelet antigens assay. After gestational hypertension and preeclampsia were excluded, investigations concerning human immunodeficiency virus, liver function, and anticardiolipin antibodies were done.

Cord platelet counts were performed in 90.1% of newborns at birth (for technical reasons, samples were not collected from or processed for all infants). Each platelet count less than 150 x 109/L in cord blood was checked by heel puncture, and only infants with confirmed thrombocytopenia were considered for further investigations. After the study was completed, antiplatelet antibodies were searched for in a control group of 100 nonthrombocytopenic pregnant women.

Laboratory Methods
Platelet counts were done in EDTA blood with a Sysmex K-1000 (Toa Medical Electronics, Kobe, Japan). Thrombocytopenia was considered present when the platelet count was less than 150 x 109/L. The monoclonal antibody–specific immobilization of platelet antigens assay was done according to Kiefel et al,15 with some modifications.17–19 Platelet antibody specificity in cases of alloimmunization was determined using a panel of selected platelet donors typed for HPA-1, HPA-3, and HPA-5 antigens. Cutoff values were determined by testing 50 healthy subjects and calculating the mean optical density plus three standard deviations.

The intra-assay coefficient of variation (n = 10) was 4.3% for anti-HPA-1a, 8.1% for anti-HPA-5b, 27.3% for negative controls for bound antibodies, and 21.8% for negative controls for circulating antibodies. Interassay variability (n = 11) was 15.6% for anti-HPA-1a and 16.6% for anti-HPA-5b. No positive result was found negative or conversely. Anticardiolipin antibodies were measured according to previous reports.20 The positivity for immunoglobulin (Ig) G and IgM isotypes was assessed with a standard curve by dilutions of a house standard. The cutoff of positivity was assessed by the 97.5th percentile of the frequency distribution of 200 sera from healthy donors.

Data were analyzed using two-sided Fisher exact test, {chi}2 test, and test for trend; 95% confidence intervals (CIs) were calculated with the exact method by means of the Confidence Interval Analysis computer program (version 1.0; BMJ, London, UK). The sensitivity, specificity, predictive values, and likelihood ratios were calculated according to standard methods for proportions. Significance level was P <= .05.


   Results
Top
Abstract
 Patients and Methods
 Results
Discussion
 References
 
Platelet counts were performed in 6770 women (87.1% were white, 5.5% Asian, 4.7% black, and 2.8% North African). Their mean age was 29.7 years, the mean gestational age at delivery was 38.8 weeks, and 594 neonates (8.8%) were premature. A total of 786 mothers (11.6%; CI 10.8, 12.4) had platelet counts less than 150 x 109/L at first measurement. The majority of cases of thrombocytopenia were mild; only 21 women (0.3% of all pregnant women) had platelet counts less than 75 x 109/L (Table 1Go).


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  		Table 1. Diagnosis in Thrombocytopenic Women
 
A second platelet count was done in 666 women and thrombocytopenia was confirmed in 566. The incidence of confirmed thrombocytopenia was therefore 8.5%. No EDTA-induced thrombocytopenia was observed. Diagnoses associated with maternal thrombocytopenia, in relation to the degree of thrombocytopenia, are detailed in Table 1Go.

A cord platelet count was obtained from 6103 newborns (90.1%): 523 infants born to mothers with confirmed thrombocytopenia and 5580 to nonthrombocytopenic mothers. Thirty (0.5%) of all newborns had confirmed thrombocytopenia, 15 of them being premature, with infections, fetal growth restriction, nonreassuring fetal heart rate patterns, bradycardia, or a combination of these. Only four cases of neonatal thrombocytopenia were severe (platelet count less than 20 x 109/L). Details of these four neonates are reported in Table 2Go. Seven thrombocytopenic infants were born to mothers with confirmed thrombocytopenia (1.3%; 95% CI 0.5, 2.7) and 23 to nonthrombocytopenic mothers (0.4%; 95% CI 0.2, 0.6; P = .012). The positive and negative likelihood ratios for predicting thrombocytopenia were 2.7 and 0.8, respectively.


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  		Table 2. Neonatal and Maternal Data for Cases of Severe Neonatal Thrombocytopenia
 
Antiplatelet antibodies were searched for in 430 (76.0%) of 566 women with confirmed thrombocytopenia. This test was not done in 136 patients, essentially because of refusals (n = 64) and technical reasons (n = 72). No specific antiplatelet antibodies were detected in 393 cases (91.4%). In 37 cases (8.6%), specific antiplatelet antibodies were detected. As shown in Table 3Go, 28 women had autoantibodies, eight alloantibodies, and one an association of autoantibody (bound to platelets) and alloantibody. Autoantibodies were detected in 6.7% and alloantibodies in 2.1% of thrombocytopenic women.


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  		Table 3. Antiplatelet Antibodies in Women With Confirmed Thrombocytopenia
 
To investigate whether maternal antiplatelet antibodies were related to maternal platelet counts, we divided the thrombocytopenic women into three groups (Table 4Go) and compared them with a control group of 100 nonthrombocytopenic women. Five of the 100 women without thrombocytopenia during their pregnancy (control group) also had antiplatelet antibodies. The difference in the prevalence of autoantibodies between the groups was not statistically significant ({chi}2 P = .24, test for trend P = .45). Among the 21 women with platelet counts less than 75 x 109/L, none of the 15 analyzed had antiplatelet antibodies. Most of them had possible explanations for their thrombocytopenia (Table 1Go).


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  		Table 4. Relationship Between Platelet Count and Antiplatelet Antibodies
 
Next we compared platelet counts of infants born to thrombocytopenic women with (n = 36) or without (n = 357) antiplatelet antibodies (the presence of maternal antiplatelet antibodies was not determined for 37 newborns). Two of 36 newborns in the former group were thrombocytopenic, compared with three of 357 in the latter (P = .068). Only one of the five thrombocytopenic neonates had a platelet count less than 20 x 109/L. The sensitivity of the assay for predicting neonatal thrombocytopenia was 40.0%, the specificity 91.2%, the predictive value of a positive test 5.6%, and the predictive value of a negative test 99.2% (95% CI 97.6, 99.8). The positive and negative likelihood ratios were 4.6 and 0.7, respectively.

Seventeen of the 431 thrombocytopenic women tested for anticardiolipin antibodies were positive (3.9%). Immunoglobulin G anticardiolipin antibodies were detected in ten women and IgM antibodies in seven women. None of the 34 women tested with antiplatelet antibodies had anticardiolipin antibodies.

Follow-up platelet counts 3 months after delivery were available for 324 mothers (57.2%) with confirmed prenatal thrombocytopenia. Only 24 (7.5%) of 324 women had persistent thrombocytopenia. Platelet antibody data were available for 275 women. Among the 24 women with persistent thrombocytopenia, five had platelet antibodies (20.8%), compared with 18 (7.2%) of 251 mothers without persisting thrombocytopenia (P = .055).


   Discussion
Top
Abstract
 Patients and Methods
 Results
 Discussion
References
 
The monoclonal antibody–specific immobilization of platelet antigens assay did not predict the risk of neonatal thrombocytopenia in our study’s unselected population of thrombocytopenic pregnant women. We found a high negative predictive value because a negative monoclonal antibody–specific immobilization of platelet antigens assay could exclude neonatal thrombocytopenia with a probability of 99.2% (95% CI 97.6, 99.8). However, the low sensitivity of this test in this particular setting does not allow such a conclusion. The high negative predictive value is attributable primarily to the low incidence of neonatal thrombocytopenia and to the fact that other causes of neonatal thrombocytopenia were present. The negative likelihood ratio is also low (0.7).

Only one previous study2 established a link between antiplatelet antibodies and neonatal thrombocytopenia. In their study, Samuels et al2 found that the incidence of neonatal thrombocytopenia in infants born to thrombocytopenic women with circulating platelet antibodies was higher than in infants born to women without such antibodies. However, this study was performed in a group of selected women with ITP or presumed ITP, and only circulating antibodies were searched for, and not with glycoprotein-specific assays.

Other findings of our study deserve comment. We found a high incidence of maternal thrombocytopenia (11.6%), compared with other studies.3 In the largest earlier series, Burrows and Kelton1 found an incidence of 6.6%, whereas in other studies higher percentages were found, such as 15% in a French study.21 When only platelet counts less than 100 x 109/L were considered, the incidence in our study was 1.1%, which is similar to that reported by Burrows et al1 (1.2%). The majority of cases of mild thrombocytopenia during pregnancy are incidental. In our study, 91.5% of women with platelet counts less than 150 x 109/L had incidental thrombocytopenia, compared with only 35% of women with platelet counts less than 75 x 109/L. Our results also indicate a relatively low prevalence of antiplatelet antibodies in women with confirmed thrombocytopenia, autoantibodies in particular being found in 6.7%. This agrees with the fact that the majority of cases of thrombocytopenia during pregnancy are of nonimmunologic origin. This is particularly true for women with platelet counts less than 75 x 109/L. None of these women had antiplatelet antibodies, but all had other causes of thrombocytopenia, mainly preeclampsia. Presence of autoantibodies in the control group could indicate either hidden autoimmune disorders or false-positive antiplatelet antibody test results.22 As in a previous report,3 only a limited percentage of women (7.5%) had postpartum persistent thrombocytopenia.

This study found that the monoclonal antibody–specific immobilization of platelet antigens assay does not predict risk of neonatal thrombocytopenia for infants born to thrombocytopenic women. A more predictive test for neonatal thrombocytopenia would be helpful, but the search for such a test might be of questionable value, not only because of the low incidence of neonatal thrombocytopenia, but also because of discordant platelet counts in twin gestations complicated by autoimmune thrombocytopenia.11 Nevertheless, the monoclonal antibody–specific immobilization of platelet antigens assay remains of value in cases of neonatal thrombocytopenia, especially when alloimmunization is suspected.18,23


   Footnotes
 
This collaborative work was partly funded by a grant from the Henri Dubois-Ferrière/Dinu Lipatti Foundation.

PII S0029-7844(98)00390-1

Received June 9, 1998. Received in revised form July 29, 1998. Accepted August 6, 1998.


   References
Top
Abstract
 Patients and Methods
 Results
 Discussion
 References
 
1. Burrows RF, Kelton JG. Fetal thrombocytopenia and its relation to maternal thrombocytopenia. N Engl J Med 1993;329:1463–6.[Abstract/Free Full Text]

2. Samuels P, Bussel JB, Braitman LE, Tomaski A, Druzin ML, Mennutti MT, et al. Estimation of the risk of thrombocytopenia in the offspring of pregnant women with presumed immune thrombocytopenic purpura. N Engl J Med 1990;323:229–35.[Abstract]

3. Burrows RF, Kelton JG. Thrombocytopenia at delivery. A prospective survey of 6715 deliveries. Am J Obstet Gynecol 1990;162: 731–4.[Medline]

4. Kaplan C, Daffos F, Forestier F, Tertian G, Catherine N, Pons JC, et al. Fetal platelet counts in thrombocytopenic pregnancy. Lancet 1990;336:979–82.[Medline]

5. Burrows RF, Kelton JG. Incidentally detected thrombocytopenia in healthy mothers and their infants. N Engl J Med 1988;319:142–5.[Abstract]

6. George JN, El-Harake MA, Raskob GE. Chronic idiopathic thrombocytopenic purpura. N Engl J Med 1994;331:1207–11.[Free Full Text]

7. Scioscia AL, Grannum PA, Copel JA, Hobbins JC. The use of percutaneous umbilical blood sampling in immune thrombocytopenic purpura. Am J Obstet Gynecol 1988;159:1066–8.[Medline]

8. Ghidini A, Sepulveda W, Lockwood CJ, Romero R. Complications of fetal blood sampling. Am J Obstet Gynecol 1993;168:1339–44.[Medline]

9. Paidas MJ, Berkowitz RL, Lynch L, Lockwood CJ, Lapinsky R, McFarland JG, et al. Alloimmune thrombocytopenia: Fetal and neonatal losses related to cordocentesis. Am J Obstet Gynecol 1995;172:475–9.[Medline]

10. Hohlfeld P, Forestier F, Kaplan C, Tissot JD, Daffos F. Fetal thrombocytopenia: A retrospective survey of 5,194 fetal blood samplings. Blood 1994;84:1851–6.[Abstract/Free Full Text]

11. Scott JR, Rote NS, Cruikshank DP. Antiplatelet antibodies and platelet counts in pregnancies complicated by autoimmune thrombocytopenic purpura. Am J Obstet Gynecol 1983;145:932–9.[Medline]

12. Hart D, Dunetz C, Nardi M, Porges RF, Weiss A, Karpatkin M. An epidemic of maternal thrombocytopenia associated with elevated antiplatelet antibody. Platelet count and antiplatelet antibody in 116 consecutive pregnancies: Relationship to neonatal platelet count. Am J Obstet Gynecol 1986;154:878–83.[Medline]

13. Matthews JH, Benjamin S, Gill DS, Smith NA. Pregnancy-associated thrombocytopenia: Definition, incidence and natural history. Acta Haematol 1990;84:24–9.[Medline]

14. Lescale KB, Eddleman KA, Cines DB, Samuels P, Lesser ML, McFarland JG, et al. Antiplatelet antibody testing in thrombocytopenic pregnant women. Am J Obstet Gynecol 1996;174:1014–8.[Medline]

15. Kiefel V, Santoso S, Weisheit M, Mueller-Eckhardt C. Monoclonal antibody-specific immobilization of platelet antigens (MAIPA): A new tool for the identification of platelet-reactive antibodies. Blood 1987;70:1722–6.[Abstract/Free Full Text]

16. Brighton TA, Evans S, Castaldi PA, Chestermann CN, Chong BH. Prospective evaluation of the clinical usefulness of an antigen-specific assay (MAIPA) in idiopathic thrombocytopenic purpura and other immune thrombocytopenias. Blood 1996;88:194–201.[Abstract/Free Full Text]

17. Boehlen F, Bulla O, de Moerloose P. Evaluation of a new antigen capture ELISA for detection and characterization of platelet alloantibodies. Thromb Res (in press).

18. de Moerloose P, Boehlen F, Extermann P, Hohlfeld P. Neonatal thrombocytopenia: Incidence and characterization of maternal antiplatelet antibodies by MAIPA assay. Br J Haematol 1998;100: 735–40.[Medline]

19. Clofent-Sanchez G, Lucas S, Laroche-Traineau J, Rispal P, Pellegrin JL, Nurden P, et al. Autoantibodies and anti-mouse antibodies in thrombocytopenic patients as assessed by different MAIPA assays. Br J Haematol 1996;95:153–60.[Medline]

20. de Moerloose P, Vogel JJ, Reber G. Anticardiolipin antibodies determination: Comparison of three ELISA assays. Clin Exp Rheumatol 1990;8:1–3.

21. Verdy E, Uzan S, Groupe de travail sur les thrombopénies maternelles et foetales. Plaquettes en cours de grossesse. Etiologie et moyens du diagnostic d’une thrombopénie maternelle. In: lère Journée parisienne obstétrico-pédiatrique. Paris: Doin, 1993:49–53.

22. Tchernia G, Morel-Kopp MC, Yvart J, Kaplan C. Neonatal thrombocytopenia and hidden maternal autoimmunity. Br J Haematol 1993;84:457–63.[Medline]

23. Dreyfus M, Kaplan C, Verdy E, Schlegel N, Durand-Zaleski I, Tchernia G, et al. Frequency of immune thrombocytopenia in newborns: A prospective study. Blood 1997;89:4402–6.[Abstract/Free Full Text]




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