ANNALS O F C L IN IC A L A N D LABORATORY S C IE N C E , Vol. 20, No. 3C opyright © 1990, In s titu te for Clinical Science, Inc.

Maternal Platelet Antibody Levels in Neonatal Isoimmune Thrombocytopenia

JOHN LAZARCHICK, M .D ., RENE RUSSELL, MLT,

and BRENDA HORN, MT

Departments o f Pathology/Laboratory Medicineand Pediatrics,

Medical University o f South Carolina, Charleston, SC 29425

ABSTRACT

A case of neonatal isoimmune thrombocytopenia (NIT) is described in which m aternal platelet antibody levels decreased during the course of pregnancy. It is suggested that if this disorder is clinically suspected, m aternal serum should be assessed for anti-platelet specificity using immunoblot analysis.

experience is presented with the use of a sensitive platelet antibody assay system and the suggestion th a t m on ito ring maternal levels of platelet antibody is not ideal.

Case R eport

In November 1987, a 27 yr G1 PO delivered a child with severe thrombocytopenia. Maternal plate­let count was normal. A post partum maternal serum sample was positive for the presence of anti-platelet antibodies up to a dilution of 1:20 of her serum, but no studies were performed to determine the platelet antigen specificity. In August 1989, at approximately 32 weeks gestation with her subsequent pregnancy, a serum sample was again analyzed for anti-platelet antibody reactivity. Although antibody could still be detected, the titer had decreased, now being positive in the neat sample only. Several anti-human leuko­cyte antigen (HLA) antibodies were detected in her serum including anti-B5, B35, B53 and B17. Approx­imately eight weeks later she delivered a child with marked thrombocytopenia, bu t her platelet count was again normal. A repeat platelet antibody assay this time showed a marked increase in her titer, being positive at a 1:40 dilution of her serum and in strength of reactivity. On the assumption that the cli-

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Introduction

N eonatal iso im m une th rom bocy to ­penia occurs in approxim ately one of every 2,000 births and is due to maternal IgG antibody directed against antigens present on the platelet of the child but absent on m aternal p latelets.1 Sensitiza­tion may occur relatively early in gesta­tion since it has been shown at PLA1 and Lek antigenic groups appear as early as 16 to 19 weeks of gestation.2,3

It has been suggested that changes in the level of m aternal antibody during the course of pregnancy may be useful in predicting the occurrence of isoimmune neonatal thrombocytopenia; however, no definitive studies are yet available. Our

Address rep rin t requests to: John Lazarchick, M .D ., D epartm ent of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425.

NEONATAL ISOIMMUNE THROMBOCYTOPENIA 201

nician was dealing with a case of isoimmune neonatal thrombocytopenia, the child received a transfusion of washed maternal platelets and had a successful out­come. There was no evidence of intracranial hemor­rhage and the child continues to do well.

Methods

An in d ire c t im m u n o flu o re sc e n c e platelet antibody assay* was performed as previously described.7 In brief, a 0.1 ml aliquot of thawed HLA type platelet pool at a final concentration of 250 X 109 per ml after washing was incubated with 0.01 ml of either normal control serum or patient serum or dilutions of each for one hour at room tem pera tu re . After centrifugation and repeated washings, the p latelet pellet was incubated with 0 .1 ml f lu o re sc e in -c o n ju g a te d goat (Fab')2 antihuman IgG, A, M antiserum for 15 m inutes at room tem peratu re . After repeated centrifugation and wash­ing, the platelet pellet was resuspended in phosphate-buffered saline to a final volume of two ml. The amount of fluo­rescein conjugate bound in each platelet suspension was th en m easured using flow cytometric analysis.

For this assay, region A on the spec­trum III was defined as consisting of all 255 fluorescence intensity channels. The intensity of fluorescence, expressed as the mean value or region A, is propor­tional to the amount of serum IgG, A, M bound to the p late le t m em brane. The m ean channel A read ing for the tes t serum plus p la te le t m ixture was then com pared to that of the control serum plus platelet mixture, and a ratio of rela­tive fluorescence was determ ined . A ratio of > 1 .3 of test serum/control serum is compatible with the presence of plate- let-directed antibody in the test serum.

Patient serum samples were obtained on th ree occasions: im m ediately post­partum in 1987, a t approxim ately 32 weeks gestation in 1989, and im m edi­

* Ortho Spectrum III flow cytometer.

ately post-partum at approximately 40 weeks gestation in 1989. The serum sam­ples were screened for the presence of anti-H LA antibodies using a panel of lymphocytes from 40 HLA-type normal donors.

P l a t e l e t P r e p a r a t io n / E l e c t r o p h o r e s is / I m m u n o b l o t t in g

P la te le t rich plasm a was p rep a re d from blood collected in ethylenediamine tetraacetic acid (EDTA) tubes by centrif­ugation at 150 g for 20 m inutes. The supernatant was then centrifuged at 1300 g for 10 minutes. The platelet button was th en resu sp e n d e d in R inger’s-ED TA containing 10 mM benzam idine H C L and 100 jJLg per ml of soybean trypsin inhibitor. After several washings, suffi­c ien t volum e was added to achieve a platelet concentration of 1 X 109 per |xl. To this final suspension was then added 1:40 volume of 40 percent Triton X-100. The platelet suspension was then incu­bated at 37°C for one hour and refriger­ated at 4°C overnight.

Sodium dodecyl sulfate po lyacry l­am ide gel electrophoresis was carried out as described by Laemmli using a five percent to 20 percent gradient gel as the separating gel and five p e rcen t poly­acrylamide as the stacking gel.6 Twenty- five |xl of platelet suspension (1 X 109 platelets) w ere m ixed w ith 25 (jlI of a solution containing 0.002 percent brom- p h e n o l b lu e , fo u r p e rc e n t so d iu m dodecyl sulfate and 0 .125M Tris, and 20 percent glycerol, pH 6.0 and incubated for five m inutes at 100°C prior to load­ing. Electrophoresis was carried out for 16 hours at 20 volts. The voltage was in c re ase d to 80V for two ad d itio n a l hours.

P roteins w ere transferred from the po lyacry lam ide gel to n itro ce llu lo se m em brane as described by Towbin et a l.10 The portion of the paper carrying th e m olecu lar w eights standards was

202 LAZARCHICK, RUSSELL, AND HORN

stained with 0.125 percen t Coomassie blue, and the rem ainder was cut into strips corresponding to the electrophore­sis lanes and placed in blocking buffer containing five percent non-fat dry milk in 0.01 M Tris, 0.17 M sodium chloride, pH 7.5.

After overnight incubation at room tem perature, the strips were extensively washed and then incubated with appro­priate antibody-containing preparations including 1:10 dilution of each of the patient’s serum samples as indicated or normal control serum. Mouse monoclo­nal anti-hum an GP I lia was diluted in 1:500 in Tris buffered saline. The strips w ere incubated at room tem peratu re overnight on a shaker bath. After wash­ing, a solution of peroxidase-conjugated goat antibody to e ither mouse IgG or hum an IgG was added to appropriate strips and the strips incubated for one hour at room tem perature. The strips were then developed using 4-chloral-l- naphthol as an indicator reagent.

Results

The anti-platelet antibody activity of the patient’s serum samples is shown on tab le I. The N ovem ber 1987 sam ple showed a significant antibody titer; how­ever, since the assay utilized a HLA-type platelet pool as target cells, no distinc­tion betw een anti-HLA or anti-platelet

T A B L E I

Maternal Serum Anti-platelet Reactivity

Sample Date MCH A Ratio* (Serum Dilution)

Neat 1:10 1:20 1:16011/87 --- 1.9 1.6 1.18/89 1-3 1.29 1.210/89 1.72 2.48 2.35 1.19

*Mean channel of region A ratio of the intensity of fluorescence of maternal serum plus target platelets to control serum plus target platelets. A value of > 1.3 is considered positive for the presence of anti-platelet antibodies.

specific antibody could be made. It is of interest to note that on the patient’s sec­ond sam ple (August 1989) th e re was actually a decrease in her antibody titer at a tim e when she was approximately 32 weeks gestation with her second p reg­nancy. Four specific anti-HLA antibodies w ere de tec tab le in h e r serum at this point. Eight weeks later, at the tim e of her delivery, the pa tien t’s serum anti­body had again increased in titer, now being strongly positive at a 1:40 serum dilution, and showed an increase inten­sity of reactiv ity w ith a M CH A ratio of 2:35.

Because of the unexpected throm bo­cytopenia in the newborn and the sud­den change in m aternal serum a n ti­p la te le t rea c tiv ity , all th re e se ru m sam ples w ere analyzed to d e te rm in e antibody specificity to normal platelet glycoproteins. These results are illus­trated in figure 1. Although not present on the Novem ber 1987 sample, there is a distinct band of reactivity with a 98 Kd glycoprotein noted in the August 1989 sam ple. The in tensity of this band is increased in the October 1989 sample. T he co n tro l lane using m ouse a n ti­hum an glycoprotein I l ia shows these bands to be the same. U nfortunately, platelet antigen typing could not be per­form ed on the m other to confirm h er PLA1 status.

Discussion

The diagnosis of neonatal isoimmune throm bocytopenia in this case would seem certain: the newborn was severely thrombocytopenic while maternal plate­let count was normal at all times. There was no indication of sepsis. M aternal antibodies w ere p resen t in increasing titer which reacted with a 98Kd platelet glycoprotein, i.e ., G PIIIa implying that she lacked an ep itope on this pro tein which resu lted in sensitization during pregnancy. Statistically, this is probably

A B C D E F

NEONATAL ISOIMMUNE THROMBOCYTOPENIA 203

F ig u r e 1. Transblot analysis following sodium dodecyl sulfate polyacrylamide gel electrophoresis of nor­mal control platelets. The electrophoretically separated platelet proteins were reacted with: (A) Normal serum; (B) Mouse anti-human platelet GPIIIa; (C ) Serum from a patient who had anti-PLA1 antibody; (D) Patient serum, 11/87; (E) Patient serum, 8/89; (F) Patient serum, 9/89.

an anti-PLA1 antibody; however, it is also known that the Yuk(Pen) antibody can also be associated with neonatal isoim­m une thrombocytopenia (NIT).5 It is also theoretically possible that the antibody developed to an as yet undefined platelet antigen group. Since platelets were not available for antigen typing from either the parents or the child, no definitive conclusion can be reached regarding the platelet antigen lacking on the maternal platelets.

This study, although only a single case, th u s suggests th a t m on ito ring m aternal an ti-p la te le t antibody titers, especially in our assay system which detects not only anti-platelet glycopro­tein bu t also anti-HLA antibodies, can be misleading. The authors were aware that this patient had several anti-HLA antibodies, including anti-B5, B35, B53, and B17. The HLA sensitization during pregnancy is relatively common; how­ever, such antibodies have only rarely been reported to cause NIT.9 The rela­tively low antibody titer on the August ’89 serum sample, compared to that of November ’87, was interpreted as indi­cating that m aternal sensitization was minimal at best.

It is known that platelet specific anti­gens are present early in gestation and

that if sensitization had occurred evi­dence of such sensitization, i.e., increas­ing antibody titers, would have been evi- d e n t by a p p ro x im a te ly 32 w ee k s gestation. O ur findings of decreasing titer is similar to report by Kaplan et al4 in a study of seven patients with NIT secondary to anti-PLAI antibody. It was shown that in two patients the titer had actually decreased during the course of pregnancy, and, in a th ird , no serum antibody was evident at any time. The basis for a decrease in tite r during a period of active sensitization is unclear. It is evident, however, from the immu- noblot analysis in our case that antibody activity against G PIIIa was evident in the August ’89 sample despite the low serum antibody activity determ ined by flow cytometric analysis. W hether or not increased sensitivity could be achieved by perform ing im m unoblot assays on suspected cases needs to be further stud­ied.

References

1. C o l e m a n , R. W ., H irsh, J., M a r d e r , B. J., and SALZMAN, E . W .: Hemostasis and Thrombosis. Basic Principals in Clinical Practice, 2nd ed. Philadelphia, J. B. Lippincott Co., 1982, pp. 476-480.

2 0 4 LAZARCHICK, RUSSELL, AND HORN

2. G r u e l , W. G ., Bo iz a r d , B ., D a f f o s , F., F o r ­e s t ie r , F ., C a e n , J . , and W a u t ie r , L . L . : Determ ination of platelet antigens and glyco­proteins in the human fetus. Blood 68:488-492,1986.

3. Ka pl a n , C ., Pa t e r e a u , C ., R e z n ik o f f -E t ie - v a n te , M . F., M u ll er , J. Y., D u m e z , W ., and Ke s s e l e r , A. : Antenatal P L A1 typing and the detection of G P Ilb-IIIa complex. Brit. J. Hae­matol. 60:586-591, 1984.

4. Ka pla n , C ., D a f f o s , F., F o r est ier , F., C ox , W. L ., Ly o n -C a e n , D . , D u pu y -M o n t b r u n , M. C ., and Sa l m o n , C .: Management of alloim- mune thrombocytopenia: Antenatal diagnosis and in utero transfusion of maternal platelets. Blood 72:340-343, 1988.

5. K u n ic k i , T., F a r ih a t a , K ., B u l l , B ., and N u g e n t , D.: The immunogenicity of platelet membrane glycoproteins. Transfus. Med. Rev. 1:21-33, 1987.

6. La e m m l i, U. K .: Cleavage of structural proteins

during the assembly of th e head of bacteria phase T4. Nature 227:680-685, 1970.

7. L a z a r c h ic k , J., J o n e s , T. J., R u s s e l l , R. J., and H a l l , S. A .: Platelet antibody detecting using frozen pooled human lymphocyte antigen- type p la te le ts as ta rg e t cells. D iagn. Clin. Immunol. 5:377-380, 1988.

8. Shibata , Y., M atsud a , I . , M iyaji, T., and Ic h i­kawa , Y.: Yuka, a new platelet antigen involved in two cases of neonatal alloimmune thrombocy­topenia. Vox Sang. 50:177-180, 1986.

9. Ste r n b a c h , M . S ., M a l e t t e , M ., N a d o n , F., and G u e n v in , R. M .: Severe alloimmune neo­natal thrombocytopenia due to specific HLA antibodies. Curr. Stud. Haematol. Blood Transf. 52:97-103, 1986.

10. T o w b in , H., S t a e h e l i n , T ., and G o r d o n , J.: E lec tro p h o res is tra n sfe r o f p ro te in s from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354, 1979.