Frequency of the HPA-1a antigen in pregnant women

Objective

The aim of the study is to determine incidence rate of Human Platelet Antigen (HPA-1a antigen) negative platelets in pregnants who applied to polyclinic and to research HPA-1a antibody existence in pregnants who were found as HPA-1a negative.

Methods

Two hundred forty pregnants are scanned in the study. HPA-1a was identified by using Enzyme-Linked Immunosorbent Assay (ELISA) method. Alloanticorps were researched in a HPA-1a negative case and HLA-DR identification was performed by polymerase chain reaction (PCR)-SSP method after DNA isolation.

Results

HPA-1a was found as negative only 1 case among 240 pregnants (0.4%). Anti-HPA-1a antibody level and HLA-DR52a result was found as negative in the pregnant found as negative in terms of HPA-1a antigen.

Conclusion

In this study, HPA-1a antigen negativity incidence rate was found as 0.4%. This rate is lower than expected level. Thus, checking newborns for platelet number instead of HPA-1a antigen and DR52a identification may be more effective. In order to determine real incidence rate of HPA-1a in pregnants in our society, a study covering a wider population is required.

Keywords

HPA-1a, neonatal alloimmune thrombocytopenia, HLA-DR52a

Introduction

Neonatal thrombocytopenia is seen at 1% of newborns (1). Though Human Platelet Antigen (HPA)-1a negativity is seen at a rate of 1/50, one alloimmune thrombocytopenia occurs in 5000 cases born in term (2). Maternal alloanticorps developed against antigens peculiar to platelets inherited from father pass placenta and causes thrombocytopenia by binding themselves to platelets. Intracerebral bleeding occurs in 10-20% of incurable thrombocytopenia cases. It is reported that 75% of intracerebral bleedings occur during intrauterine period (3). Fetal death appears in 10% of cases having intracerebral bleeding and neurological damage in 20% of them. In 50-60% of cases, most of cases can only be identified in newborn period due to alloimmunization development in first pregnancy (4-6). It is accepted that maternal immunization develops against HPA-1a approximately at a rate of 80% in general populations (7). HPA-1a alloimmunization possibility shows a strong association with the existence of human leukocyte antigen (HLA) class II DR-B3*0101(DR52a) in mother. Creating anti-HPA-1a of a mother having negative HPA-1a is controlled with HLA-DRB3*0101 allele.
There is no study in our country in which HPA-1a antigen frequency is researched on normal population or pregnants. The aim of this study is to establish HPA-1a negativity rate in pregnant women and to research the existence of anti-HPA-1a antibody and to determine its relation with DR52 genotype.

Methods

This study was performed on 240 pregnants who applied Polyclinic of the Department of Obstetrics and Gynecology, Akdeniz University consecutively. Blood samples were taken from cases for routine hemoglobin; they were informed about alloimmunization and after taking their consent, their blood samples were transferred into 2 ml of vacuumed tubes including EDTA and 10 ml of biochemical tubes.
Samples taken into full blood tubes were studied within 6 hours at Enzyme-Linked Immunosorbent Assay (ELISA); blood samples taken into biochemical tubes were first centrifuged and then kept at -80°C by transferring them into tubes. HPA-1a determination in pregnants was studied with ELISA method by using DiaMed Platelet HPA-1a identification test kit (cat.no.030011, DiaMed AG, Switzerland).
No pseudo-negativity was reported in case of the existence of enough platelet for this test. Anti-platelet antibody determination was transferred to the tube on both layers by pipette from 50µL healthy platelets and they were centrifuged for 10 minutes after 30-40 minutes of incubation. Then, DiaCell I-II-III cell mixture was transferred to both tubes by pipette about 50µL. They were centrifuged for 10 minutes and evaluated after 10 minutes of incubation.
If they were gathered on the top of both tubes, they were determined as positive; if they were gathered on the bottom of both tubes, they were determined as negative. For performing HLA-DR52 identification, DNA was isolated from full blood by using Puregene (D–5000),Gentra Systems, Switzerland) kit. HLA-DR identification was performed by using GenoVision Olerup SSP( Olerup SSP, Stockholm, Sweden) kit.

Results

As a result of HPA-1a antigen determination performed on 240 pregnants who applied Polyclinic of the Department of Obstetrics and Gynecology, Akdeniz University, HPA-1a antigen was found as negative only in one case. HPA-1a frequency rate was found as 0.4% among studied population.
Anti-platelet antibody existence was researched in pregnant found as negative in terms of HPA-1a antigen and it was found as negative.
Due to the relation between HPA-1a antigen and HLA-DR52 in neonatal alloimmunization, HLA-DR identification was done after DNA isolation in pregnant having negative HPA-1a and HLA-DR52 was found as negative as HLA-DR51 and HLA-DR52 were found as positive.

Discussion

HPA antigen frequency shows difference as to races. According to the literature, HPA-1a (%77,3), HPA-3a (%3,5), and HPA-5b (3.5%) are clinically antigens as the most important isoimmune thrombocytopenia reasons(5). Except those, approximately 16 different antigens are defined. There is no data in our country reporting HPA-1a antigen frequency. We found negativity frequency as 0.4% in our study group. This rate is between 1.6% and 2.5% in the literature (8) and it changes as to races and ethnicities. We decided that reaching a rate lower than the rates reported in the literature may be resulted from the minority of our cases. Women having negative HPA-1a constitute 2% of all pregnants and it is observed that antibody was developed in 11% of these cases (9).
The most important factor affecting the development of Anti-HPA-1a antibody is HLA class II DR52a (DRB3*0101). It can be said that antibody shall not be developed in 99% of women having negative HPA-1 and not having HLA-DR52a. It was reported that antibody was developed in 35% of women having HLA-DR52a and negative HPA-1a; however HLA-DR52a was not suggested as a routine scanning test (8). HPA-1a identification and determining real frequency of alloantibody scanning enable to determine annual affected case number, to develop health policy for preventive treatment to prevent problems related with thrombocytopenia in first pregnancies (10). Consequently, HPA-1a identification and scanning alloantibody level at routine antenatal clinics may increase unnecessary invasive initiatives and also it is not economical for our country. Instead of that, it is reported that scanning newborn platelet number would be appropriate (11).
Though alloimmune thrombocytopenia is rare, its morbidity and mortality are severe (6, 12). Even though there are publications reporting that finding anti-HPA-1a titration higher than 1/32 at antenatal observation shows severe thrombocytopenia; this test is not suggested for using routine observation (1). It is suggested to perform obstetric ultrasonography bi-weekly for cases having positive HPA antigen.
Previous fetus history is very important antenatal treatment of alloimmune thrombocytopenia. If intracerebral bleeding was observed in previous fetus, treatment can be initiated at 12th gestational week with maternal immunoglobulin (IVIG) and prednisolone (13, 14). If thrombocytopenia is found in cordocentesis performed in 24th gestational weeks in cases having medical treatment, the treatment is carried on with weekly intrauterine platelet transfusion. Fetal loss rate related with transfusion was reported as about 5.5-8.3%. Since these cases generally deliver on 32nd week, they have premature problems more than those having only medical treatments (15).

Conclusion

Consequently, even though HPA-1a antigen negativity frequency was found as 0.4% in our study; it is seen that a study is required covering a wider population for making a certain decision in our country about HPA-1a antigen frequency in our society, its relation with HLA-DR52a and alloantibody development.

References

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