|Year : 2016 | Volume
| Issue : 1 | Page : 27-30
Levels of alloantibodies to human platelet antigen in relation to blood dyscrasia occurring at various trimesters of pregnancy
EO Akanni1, AO Olayanju BMLS, MSc., FMLSCN 2, RY Akele1, AJ Olayanju2, ED Ezigbo3
1 Department of Biomedical Science, Ladoke Akintola University of Technology, Osogbo, Osun State, Nigeria
2 Department of Medical Laboratory Science, College of Medicine and Health Sciences, Afe Babalola University, Ado Ekiti, Nigeria
3 Department of Medical Laboratory Science, University of Nigeria, Enugu Campus, Enugu State, Nigeria
|Date of Submission||17-Aug-2015|
|Date of Acceptance||13-Sep-2015|
|Date of Web Publication||10-Mar-2016|
A O Olayanju
Department of Medical Laboratory Science, PMB 5454, Afe Babalola University, Ado-Ekiti, Ekiti State
Source of Support: None, Conflict of Interest: None
Context Thrombocytopenia is major complication in complicated and uncomplicated pregnancies.
Aims The aim of this study was to determine the incidence of thrombocytopenia in pregnancy and determine possible immune thrombocytopenia correlates with blood dyscrasia involving other cellular components of blood.
Settings and design The study was an institution-based cross sectional study.
Participants and methods The full blood count of 200 women attending an antenatal center was determined. Sera of 73 (36.5%) thrombocytopenic women defined by a platelet count less than 80×10 9 /l and inadequate platelet in the peripheral blood film were evaluated for circulating antiplatelet antibody directed against platelet membrane glycoprotein GPIIb/llla.
Statistical analysis Pearson and Kendall's tau-b correlation analysis was carried out on data using the SPSS (PASW version 18) statistical package.
Results The antibody was detected in two (3%), 13 (17%), and 44 (60%) of the subset of patients in the first, second, and third trimester, respectively. Fourteen (20%) of the women had no detectable antiplatelet antibody. The idiopathic immune thrombocytopenia observed in the patients was associated with anemia and leukopenia in four (9%) patients in the third trimester of pregnancy. A significant correlation was observed between thrombocytopenia and the red cell count, hemoglobin concentration, and packed cell volume of the women. Irregular attendance at an antenatal clinic and nonutilization of hematinic were evident in groups of thrombocytopenic and anemic women. There was a negative correlation between the occurrence of antiplatelet antibody and the severity of thrombocytopenia.
Conclusion There is a need to distinguish immune thrombocytopenia from gestational thrombocytopenia and assess other blood parameters in pregnancy for a proper clinical intervention.
Keywords: alloantibodies, human platelet antigen, thrombocytopenia, glycoprotein GPIIb/llla
|How to cite this article:|
Akanni E O, Olayanju A O, Akele R Y, Olayanju A J, Ezigbo E D. Levels of alloantibodies to human platelet antigen in relation to blood dyscrasia occurring at various trimesters of pregnancy. Egypt J Haematol 2016;41:27-30
|How to cite this URL:|
Akanni E O, Olayanju A O, Akele R Y, Olayanju A J, Ezigbo E D. Levels of alloantibodies to human platelet antigen in relation to blood dyscrasia occurring at various trimesters of pregnancy. Egypt J Haematol [serial online] 2016 [cited 2019 Dec 15];41:27-30. Available from: http://www.ehj.eg.net/text.asp?2016/41/1/27/178477
| Introduction|| |
Immune thrombocytopenia (ITP), which occurs in up to 10% of all pregnancies, is one of the thrombocytopenic disorders that may complicate pregnancy and its management. ITP is more likely to present with severe thrombocytopenia earlier on in pregnancy compared with gestational thrombocytopenia  .
Recent studies indicate that, besides the accelerated clearance of platelets coated by IgG antiplatelet autoantibodies, several other mechanisms contribute toward the pathogenesis of ITP, including reduced platelet production , . In such cases, the thrombocytopenia may be associated with other blood dyscrasia, with qualitative or quantitative differences in erythrocytes or leukocytes. Any effective clinical intervention must be targeted at ensuring that all the cell lines revert to physiological limits. Hence, laboratory monitoring of pregnant women should include the evaluation of all blood cells that are important markers of health.
Pregnant women with thrombocytopenia have a higher risk of bleeding excessively during or after childbirth, especially during labor, cesarean section, or other surgical intervention during pregnancy, or in the puerperium. Such bleeding complications are more likely when the platelet count is less than 50 × 10 9 /l (Gill and Kelton  ). In addition, up to 10% of infants born to thrombocytopenic women develop more severe intracranial hemorrhage or thrombocytopenia as low as 20 000/μl occurring in ~4% of infants  .
The diagnosis of ITP in pregnancy is complicated by a wide differential diagnosis that includes several other disorders that can cause thrombocytopenia (Evi S and Keith RM, 2010)  . Laboratory investigation of the pregnant patient with suspected ITP is not concluded from the platelet counts results alone  . It should include the measurement of autoantibodies and alloantibodies to platelet glycoproteins ,, . A complete blood count and examination of the peripheral blood film is essential to exclude not only pseudothrombocytopenia, but other thrombocytopenic disorders such as thrombotic thrombocytopenia purpurae or pre-eclampsia, in which the peripheral blood film may show increased numbers of fragmented red cells  .
The association of ITP with autoimmune cytopenias has been less frequently described, although some reports exist in the literature on the coexistence of pernicious anemia with autoimmune hemolytic anemia or idiopathic thrombocytopenia. Although iron deficiency is more common in Nigerian pregnant women compared with folate deficiency, in patients with iron deficiency anemia, platelets counts are almost invariably normal or increased  . According to their report, associated thrombocytopenia is rare, especially in adults.
| Participants and methods|| |
A cross sectional study was carried out to determine the prevalence of antiplatelet antibodies among pregnant women attending the antenatal clinic at the state government hospital, Osogbo. The sample size for the study was calculated using the following formula , :
where n = minimum sample size, Z = standard normal variance = 1.96 at a 95% confidence interval, d = absolute SE = 0.05, and p = prevalence = 10%.
Pearson and Kendall's tau-b correlation analysis was carried out on data using the SPSS (SPSS Inc., 233 South Wacker Drive, 11 th Floor, Chicago, IL) statistical package.
The ethics and research committee of Ladoke Akintola University Teaching Hospital, Ogbomoso, approved the experimental protocols.
Sample collection and handling
A blood specimen was withdrawn from the antecubital vein using a dry sterile disposable syringe and needle. Blood (5 ml) was collected from each participant and dispensed into an EDTA anticoagulant bottle. The sample was kept at room temperature for a manual platelet count, which was performed within 2 h of collection  . An automated full blood count was carried out on the sample, after which the plasma was separated and kept frozen at −22°C for antiplatelet antibody analysis.
Platelet count procedure
One in 20 dilution of blood was prepared by 0.02 ml of blood to 0.38 ml of diluting fluid in a test tube, the tube was tightly covered, and the content of the tube was mixed for 1 min; it was allowed to stand for 10 min for complete hemolysis of red blood cells. The cover slip was fixed on the counting chamber and charged with a pasteur pipette after thorough mixing; the chamber was placed in a Petri dish More Details containing a piece of moist filter paper and covered for 20 min to allow the cells to settle and to avoid dryness and a ×40 objective was used to count the cells present in the five small squares (measuring 0.04 mm).
Calculation from the first principle:
Length of each square = 0.2 mm.
Breadth of each square = 0.2 m.
Area = l × B = 0.2 × 0.2 = 0.04.
Area = 0.04 mm 2 .
Depth = 0.01 mm.
Volume = 0.04 mm 3 .
Volume of 5 square counted = 5 × 0.04 × 0.02 m 3 .
Number of platelets counted in the 5 square = N.
0.02 m 3 = N, 1 m 3 = N/0.02.
Total number of platelets counted = 1 m 3 = N/0.02 × dilution factor.
Dilution factor is 20.
20N/0.02 = 2000/N = 1000N.
Total platelet count = Number of platelet counted×1000.
Normal value of platelet = 150-450 × 10 9 /l.
An Platelet Antibody (APAB), ELISA kit (MyBioSource Inc., P.O. Box 3308, San Diego, CA 92195-3308, USA) was used for the detection of antiplatelet antibody, the blood was centrifuged for 20 min at 3000 rpm, and the plasma was carefully harvested and stored in aliquot at –20°C until use.
When a known antigen is attached to a solid phase (a microtiter well), it is reacted with the test sample, which may contain the antibody. The test is incubated, followed by a wash, and an enzyme-labeled antihuman globulin is reacted with the antibody in the test sample that has attached to the antigen on the solid phase. After incubation, the uncombined labeled antihuman globulin is washed off. Retention of enzyme on the surface is detected by the addition of a substrate that changes color after incubation. The reaction is stopped by changing the pH with an acid. The concentration of antibody in the test sample bound by antigen on the solid surface is proportional to the intensity of the color.
Assay procedure for antiplatelet antibody
Wash solution - one volume of wash solution was diluted in 19 volumes of deionized water.
Standard wells, sample wells, and blank (control) wells were set; 50 μl of standard was added to each standard well, 50 μl of sample was added to sample well, and 50 μl of sample diluent was added to each blank/control well. Horseradish peroxidase-conjugate reagent of 100 μl was added to each well, covered with an adhesive strip, and incubated for 60 min at 37°C. The microtiter plate was washed with an automatic washer by delivering and aspirating five times. A 50 μl of chromogen solution A and 50 μl of chromogen solution B were added to each well. It was gently mixed, protected from light, and incubated for 15 min at 37°C. Stop solution of 50 μl of was added to each well. The color in the well changed from blue to yellow. The optical density was read at 450 nm using a microelisa strip-plate reader within 15 min.
Standard operating principles and procedure for the automated full blood count (Sysmex KX-21N™ Automated Hematology Analyzer, Sysmex America, Inc., Lincolnshire, Illinois)
Whole blood was aspirated from the sample probe into the sample rotor value and 6 μl of blood measured by the sample rotor value was transferred to the WBC transducer chamber along with 1.994 ml of diluents. At the same time, 1.0 ml of WBC fluid was transferred into the chamber. Hemoglobin lysate was added to prepare a 1 : 500 dilution sample when the solution was made to react in this condition for ~10 s; RBC was hemolyzed and platelets shrank with the WBC membrane held intact. At the same time, hemoglobin was converted into red-colored methemoglobin. Of the diluted/hemolyzed sample in the WBC transducer chamber, ~1.0 ml was transferred to the hemoglobin flow cells.
A 500 μl of sample in the WBC transducer was aspirated through the aperture. The pulse of the blood cells when passing through the aperture was counted using the DC detection method.
In the hemoglobin flow cell, a 555 nm wave length beam radiating from the light-emitting diode was applied to the sample in the hemoglobin flow cell. The concentration of this absorbance was compared with that of the diluents alone that was measured before the addition of the sample, thereby calculating hemoglobin.
| Discussion|| |
This study describes the levels of alloantibodies to human platelet antigens and the relationship of these antibodies with other cellular elements of blood in various trimesters of thrombocytopenic pregnant women.
In all, 36.5% of pregnant women in this study had a low platelet count defined by a platelet count less than 80 × 10 9 /l and inadequate platelet in the peripheral blood film. The thrombocytopenia observed was associated significantly with an increased titer of antiplatelet antibody ,,, .
This immunological complication was less frequent (3%) in the first trimester of pregnancy, but occurred in 17 and 60% of pregnant women in the second  and third trimesters of pregnancy, which may be because of pre-eclampsia , .
The absence of demonstrable antiplatelet antibody in 20% of the women shows that the low platelet count may result from nonimmunological causes such as gestational thrombocytopenia or HELLP (hemolysis, elevated liver enzymes, low platelet) syndrome  . In a study on the clinicohematological features of ITP in adults to analyze the association of autoimmune disorders with ITP in Pakistani patients, of the 44 ITP patients, different hematological parameters were analyzed in the patients. There was no significant difference in hemoglobin and the total leukocyte count in patients with systemic lupus erythematosus and rheumatoid arthritis compared with the entire cohort  .
Our study found a statistically significant association between ITP and the variations observed in other cell lines ([Figure 1], [Table 1], [Table 2], [Table 3], [Table 4]).
|Figure 1 Mean ± SD of the hematological parameters of thrombocytopenic pregnant women in relation to the seroprevalence of antiplatelet antibodies. APab, antiplatelet antibody; Hb, hemoglobin; PCV, packed cell volume; PLT, platelet; RBC, red blood cell; WBC, white blood cell.|
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|Table 1 Correlation of platelet count and other hematological parameters of normal individuals|
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|Table 2 Correlation of antiplatelet antibody and other hematological parameters of normal individuals|
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|Table 3 Correlation of platelet count and other hematological parameters in thrombocytopenic patients|
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|Table 4 Correlation of antiplatelet antibody and other hematological parameters in thrombocytopenic patients|
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| Conclusion|| |
There is a low level of antihuman platelet antibody in the first trimester of pregnancy among black women. The antibody increased relative to the age of pregnancy and was present in high titers in the third trimester of pregnancy. There was an association between thrombocytopenia and anemia in pregnant black women.
The authors acknowledge the cooperation of patients, staff and students of the Department of Medical Laboratory Science, Afe Babalola University and the Department of Biomedical Science, Ladoke Akintola University of Technology, Ogbomoso.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]