Platelet antibody detection by flow cytometry: an effective method to evaluate and give transfusional support in platelet refractoriness

Abstract Background: Neonatal alloimmune thrombocytopenia (NAT) is caused by maternal alloantibodies that cross the placenta and cause increased clearance of fetal platelets. The alloantibodies are directed to incompatible human platelet antigens (HPA) inherited from the father and expressed on fetal platelet glycoproteins. Most cases of NAT are caused by HPA-1a, -5a, -5b, -15a, or -15b. Some are associated with immunization to a low frequency antigen found only in the paternal lineage. We investigated the incidence of the low frequency antigen HPA-9b in a population with suspected NAT, and compared the frequency to that found in a normal control population. HPA-9b is caused by a Val837Met polymorphism on alpha-IIb and is expressed only in a rare subset of individuals with a corresponding serine at position 843 that defines the HPA-3b antigen. Methods: We used PCR-SSP for HPA-9b genotyping. Antibody studies were performed by incubating maternal serum with intact platelets using flow cytometry, monoclonal antigen capture enzyme immunoassay, and radioimmunoprecipitation. Results: Of 294 normal control blood donors, 4 typed positive for HPA-9b, yielding a gene frequency of 0.0068. However, if only HPA-3b positive donor controls are considered (n=177) the gene frequency for HPA-9b is 0.011. Archived DNA from HPA-3b positive (HPA-3a3b or -3b3b) fathers of children with NAT were tested for HPA-9b. Of 94 fathers, 3 (3.2%) typed HPA-9a9b, yielding a gene frequency for HPA-9b of 0.016, not significantly different from the frequency for HPA-9b found in the HPA-3b-positive healthy controls (p>0.1). Similarily, when DNA from HPA-3b positive mothers was tested in tandem, 3 of 60 were positive for HPA-9b, yielding a gene frequency of 0.026. Of the 3 cases of suspected NAT (platelet count range 15,000 to 63,000) in which the mother and father were incompatible for HPA-9b, 2 had no other platelet antigen incompatibility, and one was incompatible for HPA-15b. None of the 3 maternal sera was positive for anti-platelet reactivity using radioimmunoprecipitation and paternal platelets as a target, and no anti-platelet antibody was detected either by flow cytometry or by GPIIb/IIIa antigen capture immunoassay with platelets from a HPA-9a9b donor. Interpretation: These results support the findings that HPA-9b is distributed in the general population at a frequency higher than initially suspected. However, we also found that the frequency of HPA-9b in fathers of children with suspected NAT was similar to that in the normal control population when only HPA-3b-positive (HPA-3a3b or -3b3b) targets were analyzed. Therefore, in the absence of a positive anti-platelet antibody, the significance of a maternal/paternal incompatibility for HPA-9b alone is not predictive of an increased risk of NAT.

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AET-treated platelets: their usefulness for platelet antibody detection and an examination of their altered sensitivity to immune lysis

Blood ◽

10.1182/blood.v54.5.1101.bloodjournal5451101 ◽

1979 ◽

Vol 54 (5) ◽

pp. 1101-1108

Author(s):

PL Cimo ◽

SA Gerber

Keyword(s):

Blood Cells ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Antibody Detection ◽

Enhanced Sensitivity ◽

Platelet Antibodies ◽

Different Types ◽

Drug Dependent ◽

Altered Sensitivity ◽

Bernard Soulier Syndrome ◽

Platelet Antibody

2-Aminoethylisothiouronium bromide (AET) increases the sensitivity of blood cells to complement-mediated immune lysis. We compared the sensitivities of untreated or AET-treated platelets to immune lysis induced by different types of platelet antibody in the 51Cr platelet lysis test. AET platelets were 8–16 times more sensitive to autoantibody and alloantibody, but 8–16 times less sensitive to drug- dependent antibody. AET-platelets bound similar amounts of alloantibody but less drug-dependent antibody, and they lysed at higher complement dilutions than did untreated platelets. AET-platelets detected 10 of 25 autoantibodies, 9 of 9 alloantibodies, and 5 of 8 drug-dependent antibodies. Untreated platelets detected 1 of 25, 6 of 9, and 7 of 8 of these respective platelet antibodies. The use of AET-platelets in the 51Cr platelet lysis test increases its sensitivity for detecting non- drug-dependent platelet antibodies. AET-platelets resemble paroxysmal nocturnal hemoglobinuria (PNH) platelets in their enhanced sensitivity to complement-mediated lysis. They differ from PNH platelets in their insensitivity to immune lysis induced by drug-dependent antibodies and, in this respect, are similar to Bernard-Soulier syndrome platelets.

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Progress and development of platelet antibody detection

Transfusion and Apheresis Science ◽

10.1016/j.transci.2019.102705 ◽

2020 ◽

Vol 59 (1) ◽

pp. 102705 ◽

Cited By ~ 1

Author(s):

L. Porcelijn ◽

E. Huiskes ◽

M. de Haas

Keyword(s):

Antibody Detection ◽

Platelet Antibody

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Identification of anti-HPA-1a allo-antibodies using IgG platelet antibody detection and crossmatch system assay with Galileo Echo

Platelets ◽

10.3109/09537104.2014.945409 ◽

2014 ◽

Vol 26 (5) ◽

pp. 421-424 ◽

Cited By ~ 2

Author(s):

Julie Di Cristofaro ◽

Coralie Frassati ◽

Rolande Montagnie ◽

Agnes Basire ◽

Yves Merieux ◽

...

Keyword(s):

Antibody Detection ◽

Platelet Antibody

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Platelet Antibody Detection

White cells and platelets in blood transfusion ◽

10.1007/978-1-4613-2089-0_24 ◽

1987 ◽

pp. 261-267 ◽

Cited By ~ 1

Author(s):

A. E. G. Kr. von dem Borne ◽

W. H. Ouwehand ◽

C. M. van Dalen

Keyword(s):

Antibody Detection ◽

Platelet Antibody

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Random donor platelet crossmatching: Comparison of four platelet antibody detection methods

American Journal of Hematology ◽

10.1002/ajh.2830280102 ◽

1988 ◽

Vol 28 (1) ◽

pp. 1-7 ◽

Cited By ~ 17

Author(s):

J. Freedman ◽

M. B. Garvey ◽

A. Hornstein ◽

Z. Salomon De Friedberg ◽

V. Blanchette

Keyword(s):

Detection Methods ◽

Antibody Detection ◽

Platelet Antibody

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AET-treated platelets: their usefulness for platelet antibody detection and an examination of their altered sensitivity to immune lysis

Blood ◽

10.1182/blood.v54.5.1101.1101 ◽

1979 ◽

Vol 54 (5) ◽

pp. 1101-1108 ◽

Cited By ~ 5

Author(s):

PL Cimo ◽

SA Gerber

Keyword(s):

Blood Cells ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Antibody Detection ◽

Enhanced Sensitivity ◽

Platelet Antibodies ◽

Different Types ◽

Drug Dependent ◽

Altered Sensitivity ◽

Bernard Soulier Syndrome ◽

Platelet Antibody

Abstract 2-Aminoethylisothiouronium bromide (AET) increases the sensitivity of blood cells to complement-mediated immune lysis. We compared the sensitivities of untreated or AET-treated platelets to immune lysis induced by different types of platelet antibody in the 51Cr platelet lysis test. AET platelets were 8–16 times more sensitive to autoantibody and alloantibody, but 8–16 times less sensitive to drug- dependent antibody. AET-platelets bound similar amounts of alloantibody but less drug-dependent antibody, and they lysed at higher complement dilutions than did untreated platelets. AET-platelets detected 10 of 25 autoantibodies, 9 of 9 alloantibodies, and 5 of 8 drug-dependent antibodies. Untreated platelets detected 1 of 25, 6 of 9, and 7 of 8 of these respective platelet antibodies. The use of AET-platelets in the 51Cr platelet lysis test increases its sensitivity for detecting non- drug-dependent platelet antibodies. AET-platelets resemble paroxysmal nocturnal hemoglobinuria (PNH) platelets in their enhanced sensitivity to complement-mediated lysis. They differ from PNH platelets in their insensitivity to immune lysis induced by drug-dependent antibodies and, in this respect, are similar to Bernard-Soulier syndrome platelets.

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Functional Consequences of Cellular Interactions with Heparin-PF4 Antibodies in Heparin-Induced Thrombocytopenia.

Blood ◽

10.1182/blood.v104.11.3498.3498 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3498-3498 ◽

Cited By ~ 1

Author(s):

Sarfraz Ahmad ◽

Ali Amirkhosravi ◽

Jamie M. Walker ◽

Hina Desai ◽

Mildred Amaya ◽

...

Keyword(s):

Flow Cytometry ◽

Endothelial Cells ◽

Platelet Activation ◽

Procoagulant Activity ◽

Antibody Detection ◽

Cellular Interactions ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Functional Consequences ◽

Clot Time

Abstract Heparin-induced thrombocytopenia (HIT) is a potentially devastating complication of heparin therapy. The condition is due to formation of antibodies to the heparin-platelet factor 4 (HPF4) complex that activate platelets and/or endothelial cells by binding to FcγIIa (CD32) receptors. It is not clear why only a sub-population of patients that form HPF4 antibodies also develops clinical HIT, and why only a fraction of those develop thrombosis. We hypothesized that some functionally active HPF4 antibodies (i.e. that activate platelets) may also activate leukocytes and endothelial cells by upregulating procoagulant activity; a property not shared by non-functional HPF4 antibodies. Citrated plasmas were obtained from patients clinically suspected of HIT. A commercially available ELISA (GTI, Brookfield, WI) combined with a 14C-serotonin release assay (SRA) was used to stratify patient plasmas into functional (F) and non-functional (NF) HPF4 antibody groups. Pooled normal plasma (NP) served as negative control. Some (n=20) GTI-ELISA positive samples (both F and NF) were also tested by flow cytometry using HPF4 coated microspheres (DiaMed-Caribbean, Inc., Miami, FL) for immunological antibody detection, and fluorescent annexin V binding to detect functional activity. The ability of HPF4 antibodies to cause platelet-monocyte aggregate (conjugate) formation was evaluated by two-color flow cytometry. The functional consequences of HPF4 antibody interaction with EaHy926 hybridoma endothelial cells were studied in the presence/absence of washed platelets and therapeutic heparin levels. Cell surface procoagulant activity (PCA) was measured by a single-stage clotting assay. Approximately 25% of ELISA positive plasmas were also positive by SRA. Although most ELISA positive samples were strongly positive in the microsphere flow cytometry assay (5–20 fold increase in mean fluorescence intensity [MFI] compared to NP) there was no direct correlation between the ELISA OD values and MFI. In the functional flow cytometric assay, not all the SRA positive samples were able to cause platelet activation. Platelet-monocyte aggregate formation in the presence of functional HPF4 antibodies was increased by 2–6 fold (MFI increase 50–300%). This was significantly higher than seen with the NF (p<0.05, n=6) or NP (p<0.01, n=6) plasma pools. Exposure of confluent EaHy926 cells to functional HPF4 antibodies and washed platelets caused a significant and time-dependent increase (20–35%) in cellular PCA as evidenced by the shortening of plasma clot time (235±15 vs. 330±12 sec, p<0.05). The clot time was not significantly different in samples incubated with NP or with HPF4 positive samples incubated without platelets. These observations suggest that in addition to platelet activation, a subset of functional HPF4 antibodies can cause platelet-monocyte aggregation and platelet-dependent endothelial cell interaction. This may lead to upregulation of cellular PCA (tissue factor) and thereby contribute to the pathogenesis of HIT. Non-functional HPF4 antibodies, even at comparable titer, appear to be devoid of such cellular effects. A prospective study to relate these observations to clinical findings in HIT patients is currently underway.

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