Platelet Suspension Immunofluorescence Research Articles

Clinical significance of positive platelet immunofluorescence assay in adult immune thrombocytopenia.

Immune thrombocytopenia (ITP) is a relatively common hematologic disorder manifested by low platelet count due to immune-mediated platelet destruction and/or suppression of platelet production. This study aim was to evaluate characteristics and clinical presentation of adult patients with ITP and exploring the clinical value of platelet antibodies assay in proposed cases in Iranian population. In this prospective case series 46 adult patients with ITP and platelet count of<100×10(9)/L, referred to the Taleghani Medical Center, Tehran, Iran between 2007 and 2009 were evaluated. There were 26 females and 20 males (1.3:1) with mean age of 38.9±19.7years. The platelet autoantibodies were measured by means of indirect platelet suspension immunofluorescence test. According to our results, 7 patients (15.2%) displayed a positive platelet antibody assay. There was a significant negative correlation between platelet count and antibody level (r=-0 0.59; p<0.001). Additionally, a positive correlation between platelet count and patients' age (r=0.302; p=0.042) was detected. 20 patients (56.5%) were symptomatic at presentation and the most common bleeding signs were petechia, purpura and epistaxis. Results indicated no significant correlation between increased platelet antibody level and bleeding manifestations except for hematuria (r=0.435; p=0.02) and epistaxis (r=0.382; p=0.015). Disclosure of platelet autoantibodies and the consequential thrombocytopenia associated to some extent but not completely with the propensity to bleed which necessitates more factors to be evaluated.

Antigen expression of frozen platelets

Platelet antigens of platelet samples from 36 donors, frozen for different intervals, were evaluated by the platelet suspension immunofluorescence test (PSIFT). A, B, PLA1(HPA-1a) and various HLA antigens were tested by their corresponding antisera. The antigen could be detected in almost all the samples after one month of freezing. After 3 and 6 months, the platelet antigens could only be detected in 29.2% and 3.7% of the samples, respectively. There was no difference in decay of antigen expression among A, B, PLA1 and HLA antigens. When compared with the freshly prepared platelets, frozen platelets presented stronger antigen expression after 2 to 4 weeks of storage. This may suggest that the frozen platelets could be used for platelet crossmatching procedures without loss of their antigenicity within one month.

Platelet autoantibodies and lupus-associated thrombocytopenia.

To characterize the antigenic targets of anti-platelet antibodies (APA) found in systemic lupus erythematosus (SLE)-associated thrombocytopenia, 48 patients with immune thrombocytopenia and SLE were compared with 20 patients with SLE who had never been thrombocytopenic. Both cases and controls were tested for circulating APA by an indirect platelet suspension immunofluorescence assay (PSIIFT) and by indirect monoclonal antibody specific immobilization of platelet antigens (MAIPA). A direct platelet suspension immunofluorescence assay (PSIFT) was also used for antibodies bound to platelets in vivo in thrombocytopenic patients; 13 of them with high titres of platelets-bound APA were investigated by direct and indirect MAIPA and platelet eluate analysis. Circulating APA were detected by PSIIFT in 88% of cases and 55% of controls (P = 0.0066) and platelet-bound antibodies were detected by PSIFT in 90% of cases. Indirect MAIPA detected specific APA (mainly directed against GpIIbIIIa) in 36% of cases and only 5% of the controls (P = 0.0076). Nine out of the 13 fully investigated thrombocytopenic patients (69%) had a positive direct MAIPA and/or APA detected in platelet eluates. In conclusion, the production of specific anti-platelet autoantibodies, mainly directed against GpIIb/IIIa, and their binding to platelet membrane plays an important role in the pathogenesis of SLE-associated thrombocytopenia.

Neonatal alloimmune thrombocytopenia in the Irish population: a discrepancy between observed and expected cases.

To estimate the rate of detection of neonatal alloimmune thrombocytopenia (NAITP) in the Irish population, to investigate clinical presentation and outcome in affected infants, and to determine the extent, if any, to which this condition is underdiagnosed at present. Cases were collected in a retrospective fashion from a review of platelet serology laboratory records from January 1992 to December 2000. Clinical data were obtained from hospital records. Testing for maternal antiplatelet antibody was by one or more of the following: the platelet suspension immunofluorescence test, a commercial antigen capture enzyme linked immunosorbent assay (GTI-PakPlus), and the monoclonal antibody immobilisation of platelet antigens assay. Platelet antigen typing was by the polymerase chain reaction technique with sequence specific primers. Twenty seven serologically verified cases of NAITP were identified in 18 families. Maternal antibody to human platelet antigen 1a accounted for 25 of the 27 confirmed cases. Twenty one of 26 infants were born with severe thrombocytopenia. Nineteen of 27 infants had bleeding manifestations at birth. Petechiae and bruising were most commonly observed (n = 17). There were no documented cases of intracranial haemorrhage in this group but systematic cranial ultrasound was not performed. Screening studies in predominantly white populations have estimated the incidence of NAITP to be between 1 in 1000 and 1 in 2000 live births. With 50 000 births each year in Ireland, these results give a clinical detection rate for NAITP of just 1 case in 16 500 live births, strongly suggesting that NAITP is currently underdiagnosed. Antenatal screening to detect women at risk of having babies with NAITP is now scientifically feasible and should be considered.

INCIDENTAL DIAGNOSIS AND TEMPESTIVE THERAPY IN A CASE OF NEONATAL ALLOIMMUNE THROMBOCYTOPENIA DUE TO ANTI-HPA-5b

Neonatal alloimmune thrombocytopenia (NAIT) is usually attributable to HPA-1a antibodies. The authors report a case of incidentally diagnosed thrombocytopenia in a small for gestational age infant. A NAIT was suspected and she was successfully treated with intravenous IgG. The direct and indirect platelet suspension immunofluorescence test (PSIFT) in maternal blood suggested alloimmunization to HPA-5b. Empiric treatment with IgG could be useful in case of clear suspect even in absence of confirmed diagnosis.

Effect of paraformaldehyde on platelet size and on measurement of surface IgG

In many platelet assays, as in measurement of platelet-adherent IgG (PAIgG), platelets are fixed in paraformaldehyde (PFA). To better clarify the effect of PFA on platelet size and on PAIgG measurement we compared PAIgG levels in a series of 40 samples, with or without PFA fixing. We used an ELISA which was set up on unfixed platelets and gave excellent results in terms of linearity ( r = 0.923), precision (mean CV = 5%) and correlation with a platelet suspension immunofluorescence test. We found PAIgG values in unfixed platelets were about 10-fold higer than in PFA-fixed (0.411 - 0.172 fg/platelet vs. 0.035 - 0.019 fg/platelet). This discrepancy could be a consequence of the smaller mean platelet volume (MPV) of washed platelets when fixed in PFA (8.0 - 0.8 fl as compared to 10.1 - 1.07). This effect of PFA could decrease the amount of binding sites for IgG exposed on the platelet membrane and hence explain the significantly lower PAIgG values observed in fixed platelets. The PAIgG measurements on unfixed platelets from 200 healthy subjects displayed a Gaussian distribution with a mean - SD of 0.32 - 0.13 fg/platelet, i.e., 1200 - 500 molecules/platelet.

Congenital Macrothrombocytopenic Thrombopathy and Nephritis (Epstein Syndrome–Variant)

Congenital Macrothrombocytopenic Thrombopathy and Nephritis (Epstein Syndrome–Variant)

Provision of HPA-1a (PlA1)-negative platelets for neonatal alloimmune thrombocytopenia: screening, testing, and transfusion protocol

HPA-1a-negative platelet products are not routinely available for newborns with alloimmune thrombocytopenia. In this article we describe a program established to identify normal pheresis donors who are HPA-1a-negative and to organize their future donations so that our regional blood center would always have an HPA-1a-negative platelet product available. The solid phase red cell adherence assay was used for initial screening of platelet pheresis products. HPA-1a-negative donors were confirmed with the platelet suspension immunofluorescence test using three anti-HPA-1a sera. Screening of 2600 platelet-pheresis donor samples identified 40 HPA-1a-negative donors. Of these, 36 are active and are coded for recognition on the daily pheresis inventory sheet. Theoretically, assuming four donations per year and donors’ cooperation with scheduling, these 36 donors would enable us to have at least one HPA-1a-negative product available every day. In addition, a decision tree for patient management using platelet serology and availability of HPA-1a-negative products was developed. The GTI-PAK™12 is the major technique used for serologic screening of mothers of patients thought to have neonatal alloimmune thrombocytopenia. By screening pheresis donors and developing a clinical decision tree, HPA-1a-negative products, a rare resource, can be fully utilized. Immunohematology 1999;15:71–74.

Laboratory abnormalities in thrombotic thrombocytopenic purpura. Canadian Apheresis Group.

Thrombotic thrombocytopenic purpura is an uncommon disorder that requires prompt recognition and intervention to prevent death. To date, information regarding the classic laboratory abnormalities in the disease has been derived from small numbers of patients whose laboratory tests have been done at many different sites. We report the laboratory findings in 135 patients who presented with thrombotic thrombocytopenic purpura to 17 Canadian centres. 50 men and 85 women had a mean platelet count of 25.3+/-19.4x10(9)/l. The initial platelet count correlated with mortality; 32% of patients with a platelet count of 20x10(9)/l or less died compared with 18% of patients with a platelet count >20x10(9)/l (P=0.058). The platelet-associated IgG was elevated in 88% at presentation whereas the indirect platelet suspension immunofluorescence test was positive in only 18%, 93% of the sera showed reactivity against platelets following protein blotting. All sera tested also showed reactivity against endothelial cells. Immune complexes were seen in all patients, whereas the platelet aggregating factor was detected in 59%. Although the von Willebrand factor was elevated in the majority of patients at entry, the multimer pattern was variable and showed no predictive pattern. Renal dysfunction was common (18%).

Three Cases of Platelet Alloimmunisation Associated with the Presence of a Novel Platelet Specific Antibody

In three cases of platelet alloimmunisation, a platelet-specific alloantibody was detected which could not be classified within the known human platelet alloantigen or HLA systems. The first case was of a family in which two siblings suffered neonatal alloimmune thrombocytopenia at birth. In the second case, the newborn was suffering from phocomelia with hypoplastic thrombocytopenia. The third case was a male who became refractory to transfusions of HLA-matched platelets after a related bone marrow transplantation. The serum samples were investigated by: enzyme-linked immunosorbent assay, platelet suspension immunofluorescence test (PSIFT), monoclonal antibody immobilisation of platelet antigens assay (MAIPA), and by the lymphocytotoxicity test. The antibody gave positive reactions with 26% of normal donor platelets. Surprisingly, no platelet-specific antibody was detected by PSIFT or by MAIPA and there was no evidence found to support classifying the antibody within the HLA system. The reactivity pattern of the antibody detected and the clinical presentation of the three cases described, strongly suggest the presence of an additional platelet-specific alloantigen system.

Three Cases of Platelet Alloimmunisation Associated with the Presence of a Novel Platelet-Specific Antibody

Background and Objectives: In three cases of platelet alloimmunisation, a platelet-specific alloantibody was detected which could not be classified within the known human platelet alloantigen or HLA systems. The first case was of a family in which two siblings suffered neonatal alloimmune thrombocytopenia at birth. In the second case, the newborn was suffering from phocomelia with hypoplastic thrombocytopenia. The third case was a male who became refractory to transfusions of HLA-matched platelets after a related bone marrow transplantation. Materials and Methods: The serum samples were investigated by: enzyme-linked immunosorbent assay, platelet suspension immunofluorescence test (PSIFT), monoclonal antibody immobilisation of platelet antigens assay (MAIPA), and by the lymphocytotoxicity test. Results: The antibody gave positive reactions with 26% of normal donor platelets. Surprisingly, no platelet-specific antibody was detected by PSIFT or by MAIPA and there was no evidence found to support classifying the antibody within the HLA system. Conclusion: The reactivity pattern of the antibody detected and the clinical presentation of the three cases described, strongly suggest the presence of an additional platelet-specific alloantigen system.

Platelet-specific antibodies in transfused patients.

Sera of 104 patients with haematological diseases, transfused with platelets and sometimes also with red cells, were prospectively investigated in: the lymphocytotoxicity test (LCT), the platelet suspension immunofluorescence test (PSIFT) and the monoclonal antibody immunobilization of platelet antigens (MAIPA) technique. All tests on admission were negative. The overall incidence of alloantibodies after transfusions was 30.8%. Platelet-specific antibodies were, however, detected much more rarely (9.6%) than anti-HLA (21.3%). The specificity against HPA antigens was defined in 4 (3.9%) patients: anti-HPA-la, 2b, 3a and 5b. In the remaining 6 (5.7%) cases only the glycoproteins on platelets were identified (GPIb and GPIIIa) which gave positive reaction with the antibodies. Our observations indicate a rather low incidence of platelet-specific antibodies after transfusions.

Flow cytometric analysis in platelet crossmatching using a platelet suspension immunofluorescence test.

The sensitivity of flow cytometric measurement of platelet antibodies in a crossmatch technique was investigated. The corrected count increment after platelet transfusion was compared with the fluorescence ratio determined by flow cytometric measurement. When crossmatching was performed before transfusion(s) in alloimmunized patients, a fluorescence ratio < or = 1.7 was associated with satisfactory responses (corrected count increment > or = 7.5), and the predictive values for negative and positive crossmatch results were 94 and 87 percent, respectively. Analysis of antigen preservation during platelet storage with antibodies to HLA alpha-chain, HLA-B27, HPA-1a, and HPA-3a showed that platelets can be stored, refrigerated, for up to 4 weeks without significant loss of HLA class I and HPA-1a. There was a slight but continuous loss of HPA-3a upon storage. Flow cytometric measurement of fluorescence in the platelet suspension immunofluorescence test can be used for platelet crossmatching, with sensitivity and predictive values comparable to those of previously described techniques and with the advantage of automation.

High prevalence of platelet autoantibodies in patients with systemic lupus erythematosus.

The aim of this study was to determine the prevalence of platelet autoantibodies (PAA) in patients with systemic lupus erythematosus (SLE) and its correlation with clinical and other laboratory manifestations of the disease, as well as to evaluate the influence of platelet count and disease activity on the result of the test for PAA. Ninety SLE patients, 29 with thrombocytopenia, were evaluated. The presence of PAA was determined using the direct and indirect platelet suspension immunofluorescence test. A total of 166 PAA determinations were performed in the 90 patients upon entry into the study. Fifty-six of the 90 patients (62%) with SLE were positive for PAA. There were no statistically significant correlations between the presence of PAA and the different disease manifestations except for thrombocytopenia (P = 0.0005). The presence of PAA in the same patient was significantly associated with current thrombocytopenia and disease activity. It was concluded that the prevalence of PAA in SLE patients is high. All SLE patients with thrombocytopenia had PAA, although some patients with PAA had a normal platelet count. The presence of PAA in SLE patients is not synonymous with thrombocytopenia but should instead be regarded as one of the multiple serological abnormalities which may develop in these patients.

Lack of transmission of neutrophil and platelet antibodies by intravenous immunoglobulin in bone marrow transplant patients

Several studies have demonstrated that the administration of intravenous immunoglobulin (IVIG) may be followed by the transient appearance of positive red cell antibody screens, positive direct antiglobulin tests, and, occasionally, frank hemolysis. However, little information is available regarding the possibility that IVIG could transmit neutrophil and/or platelet antibodies. Serum samples were obtained both immediately before and immediately after the administration of 12 separate lots of commercially available IVIG to bone marrow transplant patients. None of the patients were shown by standard granulocyte immunofluorescence testing to have acquired neutrophil antibodies. Four of the 12 postinfusion sera were positive for platelet antibodies in standard platelet suspension immunofluorescence testing, but in all four instances the corresponding preinfusion serum was positive as well. The risk of acquiring neutrophil and/or platelet antibodies after the administration of commercially available IVIG appears to be low.

Frozen Platelet Plates for Platelet Antibody Detection and Cross-Match

We performed 2 studies aimed at developing a frozen platelet panel suitable for platelet cross-matching. The stability of the most important platelet membrane glycoproteins and the reactivity of antigens of the human platelet antigen (HPA) and of the human leukocyte antigen (HLA) systems were evaluated with the platelet suspension immunofluorescence test (PSIFT) in a panel of platelets frozen in microplates with 6% dimethylsulfoxide. In study No. 1 we evaluated platelet reaction with a broad-spectrum weak anti-HLA and a potent anti-HPA-la antiserum and the expression of glycoproteins lb and Ilb/IIIa complex on platelet membrane before freezing and after 0.5,1,2, 3,4,5,6 and 12 months of storage at -80°C. In study No. 2 we examined platelet reactivity with anti-HPA-lb, -HPA-2a, -HPA-3a, -HLA-A2, -HLA-A3 of platelets stored frozen for 12 months in parallel with fresh platelets from the same donors. Study No. 1 showed that glycoprotein expression was stable and that the weak anti-HLA and the potent anti-HPA-la antibodies were clearly detected during 12 months at -80°C. Of the 35 paired PSIFT performed in study No. 2 with fresh and frozen/thawed platelets incubated with anti-HPA-lb, -HPA-2a, -HPA-3a -HLA-A2, -HLA-A3 antisera and AB serum, concordant reactions were obtained in all cases with the exception of 1 case of HLA-A3-positive platelets incubated with anti-HLA-A3 antiserum, that was reactive with frozen/thawed platelets but nonreactive with fresh platelets from the same donor. The discrepant finding obtained with fresh platelets from 1 donor could be due to the well-known variable and weak association of HLA antigens to platelet membrane. We conclude that frozen platelet plates can be stored and used for at least 12 months for detecting platelet-reactive antibodies in patients’ sera.

HLA antibodies--the cause of platelet alloimmunization in Chinese.

Lymphocytotoxicity test (LCT) and platelet suspension immunofluorescence test (PSIFT) were used together to screen platelet-associated antibodies in patients who received long-term platelet transfusion. Twenty-four of 53 patients (45.3%) were immunized subsequently. Since the concordance of LCT and PSIFT was 100%, most of the platelet associated antibodies were of HLA specificity, and platelet specific antibody alone (in absence of HLA) was not detected. The identified antibodies were anti-A2, A11, A24, B5, B46, B57, B60, and B62. The majority of them were against the high frequency HLA antigens in the Chinese population. The development of antibody could not be correlated with the number of platelet-donors exposed, the time interval after the initiation of platelet transfusion, or the percentage of reactive lymphocytotoxic panels. HLA antibody was the major factor in causing platelet alloimmunization in the Chinese patients. However, some other unknown factors should be looked for. In addition, ABO incompatibility did not affect the posttransfusional increment while the platelet was compatible with LCT crossmatching.

An in‐vitro direct chemiluminescence assay for assessment of platelet‐bound antibody in thrombocytopenic patients

A direct chemiluminescence test (CLT) employing autologous monocytes and granulocytes was used to assay platelet-bound antibody in comparison to the direct platelet suspension immunofluorescence test (PSIFT) in 50 patients. Twenty-six of the 30 immune thrombocytopenic purpura (ITP) patients, including all 21 chronic ITP patients with platelet counts below 100 x 10(9)/l, showed positive results with at least one type of phagocytic cell; so did seven of the eight patients with other presumed autoimmune thrombocytopenias, and three out of 12 patients with thrombocytopenia associated with non-immune disorders. This test appears potentially valuable in the clinical diagnosis of immune thrombocytopenias.

Platelet crossmatching with lymphocytotoxicity test: an effective method in alloimmunized Chinese patients

Fifty-three patients receiving long-term platelet transfusions were regularly screened for platelet-associated antibodies by a platelet suspension immunofluorescence test (PSIFT) and a lymphocytotoxicity test (LCT). Subsequently, 24 patients became alloimmunized; all of their antibodies were of HLA specificity. Eighty-two single-donor platelet transfusions were given, and the clinical responses were considered satisfactory if the 18-hour corrected count increment was 7.5 x 10(3) per microL or higher. In the meantime, 82 pairs of patient sera and donor lymphocytes were crossmatched. Among 63 crossmatched transfusions, 53 (84%) resulted in a satisfactory increment, with a mean (+/- SEM) of 17.71 +/- 1.96 (x 10(3)/microL), and 10 did not result in a satisfactory increment. The increments after 19 unmatched transfusions and 25 random-donor (uncrossmatched) transfusions were 0.7 +/- 0.3 and 2.39 +/- 0.66, respectively. The difference was not significant (p greater than 0.05). The agreement between the LCT results and clinical response was 88 percent. Retrospectively, the corrected count increments showed no significant differences (p greater than 0.05) among three groups of HLA grading: the increments for A/BU/BX, C/D, and random HLA matches were 22.97 +/- 4.07, 15.1 +/- 1.97, and 14.85 +/- 2.04, respectively. These results suggest that platelet crossmatching by LCT is an effective method for use in alloimmunized patients, especially Chinese patients.

Evaluation of a solid phase red cell adherence technique for platelet antibody screening.

Solid-phase red-cell adherence (SPRCA) techniques in platelet serology are used mainly for crossmatching. A SPRCA method for general diagnostic application was evaluated in parallel with the platelet suspension immunofluorescence test (PIFT). Of 149 patient sera sent for investigation of thrombocytopaenia, 76 were negative and 59 positive when studied by both methods, eight positive by PIFT only and six positive by SPRCA only. The reactivity observed for 24 sera containing HLA antibodies tested with chloroquine-treated and untreated platelets was similar for both methods. All of 14 sera containing quinine-associated antibodies reacted strongly to both techniques in the presence of added quinine. In comparison, however, whereas all sera were nonreactive to SPRCA in the absence of added quinine, and with PIFT, seven of the sera reacted weakly. Titration studies with three examples of anti-PlA1 and five sera containing HLA antibodies generally showed a one doubling dilution lower titre with the SPRCA procedure. End-point interpretation, however, was more readily achieved with the SPRCA method. The SPRCA technique displays similar sensitivity and specificity to the PIFT and is recommended for use by routine hospital laboratories to screen platelet antibodies.