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Background: Drug-induced immune thrombocytopenia (DITP) is a serious, life-threatening clinical syndrome, the diagnosis of which is consistently difficult. In this report, we present a case of DITP caused by meropenem that was confirmed by laboratory tests., Case Report: A 59-year-old male patient developed severe thrombocytopenia 8 days after the administration of meropenem and cefoperazone-sulbactam. After other causes were ruled out, DITP was suspected. Drug-induced platelet (PLT) antibodies were detected by enzyme immunoassay, flow cytometry, and monoclonal antibody immobilization of PLT antigens (MAIPA). All these tests were performed in the presence and absence of the associated drugs., Results: PLT antibodies were detected in the patient's serum only in the presence of meropenem. MAIPA experiments demonstrated that glycoprotein IIb/IIIa was the binding site of the meropenem-induced PLT antibodies., Conclusions: Drug-induced immune thrombocytopenia should be considered in cases of acute thrombocytopenia in patients undergoing meropenem treatment. Clinicians should be cognizant of DITP, and a definitive diagnosis should be pursued, if feasible., (© 2017 AABB.)

Background: Fetal-neonatal alloimmune thrombocytopenia (FNAIT) diagnosis relies on maternofetal incompatibility and alloantibody identification. Genotyping for rare platelet (PLT) polymorphisms allowed the identification of three families with suspected or confirmed maternofetal incompatibility for the αIIb-c.2614C>A mutation (Halle et al., Transfusion 2008;48:14-15)., Study Design and Methods: A polymerase chain reaction-sequence-specific primers amplification assay was designed to genotype the αIIb-c.2614C>A mutation. HEK293 cells expressing αIIb-Leu841 or αIIb-Met841 αIIbβ3 forms were used to probe the reactivity of maternal sera from these families and to study the effects of the substitution on αIIbβ3 expression and functions., Results: Tested by flow cytometry (FCM), one serum sample specifically reacted with αIIb-Met841 but not with αIIb-Leu841 αIIbβ3. This specificity revealed the αIIb-Leu841 polymorphism as a new alloantigen named Cab3(a+) . Cross-match testing using FCM also showed the Cab3(a+) antigen to be expressed at the PLT surface. As for anti-human PLT alloantigen (HPA)-3a (or -3b) and anti-HPA-9bw, detection of anti-Cab3(a+) alloantibodies appeared difficult and required whole PLT assays when classical monoclonal antibody-specific immobilization of PLT antigen test failed. In our FNAIT set, the immune response to Cab3(a+) maternofetal incompatibility could induce severe thrombocytopenias and life-threatening hemorrhages. The p.Leu841Met substitution has limited effects, if any, on local αIIb structure, preserving both αIIbβ3 expression and functions., Conclusion: The Cab3(a+) polymorphism is a new rare alloantigen (allelic frequency <1%) carried by αIIb that might result in severe life-threatening thrombocytopenias. In Sub-Saharan African populations, higher Cab3(a+) gene frequencies (up to 8.2%; Halle et al., Transfusion 2008;48:14-15) and homozygous people are observed., (© 2012 American Association of Blood Banks.)

Background: Blood group A and B antigens are expressed only weakly on platelets (PLTs) of most individuals but are very strongly expressed on PLTs from approximately 1 percent of normal subjects (Type II high expressers). The implications of this trait for transfusion medicine are undefined., Study Design and Methods: A family was studied in which two Group B infants were born with neonatal thrombocytopenia, whereas a third infant whose blood group was A(2) had a normal PLT count at birth., Results: Serologic studies demonstrated a maternal antibody that reacted strongly with PLTs from the father and the two group B children in flow cytometry and with GPIIb/IIIa from their PLTs in solid-phase assays. No PLT-specific antibodies were detected in maternal serum sample, but it contained a high-titer immunoglobulin G antibody specific for blood group B. All PLT-reactive antibody in the mother's serum was removed by absorption with pooled, washed group A and B red cells (RBCs). Studies with monoclonal anti-B and measurement of serum B-glycosyltransferase activity showed that the father and both group B children were Type II high expressers of blood group B., Conclusions: The findings indicate that high-titer blood group antibodies acquired from the mother can cause thrombocytopenia in infants possessing the Type II high-expresser phenotype despite competition for antibody binding by blood group antigens expressed on RBCs and other tissues.

Background: Cold hemagglutinins are generally immunoglobulin M (IgM) kappa antibodies reactive at temperatures below 37 degrees C and if of high titer may cause hemolysis. Platelet (PLT) cold agglutinins (CAs) are rare and poorly characterized. A detailed molecular characterization of the variable domains of a pathologic, PLT-reactive, CA is presented., Case Report: A 70-year-old woman was admitted with rectal bleeding accompanied by widespread petechiae, bruising, tongue and buccal mucosa bleeding, and epistaxes and proved refractory to HLA- and HPA-matched PLTs. Detailed investigation showed monoclonal heavy-chain gene rearrangement with an IgM paraprotein of 3.3 g per L and a trace of kappa Bence Jones protein in the urine, compatible with a diagnosis of secretory B-cell non-Hodgkin's lymphoma (B-NHL). PLT antibody (PAIg) investigations revealed a potent IgM kappa PLT CA. Sequencing of the rearranged variable domain genes of the malignant clone together with idiotype-specific antibodies obtained by DNA-based immunization of rabbits and matrix-assisted laser desorption/ionization-time-of-flight analysis of the PAIgM provided a irrefutable link between the thrombocytopenia, the IgM paraprotein, and the PAIgM against alphaIIbbeta3. The thrombocytopenia and bleeding were refractory to standard treatment and PLT transfusion, but treatment with rituximab resulted in a recovery of the PLT count and a complete remission of B-NHL., Conclusion: The IgM kappa paraprotein derived from the malignant B-cell clone was a potent and clinically significant CA against alphaIIbbeta3. The testing for PLT CAs in patients with a paraprotein and refractory to matched PLTs may aid the selection of appropriate treatment.

Background: The aims were to characterize the helper T-cell response to platelet (PLT) glycoprotein (GP) IIIa, which stimulates the alloimmune antibody response to human PLT antigen (HPA)-1a, to identify immunodominant epitopes and to examine the HLA Class II associations., Study Design and Methods: Peripheral blood mononuclear cells (PBMNCs) were obtained from 21 HPA-1b1b women who had an HPA-1a-mismatched pregnancy, 14 of whom developed anti-HPA-1a, and 11 control donors. PBMNCs were stimulated with two panels of 15-mer peptides corresponding to the HPA-1a/1b polymorphic region, with either Leu33 (-1a) or Pro33 (-1b) at each possible position, and the proliferative responses were measured. HLA Class II and HPA genotyping was by conventional polymerase chain reaction-sequence-specific priming., Results: Peptides with Leu33 at, or near, the C-terminus contained an immunodominant epitope, stimulating proliferation by helper T cells from all nine women who had anti-HPA-1a at the time of testing; peptide L1 (Val19-Leu33) stimulated a response in 50 percent of these women. Their T cells did not respond to the corresponding HPA-1b Pro33 peptides, and responses to either peptide panel were rare in unimmunized women and controls. HLA-DRB3*01+ was significantly overrepresented (p = 0.014) in alloimmunized women whose T cells responded to the major HPA-1a Leu33-containing epitope. Conversely, HLA-DRB1*15 was negatively associated (p = 0.014) with this response., Conclusions: The HPA-1a polymorphic region of GPIIIa contains both the linear T-cell and the conformational B-cell epitopes. The immunodominant T-cell epitope is constrained by HLA-DRB3*01+, and if presented by a tolerogenic route, a peptide containing this epitope may form the basis for the prevention or reversal of the alloimmune response to HPA-1a.

Background: Ceftriaxone, a third-generation cephalosporin, has been reported to occasionally cause fatal drug-induced immune hemolytic anemia (DIHA). A clinical and serologic analysis of the first two patients with severe drug-induced thrombocytopenia (DITP) due to ceftriaxone and one patient with fatal DIHA is reported., Study Design and Methods: Sera were assessed by the IAT, EIA, glycoprotein (GP)-specific immunoassay, flow cytometry, and immunoprecipitation using transfectants expressing GPIIb/IIIa and GPIb/IX and with different cephalosporins., Results: Sera from Patients 1 and 2 reacted strongly with PLTs in the presence of the drug, but not with RBCs. The binding sites of the drug-dependent antibodies (DDAbs) could be localized to GPIIb/IIIa and GPIb/IX, respectively. Inhibition studies indicated that DDAbs recognized epitopes residing on the GPIIb/IIIa complex and on the GPIX subunit, respectively. No cross-reactivity was observed with other cephalosporin derivatives. Serum 3 showed strong agglutination with RBCs of Rh(null) phenotype in the presence of ex-vivo metabolites of ceftriaxone, but no cross-reactivity with PLTs., Conclusions: The first two cases of severe DITP and a third patient with DIHA are reported. DDAbs from all patients showed individual reaction patterns and clear cell lineage specificity. In addition, the DDAbs were dependent on the substitution at position 3 of the ceftriaxone molecule. Epitopes on GPIIb/IIIa and GPIX were involved on PLTs. The Rh protein was not the only target of DDAbs on RBCs.

Background: Filtered PLT concentrates (PCs) were prepared in plasma pooling three (for children) or six buffy coats (BCs; for adults) after holding them a maximum of 4 hours (blood bags collected in the afternoon) or 18 hours (blood bags collected in the morning)., Study Design and Methods: With flow cytometry, PCs prepared after holding BCs 4 or 18 hours were compared. BCs removed from whole-blood donations in quadruple bag packs ("top-top") were held 4 or 18 hours before pooling them with a sterile connecting device. After the BCs were centrifuged, the supernatant was transferred through a BC filter (Autostop, Pall Medical) to a CLX bag. Samples for analysis were collected from the whole-blood bag, BCs, and PCs immediately after preparation and after 1, 3, 5, and 7 days of storage on a flat-bed agitator at 22 +/- 2 degrees C. The main PLT membrane glycoproteins (GPs, IIb-IIIa, IV, and Ibalpha), some of their ligands (fibrinogen, fibronectin, and VWF), activation-dependent antigens (CD62P and CD63), and procoagulant activity markers (annexin V and bound coagulation FV-Va) have been studied., Results: In the 12 PCs (six pools of 3 units each group) studied, a minor increase in activation markers during preparation was observed. During the storage, a significant increase in the expression of GPIIb-IIIa, CD62P, CD63, annexin V, and FVa was measured. After 5 days of storage, only the percentage of PLTs with bound fibrinogen was significantly greater in PCs prepared after holding BCs for 4 hours., Conclusion: In PCs prepared after holding BCs 4 or 18 hours before pooling and filtering, only a minor significant difference in the percentage of PLTs with bound fibrinogen was found after 5 days of storage. This difference is probably of little, if any, transfusional significance.

Background: Severe neonatal alloimmune thrombocytopenia is often due to antibodies against human platelet antigen type 1a (HPA-1a). The aim of this study was to develop a quantitative ELISA for the measurement of antibodies against HPA-1a., Study Design and Methods: HPA-1a glycoprotein (GP) IIb-IIIa was immobilized and mixed with recalcified anti-HPA-1a-positive plasma overnight at 4 degrees C. The beads were washed, the antibodies against HPA-1a were eluted, and the eluate pH level was promptly adjusted. The purified antibodies were dialyzed and used for the development of an ELISA incorporating HPA-1a-coated plates., Results: Serial doubling dilutions of the purified antibodies resulted in consistent sigmoid standard curves with a sensitivity of 0.5 microg per mL. To determine the reproducibility of the ELISA, antibodies against HPA-1a in five plasma samples (Samples A-E) were measured at serial doubling dilutions in four separate assays. Three of the samples (Samples A-C) contained antibodies against HPA-1a. The mean amounts in microg per mL (+/- SD, percentage of CV) obtained in the four assays were as follows: Sample A, 133 (9.4, 7.1%); Sample B, 16.5 (1.7, 10%); and Sample C, 8 (0.8, 10%). The amounts in the two antibody-negative controls (Samples D and E) were consistently less than 0.2 microg per mL., Conclusion: Using immobilized HPA-1a1a, antibodies against HPA-1a has been purified, and a quick and simple quantitative assay has been developed.

Background: Diclofenac has frequently been implicated as the cause of immune hemolytic anemias and less frequently of immune thrombocytopenia. The presence of the causative antibodies has only been demonstrated in patients with immune hemolytic anemia, but not yet in patients with thrombocytopenia. The cases of two patients in whom diclofenac simultaneously induced antibodies against platelets and RBCs are reported., Study Design and Methods: The investigation was carried out with standard serologic tests for detection of antibodies against platelets and RBCs. The patients' sera were tested in the presence and absence of diclofenac and its metabolites., Results: One of the two patients developed severe hemolysis and significant thrombocytopenic purpura. The other patient developed significant thrombocytopenia but no hemolysis. Both patients had a positive DAT and drug- and/or metabolite-dependent antibodies against RBCs and platelets., Conclusion: Based on our findings and those of other investigators, we believe that diclofenac leads to the production of antibodies against RBCs and/or platelets.

Background: Solid-phase RBC adherence (SPRCA) assays (Immucor) detect HLA and/or platelet-specific antibodies. Pretreatment of reagent platelets with enzymes was investigated to determine whether the sensitivity of the assay could be increased., Study Design and Methods: SPRCA testing, following the manufacturer's directions, was performed on 51 sera from patients with either a positive SPRCA antibody screen, suboptimal clinical responses to platelet transfusions, and/or suspected immune thrombocytopenic purpura; testing was also performed following pretreatment of the reagent platelets with bromelin, papain, or ficin. Sera from 23 patients having negative routine SPRCA antibody screens and good clinical responses to transfusion were tested as controls. Lymphocytotoxic antibody testing was also performed on selected samples. The effectiveness of enzyme treatment was judged by the increase in the proportion of reagent platelets reacting with the sample and the observed reaction strengths., Results: Pretreatments of reagent platelets with all three enzymes increased the reactivity of known antibodies and detected some HLA and platelet-specific antibodies that had not reacted in routine testing. The clinical significance of the antibody specificities detected only after enzyme pretreatment was verified by a correlation with results from transfusing antigen-negative units. Only occasional false-positive results after enzyme pretreatment were observed., Conclusions: The use of enzyme pretreatment of SPRCA screening strips can provide information that is useful in selecting appropriate units for transfusion.

Background: Posttransfusion purpura (PTP) is characterized by severe thrombocytopenia following blood transfusion that results from alloimmunization to platelet-specific alloantigens. Most cases involve antibodies against HPA-1a in homozygous HPA-1b persons., Case Report: A patient developed PTP after cardiopulmonary bypass associated with a platelet-specific antibody with strong reactivity against HPA-5a (Br(b)). Geno-typing confirmed that the patient was homozygous for HPA-5b., Conclusion: This is the first well-documented occurrence of PTP associated with isolated allosensitization to HPA-5a or Br(b). The case highlights the importance of maintaining a high level of suspicion for PTP in the appropriate clinical setting, even in an atypical patient.

Background: A study in humans showed that the transfusion of previously frozen human platelets after cardiopulmonary bypass, despite decreased survival, resulted in better hemostatic function than that of liquid-preserved platelets stored at 22 degrees C for 3 to 4 days., Study Design and Methods: In this study, fresh, 3- to 4-day-old liquid-preserved, and cryopreserved human platelets were studied by the use of monoclonal antibodies directed against p-selectin, glycoprotein (GP)Ib, activated GPIIb/IIIa, and coagulation factor V in a three-color flow cytometric method., Results: The fresh and liquid-preserved platelets had normal surface levels of GPIb, while the cryopreserved platelets were composed of distinct subpopulations of GPIb-normal and GPIb-reduced platelets. On the basis of the binding of factor V, both subpopulations of cryopreserved platelets exhibited greater surface binding of factor V than did fresh and liquid-preserved platelets. Activated GPIIb/IIIa was elevated on GPIb-normal platelets, but not on GPIb-reduced platelets. Baboon platelets frozen by a procedure identical to that used to freeze human platelets also had GPIb-normal and GPIb-reduced subpopulations after the freezing-thawing-washing procedure. Autologous cryopreserved baboon platelets labeled with biotin-X-N-hydroxysuccinimide showed a rapid removal of GPIb-reduced platelets during the 5-minute postinfusion period, whereas GPIb-normal platelets had an in vivo recovery of 48 percent and a lifespan of slightly less than 6 days., Conclusions: Improved in vivo function of cryopreserved platelets may be related to the rapid hemostatic effect of the GPIb-reduced subpopulation secondary to increased binding of factor V and expression of p-selectin.

Background: Whole blood-derived buffy coat (BC) has become an alternative source from which to prepare random-donor platelet concentrates. The influence of prolonged storage of BC prior to platelet concentrate preparation is a matter of controversy. The impact of BC storage on cytokine release was evaluated and the platelet activation quantified., Study Design and Methods: BCs were prepared from whole-blood donations after hard-spin centrifugation. After 1, 3, 6, 12, and 24 hours of storage at 22 degrees C without agitation, samples were withdrawn for cell count and blood gas analysis and measurement of interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor alpha and platelet factor 4. Platelet surface markers CD41a, CD42b, CD62P, and CD63 were analyzed by flow cytometry, and the antibody-binding sites were quantified by using microbeads., Results: Inflammatory cytokines IL-1beta, IL-6, and tumor necrosis factor alpha were hardly detectable in stored BCs but levels of IL-8 increased in 25 percent of BCs after 24 hours. A constant increase in platelet factor 4 was observed, which accelerated after 12 hours of storage. Analysis of platelet surface markers showed an initial decrease of platelet activation, followed by an increase after storage for 12 to 24 hours., Conclusion: Storage of BCs for up to 12 hours without agitation showed a good preservation of platelets but storage of BCs for 24 hours resulted in increased platelet activation and significantly higher release of platelet factor 4 and IL-8. Stored BCs might well be suitable for platelet preparation, but their storage time should not greatly exceed 12 hours.

Background: Diagnosis, and occasionally treatment, of disorders involving platelet-specific alloimmunization, including neonatal alloimmune thrombocytopenia and posttransfusion purpura, requires platelet allotyping. Allele-specific restriction analyses for convenient genotyping are available for the major human platelet antigen (HPA) systems except HPA-4., Study Design and Methods: An allele-specific restriction analysis for HPA-4 was developed by designing a polymerase chain reaction primer containing a single-base substitution (A for T) at glycoprotein IIIa cDNA position 529. The resulting mismatch did not interfere with DNA amplification. Digestion of the polymerase chain reaction product with BsmI allowed differentiation of HPA-4a and HPA-4b alleles., Results: A 126-bp polymerase chain reaction product was amplified with the novel primer. BsmI endonuclease cleaved product encoding HPA-4a into 104- and 22-bp fragments and left DNA encoding HPA-4b intact., Conclusion: This rapid allele-specific restriction analysis for genotyping the HPA-4 system complements similar methods for other platelet alloantigenic determinants.

Background: Polymorphism of glycoprotein IIIa on human platelets is one of the factors in alloimmunization that causes neonatal alloimmune thrombocytopenia and refractoriness to platelet transfusion., Study Design and Methods: DNA typing methods were originally developed to determine the genotypes of five human platelet antigen (HPA) systems located on glycoprotein IIIa: HPA-1, HPA-4, HPA-6W, HPA-7W and HPA-8W. The gene frequencies of these platelet antigens were determined by DNA typing of 331 unrelated Japanese donors., Results: The gene frequencies of the low-frequency antigens were 0.002, 0.011, and 0.027 for HPA-1b, HPA-4b, and HPA-6W(b), respectively. All 331 Japanese donors tested were HPA-7W(a/a) and HPA-8W(a/a). Moreover, in the present study, none of the donors tested had two or more of these low-frequency antigens., Conclusion: The risk of neonatal alloimmune thrombocytopenia and refractoriness to platelet transfusion induced by the antigens of the HPA-1, HPA-7W, and HPA-8W systems was extremely rare in Japanese. However, attention must be paid to the involvement of the HPA-4 and HPA-6W systems in these clinical disorders.

Background: Alloantibodies to HPA-1a (PlA1) are the major cause of neonatal alloimmune thrombocytopenia and posttransfusion purpura and have been implicated in refractoriness to random-donor platelet transfusions. However, most assays used to phenotype platelets are cumbersome or time-consuming for large numbers of samples., Study Design and Methods: A simple, competitive (inhibition) enzyme-linked immunosorbent assay for HPA-1a phenotyping of donor platelets was developed. A segment from the donor platelet unit transfer line was sealed to obtain a small aliquot of platelets. These platelets were washed once and added to a predetermined dilution of serum containing alloantibodies to HPA-1a. Residual anti-HPA-1a binding to the glycoprotein IIb/IIIa purified by lectin and high-performance liquid chromatography and coated on microtiter wells was detected with a conjugated antihuman IgG. A lack of inhibition equivalent to control (no platelets) was used to determine that the platelets were HPA-1b/b., Results: Of the 557 platelet units tested, 14 (2.5%) were found to be HPA-1a negative, and they were confirmed to be HPA-1b/b by DNA genotyping. Two of the 14 HPA-1b/b units were also HPA-3b/b (approx. 0.35% of the random population). Use of the microtiter format allows 100 to 200 samples to be processed per day., Conclusion: This simple and inexpensive assay is useful for identifying HPA-1b/b units for platelet-compatible transfusions or for platelet antibody investigations.