Gregory A. Denomme

High‐throughput multiplex single‐nucleotide polymorphism analysis for red cell and platelet antigen genotypes

BACKGROUND: Transfusion recipients who become alloimmunized to red cell or platelet (PLT) antigens require antigen‐negative blood to limit adverse transfusion reactions. Blood collection facilities use regulated and unregulated antibodies to phenotype blood, the cost of which can be prohibitive depending on the antisera and demand. An alternative strategy is to screen blood for these antigens with genomic DNA and the associated single‐nucleotide polymorphisms (SNPs).

A diagnostic test for heparin-induced thrombocytopenia: detection of platelet microparticles using flow cytometry.

Based on our previous observation that heparin‐induced thrombocytopenia (HIT) sera can generate platelet microparticles from washed platelets in a heparin‐dependent fashion, we developed a test for HIT using flow‐cytometry to measure heparin‐dependent platelet microparticle formation. During the developmental phase of the assay the optimal physical conditions for microparticle generation were defined. 133 sera were then evaluated using the microparticle assay and the serotonin release assay to determine the threshold for defining a positive result that gave optimal sensitivity and specificity. The microparticle assay was then prospectively evaluated against the serotonin release assay in 202 sera referred to our laboratory for HIT testing. Overall agreement between the two assays was 96% (Cohen‘s kappa = 0.91). When the clinical data were reviewed on patients whose sera gave discrepant results between the two assays, no case of HIT was detected by one assay and missed by the other. The platelet microparticle assay is as accurate as the serotonin release assay and may be a useful non‐radioactive test for HIT.

Partial D, weak D types, and novel RHD alleles among 33,864 multiethnic patients : implications for anti-D alloimmunization and prevention

BACKGROUND: The D antigen includes category D, partial D, and weak D types, which are important because anti‐D alloimmunization can occur in some but not all persons that express a variant RHD allele. At present, there is little prospective information on the prevalence of D variants among obstetric patients and potential transfusion recipients.

Fcγ receptor IIa and IIIa polymorphisms in childhood immune thrombocytopenic purpura

Summary. Fcγ receptor‐mediated destruction of autoantibody‐sensitized platelets is central to the immune pathophysiology of childhood immune thrombocytopenic purpura (ITP). Allelic variants exist among the random population for some Fcγ receptors. The variants represent single nucleotide polymorphisms, leading to functional differences in the ability to bind immunoglobulin (Ig)G or IgG subclasses. The genotypic frequencies for two Fcγ receptor single nucleotide polymorphisms, FcγRIIa‐131 arginine (R) versus histidine (H) and FcγRIIIa‐158 valine (V) versus phenylalanine (F) were examined in 98 children diagnosed with childhood ITP. The genotype frequencies were compared with those of 130 healthy control subjects. Chi‐square analysis was used to determine whether the allelic frequencies of the high‐affinity receptor variants were associated with childhood ITP. Both the FcγRIIa‐131H and the FcγRIIIa‐158V were significantly over‐represented in children with ITP versus the control subjects (P‐values 0·03). The same statistical difference was noted with the combined FcγRIIa‐131H and FcγRIIIa‐158V allelic gene frequencies. There was no statistical difference between children who later developed chronic ITP compared with children with acute ITP, suggesting that additional factors are responsible for the development of the chronic form of the disease. These observations underscore the importance of Fcγ receptor‐mediated cell clearance in childhood ITP.

It's time to phase in RHD genotyping for patients with a serologic weak D phenotype

In 2014, the College of American Pathologists (CAP) Transfusion Medicine Resource Committee (TMRC) reported the results of a survey of more than 3100 laboratories concerning their policies and procedures for testing serological weak D phenotypes and administration of Rh immune globulin (RhIG).1 Among the findings of this survey is the observation that there is a lack of standard practice in the United States for interpreting the RhD type when a serological weak D phenotype is detected. In some laboratories, an individual with a serological weak D phenotype, especially if a blood donor, is interpreted to be RhD-positive. In the same or other laboratories, especially if a serological weak D phenotype is detected in a female of child-bearing potential, the individual is likely to be managed as RhD-negative for transfusions and, if pregnant, considered a candidate for RhIG. Also, the performance characteristics of serological typing methods for RhD vary. For patients, including pregnant women, the majority of laboratories have policies and procedures that do not use the indirect antiglobulin (weak D) test, thereby avoiding detection of a serological weak D phenotype so that the RhD type will be interpreted to be RhD-negative. Other laboratories typically perform a weak D test for the same category of patients. For blood donors and newborns, it is standard practice for laboratories to have policies and procedures for RhD typing to ensure that serological weak D phenotypes are detected and interpreted as RhD-positive.1 The goal of these RhD typing practices is to protect RhD-negative persons from inadvertent alloimmunization to the D antigen by exposure to RhD-positive RBCs, including RBCs expressing a serological weak D phenotype. Although there has not been a recent prospective study in the United States, it is estimated that current RhD typing practice, together with contemporary obstetrical practices for administration of antepartum and postpartum RhIG, is 98.4 to 99 percent successful in preventing RhD alloimmunization and RhD hemolytic disease of the fetus/newborn.2 However, there are unwarranted consequences associated with the practice of not determining the RHD genotype of persons with a serological weak D phenotype, including unnecessary injections of RhIG and transfusion of RhD-negative RBCs -- always in short supply -- when RhD-positive RBCs could be transfused safely. CAP’s TMRC reviewed the current status of RHD genotyping and proposed that selective integration of RHD genotyping in laboratory practices could improve the accuracy of RhD typing results, reduce unnecessary administration of RhIG in women with a serological weak D phenotype, and decrease unnecessary transfusion of RhD-negative RBCs to recipients with a serological weak D phenotype.1 In response to the findings of the CAP TMRC survey, AABB and CAP convened a Work Group on RHD Genotyping and charged it with developing recommendations to clarify clinical issues related to RhD typing in persons with a serological weak D phenotype. As an initial step for formulating recommendations, the Work Group reviewed the current state of molecular science of RHD, including more than 140 publications covering background;1-12 D variants with anti-D;13-29 molecular basis of serological weak D phenotypes;30-92 reviews, editorials and commentaries;93-129 technical resources;130-142 and standards and guidelines.143-149 This Commentary summarizes the proceedings and recommendations of the Work Group.

Applying molecular immunohematology discoveries to standards of practice in blood banks: now is the time

Lessons from more than 100 years of immunohematology exemplify that many critical discoveries were made serendipitously and their more rapid implementation could have benefited transfusion recipients and pregnancies. Constituents of blood that are not essential for the attempted therapeutic benefit of a transfusion are largely removed from todays blood products. We are now moving on to avoid unnecessary exposure to potentially harmful constituents of the therapeutically required cells, like blood group antigens that are foreign to the patient. Cost efficacy needs to be kept in mind but may eventually prove much better than anticipated, once hidden benefits are captured, as we show by examples from past immunohematologic developments. Here, we detail clinical applications for molecular immunohematology advances including “dry‐matching” that will improve transfusion outcomes and argue for their widespread implementation by rapid timelines through standards of practice.

Maternal immunization to Gov system alloantigens on human platelets

BACKGROUND: Immunization to platelet alloantigens can occur during pregnancy or after the transfusion of blood components. Platelet alloantibodies can cause neonatal alloimmune thrombocytopenia and posttransfusion purpura. Transfusion‐induced alloimmunization to a novel platelet alloantigen system, Gov, expressed on the 175‐kDa glycosyl phosphatidylinositol‐anchored platelet glycoprotein, CD109, was previously described. This report describes three unrelated patients who were alloimmunized to Gov(a) or Gov(b) during pregnancy. STUDY DESIGN AND METHODS: Platelets were typed by using radioimmunoprecipitation for HPA‐1a, −3a, −5a, −5b, Gov(a), and Gov(b) and by polymerase chain reaction‐restriction fragment length polymorphism for HPA‐1a, −1b, −3a, and −3b. Maternal sera were screened for platelet antibodies by using radioimmunoprecipitation and the antigen capture assay. RESULTS: Patients 1 and 2 were investigated after the diagnosis of neonatal alloimmune thrombocytopenia in their children, and alloantibodies specific for Gov(b) and Gov(a), respectively, were detected in maternal serum. Serum from patient 3, who had mild idiopathic thrombocytopenia purpura with no detectable autoantibody, was found to contain alloantibodies to Gov(b) and to HPA‐ 5b, presumably as a result of immunization during pregnancy. Platelet typings confirmed that the patients were at risk for alloimmunization to the respective antigen. CONCLUSION: This report of three cases of maternal alloimmunization to antigens in the Gov system indicates that immunization can occur via placental transfer of antigen and that Gov system alloantibodies may be associated with neonatal alloimmune thrombocytopenia.

Novel 3'Rhesus box sequences confound RHD zygosity assignment.

BACKGROUND: Paternal RHD zygosity is required for genetic counseling and management of HDN caused by anti‐D. The most common D− haplotype is due to the deletion of RHD, which results in the formation of a hybrid Rhesus box, theoretically through the recombination of 5′ and 3′Rhesus boxes.

Chemical compounds that target thiol-disulfide groups on mononuclear phagocytes inhibit immune mediated phagocytosis of red blood cells

BACKGROUND: Patients having immune cytopenias produce antibodies that target hematopoietic cells resulting in their phagocytosis and intracellular destruction. Early reports suggested that phagocytosis could be inhibited by interfering with membrane thiol (SH) groups on phagocytes. Thus, whether chemical compounds that interact with SH or disulfide (SS) groups on mononuclear phagocytes can inhibit phagocytosis of antibody‐coated cells was examined.

Rh discrepancies caused by variable reactivity of partial and weak D types with different serologic techniques

BACKGROUND: RhD discrepancies between current and historical results are problematic to resolve. The investigation of 10 discrepancies is reported here.