Mia J. Sullivan

The neutrophil alloantigen HNA-3a (5b) is located on choline transporter-like protein 2 and appears to be encoded by an R>Q154 amino acid substitution

The molecular basis of the HNA-3a/b (5b/a) leukocyte antigen system has not yet been defined despite evidence that HNA-3a-specific antibodies are particularly prone to cause severe, often fatal, transfusion-related lung injury. We used genome-wide single nucleotide polymorphism scanning and sequencing of DNA from persons of different HNA-3a/b phenotypes to identify a single single nucleotide polymorphism in exon 7 of the CLT2 gene (SLC44A2) that predicts an amino acid substitution in the first extracellular loop of choline transporter-like protein 2, a member of the choline transporter-like protein family of membrane glycoproteins, and correlates perfectly with HNA-3a/b phenotypes (R154 encodes HNA-3a; Q154 encodes HNA-3b). Mass spectrometric analysis of proteins immunoprecipitated from leukocytes by anti-HNA-3a provided direct evidence that anti-HNA-3a recognizes choline transporter-like protein 2. These findings will enable large-scale genotyping for HNA-3a/b to identify blood donors at risk to have HNA-3a-specific antibodies and should facilitate development of practical methods to detect such antibodies and prevent transfusion-related lung injury.

HNA-3a–specific antibodies recognize choline transporter–like protein-2 peptides containing arginine, but not glutamine at Position 154

BACKGROUND: Antibodies specific for the neutrophil antigen HNA‐3a cause severe, sometimes fatal transfusion‐related acute lung disease (TRALI) when transfused, but it has not been possible to screen blood donors for anti‐HNA‐3a because using neutrophils as targets was impractical and molecular properties of the antigen were unknown. Recently it was shown that HNA‐3a is carried on choline transporter–like protein‐2 (CTL2) and that the HNA‐3a/b phenotype is closely correlated with an R154Q amino acid polymorphism in CTL2. However, it has not been shown by direct experiment that R154 is essential for the HNA‐3a epitope.

Protamine‐induced immune thrombocytopenia

Protamine is widely used to reverse the anticoagulant effects of heparin. Although mild thrombocytopenia is common in patients given protamine after cardiac procedures, acute severe thrombocytopenia has not been described. We encountered a patient who experienced profound thrombocytopenia and bleeding shortly after administration of protamine and performed studies to characterize the responsible mechanism.

Determination of neutrophil antigen HNA‐3a and HNA‐3b genotype frequencies in six racial groups by high‐throughput 5′ exonuclease assay

BACKGROUND: People with the human neutrophil antigen (HNA)‐3b/3b type can make HNA‐3a antibodies, which have been reported to cause immune neutropenia disorders and are especially prone to cause severe cases of transfusion‐related acute lung injury. However, knowledge of HNA‐3 allele frequencies outside Caucasian populations is limited. We developed a high‐throughput genotyping assay and determined the HNA‐3a/3b genotype frequencies in six different racial and ethnic groups.

Two cases of maternal alloimmunization against human neutrophil alloantigen-4b, one causing severe alloimmune neonatal neutropenia

Human neutrophil antigen (HNA)−4a/4bw is encoded by 230G>A in ITGAM, which results in an Arg61His substitution of the αM chain (CD11b) of complement receptor 3 (CR3; CD11b/18 or Mac‐1). HNA‐4a antibodies have been detected in the sera of female blood donors and in maternal sera that caused alloimmune neonatal neutropenia (ANN), in which maternal immunoglobulin (Ig)G antibodies against a paternally inherited HNA cross the placenta and destroy fetal and neonatal neutrophils. However, to date, antibodies specific for HNA‐4b have not been reported. Here, we report the first two examples of HNA‐4b antibodies.

Full-length recombinant choline transporter–like protein 2 containing arginine 154 reconstitutes the epitope recognized by HNA-3a antibodies

BACKGROUND: Recent reports have shown that the HNA‐3a leukocyte antigen, a target for antibodies that cause severe transfusion‐related acute lung injury, correlates with an arginine 154 (rather than glutamine) polymorphism in choline transporter–like protein 2 (CTL2) but did not show directly that R154 determines HNA‐3a. CTL2 peptides containing R154 are recognized by only half of HNA‐3a antibodies studied to date. Constructs that react with all HNA‐3a antibodies are needed to fully define the HNA‐3a epitope.

A new low‐frequency alloantigen (Khab) located on platelet glycoprotein IIIa as a cause of maternal sensitization leading to neonatal alloimmune thrombocytopenia

A total of 34 human platelet (PLT) alloantigens (HPAs) have been identified to date, and all have been implicated in neonatal alloimmune thrombocytopenia (NAIT), in which maternal antibodies destroy fetal and neonatal PLTs that carry an incompatible HPA inherited from the father. All of the recently discovered HPA occur with low frequency (typically ≤0.1%) in Caucasian populations. We report a case of NAIT, in which maternal serum contained strong immunoglobulin (Ig)G antibodies reactive with a new low-frequency HPA localized on paternal PLT glycoprotein (GP)IIIa. A gravida 7 para 4 Latino/Hispanic female gave birth at 29 weeks’ gestation due to decreased fetal movement to an infant with prominent petechiae and 43 × 10 PLTs/L. The infant was treated with intravenous immune globulin and three PLT transfusions over a 2-week period. The third transfusion increased the PLTs from 11 × 10 to 240 × 10/L, and the counts remained normal thereafter. One of the previous three live-born infants delivered was also reported to have had thrombocytopenia, indicating that the mother was probably immunized from a prior pregnancy. METHODS AND RESULTS

Severe neonatal alloimmune thrombocytopenia caused by maternal sensitization against a new low‐frequency alloantigen (Domb) located on platelet glycoprotein IIIa

A total of 35 human platelet (PLT) alloantigens (HPAs) have been identified to date, and all have been implicated in fetal and neonatal alloimmune thrombocytopenia (FNAIT), in which maternal antibodies clear fetal and neonatal PLTs that carry an incompatible HPA inherited from the father. All of the most recently discovered HPAs occur with low frequency ( 0.1%). We report a case of FNAIT, in which maternal serum contained immunoglobulin (Ig)G antibodies reactive with a new low-frequency HPA localized on paternal PLT glycoprotein (GP)IIIa. The first pregnancy of Latino or Hispanic parents resulted in an infant born with 6 3 10 PLTs/L and intracranial hemorrhage. The infant was treated with a single PLT transfusion and single dose of intravenous immunoglobulin G (IVIG) and PLT counts recovered to normal levels.