M. De Haas

Soluble Fc gamma receptor III in human plasma originates from release by neutrophils.

FcRIII (the CD16-antigen), a low affinity receptor for IgG, is expressed by neutrophils, natural killer lymphocytes, and macrophages. We have developed a sensitive radioimmunoassay to quantify FcRIII. A soluble form of FcRIII was identified in human plasma. Immunoprecipitation of FcRIII from plasma showed that the plasma form of FcRIII has an identical electrophoretic mobility as the FcRIII expressed by neutrophils. Moreover, the plasma form of FcRIII exhibited the same polymorphism as does the neutrophil FcRIII. The neutrophil expresses the phosphatidylinositol-linked form of FcRIII, encoded by the gene FcRIII-1. Because it is not known whether this gene is also active in nonhematopoietic cells, we analyzed patients with an acquired clonal disorder of their hematopoietic cells, paroxysmal nocturnal hemoglobinuria (PNH). PNH patients appeared to have a strongly reduced expression of FcRIII on their neutrophils. The concentration of FcRIII in the plasma of these patients was also reduced, indicating that plasma FcRIII originates from neutrophils. A patient deficient in FcRIII-1 but with a normal expression of FcRIII-2 had no soluble FcRIII in her plasma, also indicating that plasma FcRIII originates from neutrophils. The electrophoretic mobility of the protein backbone of plasma FcRIII and FcRIII released by activated neutrophils was identical, whereas deglycosylated FcRIII obtained from a lysate of neutrophils migrated slower. This indicates that plasma FcRIII originates from activation-induced release by neutrophils. Stimulation of neutrophils or neutrophil cytoplasts (closed membrane vesicles filled with cytoplasm) with low concentrations of FMLP (10(-9)-10(-8) M) or phorbol myristate acetate (1-10 ng/ml) induced a dose-dependent release of FcRIII. The plasma concentration of FcRIII was relatively constant (range 40-280% of the mean). Soluble FcRIII was also detected in inflamed joint fluids of arthritis patients, suggesting that FcRIII is also released by activated neutrophils in vivo.

Effect of screening for red cell antibodies, other than anti‐D, to detect hemolytic disease of the fetus and newborn: a population study in the Netherlands

BACKGROUND: Hemolytic disease of the fetus and newborn (HDFN) is a severe disease, resulting from maternal red cell (RBC) alloantibodies directed against fetal RBCs. The effect of a first‐trimester antibody screening program on the timely detection of HDFN caused by antibodies other than anti‐D was evaluated.

International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology: Berlin report.

The International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology convened during the International congress in Cancun, July 2012. This report details the newly identified antigens in existing blood group systems and presents three new blood group systems.

Noninvasive fetal blood group genotyping of rhesus D, c, E and of K in alloimmunised pregnant women: evaluation of a 7‐year clinical experience

Please cite this paper as: Scheffer P, van der Schoot C, Page‐Christiaens G, de Haas M. Noninvasive fetal blood group genotyping of rhesus D, c, E and of K in alloimmunised pregnant women: evaluation of a 7‐year clinical experience. BJOG 2011;118:1340–1348.

Screening in pregnancy for fetal or neonatal alloimmune thrombocytopenia: systematic review

Please cite this paper as: Kamphuis M, Paridaans N, Porcelijn L, De Haas M, van der Schoot C, Brand A, Bonsel G, Oepkes D. Screening in pregnancy for fetal or neonatal alloimmune thrombocytopenia: systematic review. BJOG 2010;117:1335–1343.

Blood group genotyping: from patient to high‐throughput donor screening

Blood group antigens, present on the cell membrane of red blood cells and platelets, can be defined either serologically or predicted based on the genotypes of genes encoding for blood group antigens. At present, the molecular basis of many antigens of the 30 blood group systems and 17 human platelet antigens is known. In many laboratories, blood group genotyping assays are routinely used for diagnostics in cases where patient red cells cannot be used for serological typing due to the presence of auto‐antibodies or after recent transfusions. In addition, DNA genotyping is used to support (un)‐expected serological findings. Fetal genotyping is routinely performed when there is a risk of alloimmune‐mediated red cell or platelet destruction. In case of patient blood group antigen typing, it is important that a genotyping result is quickly available to support the selection of donor blood, and high‐throughput of the genotyping method is not a prerequisite. In addition, genotyping of blood donors will be extremely useful to obtain donor blood with rare phenotypes, for example lacking a high‐frequency antigen, and to obtain a fully typed donor database to be used for a better matching between recipient and donor to prevent adverse transfusion reactions. Serological typing of large cohorts of donors is a labour‐intensive and expensive exercise and hampered by the lack of sufficient amounts of approved typing reagents for all blood group systems of interest. Currently, high‐throughput genotyping based on DNA micro‐arrays is a very feasible method to obtain a large pool of well‐typed blood donors. Several systems for high‐throughput blood group genotyping are developed and will be discussed in this review.

The role of Fcγ receptor polymorphisms and C3 in the immune defence against Neisseria meningitidis in complement-deficient individuals

Individuals with either a late (C5–9) complement component deficiency (LCCD) or properdin deficiency are at increased risk to develop meningococcal disease, often due to serogroups W135 and Y. Anti‐meningococcal defence in both LCCD persons and properdin‐deficient individuals without bactericidal antibodies depends mainly on phagocytosis. Three types of opsonin receptors are involved in phagocytosis by polymorphonuclear cells (PMN). These represent the polymorphic FcγRIIa (CD32) and FcγRIIIb (CD16b) receptors, and the C3 receptor CR3 (CD11b/CD18). When the distribution of FcγRIIa and FcγRIIIb allotypes was assessed in 15 LCCD and in 15 properdin‐deficient patients with/without previous meningococcal disease, we found the combination of FcγRIIa‐R/R131 with FcγRIIIb‐NA2/NA2 allotypes to be associated with previous meningococcal disease (odds ratio 13·9, Fisher’s test P = 0·036). No such relation was observed in the properdin‐deficient patients. The importance of FcγRIIa allotypes was also demonstrated using in vitro phagocytosis assays. PMN from FcγRIIa‐R/R131 homozygous donors internalized IgG2 opsonized meningococci W135 significantly (P < 0·05) less than PMN from FcγRIIa‐H/H131 donors. When properdin‐deficient serum was tested, it was observed that reconstitution with properdin resulted in enhanced PMN phagocytosis of the W135 meningococci (P = 0·001). This enhanced phagocytosis was parallelled by an increase in C3 deposition onto the opsonized meningococci W135 (r = 0·6568, P = 0·01). We conclude that the occurrence of meningococcal disease in LCCD patients is associated with certain FcγR allotypes. Properdin‐deficient individuals are susceptible to meningococcal disease because of an insufficient C3 deposition on the surface of meningococci, resulting in insufficient phagocytosis.

Analysis of CD20-dependent cellular cytotoxicity by G-CSF-stimulated neutrophils

Rituximab, a chimeric CD20 monoclonal antibody (mAb), is widely used in the treatment of patients with low-grade non-Hodgkins lymphoma. Possible anti-tumour mechanisms involve complement-mediated lysis and/or antibody-dependent cellular cytotoxicity (ADCC). Because G-CSF greatly enhances the cytotoxicity of neutrophils (PMN) in ADCC, the clinical efficacy of rituximab might be enhanced by the addition of G-CSF. Therefore, we investigated the neutrophil-mediated CD20-dependent cellular cytotoxicity in B cell lines. In contrast to previous studies by others, we found that G-CSF-primed PMN are capable of functioning as effector cells in CD20-dependent cellular cytotoxicity. However, HLA class II mAbs were far more effective. The differences between HLA class II- and CD20-mediated PMN-ADCC were not due to: (1) the use of chimeric (hIgG1) mAbs vs mIgG2a mAbs; (2) HLA class II-induced apoptosis as an ‘ADCC-sensitising’ mechanism; (3) CD20-induced inhibition of ADCC; (4) inferior membrane mobility of CD20. Analysis of Fcγreceptor (FcγR) involvement showed that although CD20-induced ADCC was mediated mainly via FcγRI, for optimal lysis FcγRI and FcγRII were both required. In contrast, in HLA class II-dependent ADCC both FcγRI and II were capable of independently inducing maximum lysis. The mechanism underlying these differences in FcγR-binding and activation remains to be elucidated.

Clearance of red cells by monoclonal IgG3 anti-D in vivo is affected by the VF polymorphism of Fc gamma RIIIa (CD16)

Human red cells (RBC) coated with IgG anti‐D are cleared from the circulation to the spleen by macrophages which express IgG receptors (Fcγ R). Polymorphisms of Fcγ RIIa and Fcγ RIIIa affect IgG binding in vitro, and may alter the efficiency of clearance of immune complexes in vivo. In a RBC clearance study, 22 Rh D‐negative subjects were given 100–400 µg human monoclonal or polyclonal IgG anti‐D i.m. followed 48 h later by 51Cr‐labelled D+ RBC. The half lives of the infused D+ RBC were determined, together with the coating levels of anti‐D on the D+ RBC. Fcγ RIIA and FcγRIIIA genotyping was performed. Large ranges of phagocytosis and extracellular lysis of RBC in vitro, and of half lives of RBC in vivo, were observed. Clearance of RBC coated with monoclonal IgG3 anti‐D (BRAD‐3) was more rapid in five subjects homozygous for Fcγ RIIIa‐F/F158 than in three subjects expressing the Fcγ RIIIa‐V158 allele (P = 0·024). This effect was not observed, however, for those individuals given polyclonal anti‐D. There was also no significant difference in the efficiency of RBC destruction in vitro or of RBC clearance in vivo between the subjects analysed for individual genotypes or alleles or combinations of alleles. In conclusion, the presence of the Fcγ RIIIa‐V158 allele compromised the efficiency of removal of RBC coated with IgG3 anti‐D.

Noninvasive fetal genotyping of human platelet antigen-1a

Please cite this paper as: Scheffer P, Ait Soussan A, Verhagen O, Page‐Christiaens G, Oepkes D, de Haas M, van der Schoot C. Noninvasive fetal genotyping of human platelet antigen‐1a. BJOG 2011;118:1392–1395.