Edwin Massey

Ten years of hemovigilance reports of transfusion‐related acute lung injury in the United Kingdom and the impact of preferential use of male donor plasma

BACKGROUND AND METHODS: From 1996 through 2006, 195 cases were reported as transfusion‐related acute lung injury (TRALI) to the Serious Hazards of Transfusion scheme and from 1999 onward classified by probability, using clinical features and HLA and/or HNA typing. From late 2003, the National Blood Service provided 80 to 90 percent of fresh‐frozen plasma (FFP) and plasma for platelet (PLT) pools from male donors.

Effect of high throughput RHD typing of fetal DNA in maternal plasma on use of anti-RhD immunoglobulin in RhD negative pregnant women: prospective feasibility study.

Objectives To assess the feasibility of applying a high throughput method, with an automated robotic technique, for predicting fetal RhD phenotype from fetal DNA in the plasma of RhD negative pregnant women to avoid unnecessary treatment with anti-RhD immunoglobulin. Design Prospective comparison of fetal RHD genotype determined from fetal DNA in maternal plasma with the serologically determined fetal RhD phenotype from cord blood. Setting Antenatal clinics and antenatal testing laboratories in the Midlands and north of England and an international blood group reference laboratory. Participants Pregnant women of known gestation identified as RhD negative by an antenatal testing laboratory. Samples from 1997 women were taken at or before the 28 week antenatal visit. Main outcome measures Detection rate of fetal RhD from maternal plasma, error rate, false positive rate, and the odds of being affected given a positive result. Results Serologically determined RhD phenotypes were obtained from 1869 cord blood samples. In 95.7% (n=1788) the correct fetal RhD phenotype was predicted by the genotyping tests. In 3.4% (n=64) results were either unobtainable or inconclusive. A false positive result was obtained in 0.8% (14 samples), probably because of unexpressed or weakly expressed fetal RHD genes. In only three samples (0.2%) were false negative results obtained. If these results had been applied as a guide to treatment, only 2% of the women would have received anti-RhD unnecessarily, compared with 38% without the genotyping. Conclusions High throughput RHD genotyping of fetuses in all RhD negative women is feasible and would substantially reduce unnecessary administration of anti-RhD immunoglobulin to RhD negative pregnant women with an RhD negative fetus.

Noninvasive prenatal diagnosis of fetal blood group phenotypes: current practice and future prospects

Fetuses of women with alloantibodies to RhD (D) are at risk from hemolytic disease of the fetus and newborn, but only if the fetal red cells are D‐positive. In such pregnancies, it is beneficial to determine fetal D type, as this will affect the management of the pregnancy. It is possible to predict, with a high level of accuracy, fetal blood group phenotypes from genotyping tests on fetal DNA. The best source is the small quantity of fetal DNA in the blood of pregnant women, as this avoids the requirement for invasive procedures of amniocentesis or chorionic villus sampling (CVS). Many laboratories worldwide now provide noninvasive fetal D genotyping as a routine service for alloimmunized women, and some also test for c, E, C and K.

Maturing reticulocytes internalize plasma membrane in glycophorin A–containing vesicles that fuse with autophagosomes before exocytosis

The erythrocyte is one of the best characterized human cells. However, studies of the process whereby human reticulocytes mature to erythrocytes have been hampered by the difficulty of obtaining sufficient numbers of cells for analysis. In the present study, we describe an in vitro culture system producing milliliter quantities of functional mature human adult reticulocytes from peripheral blood CD34(+) cells. We show that the final stage of reticulocyte maturation occurs by a previously undescribed mechanism in which large glycophorin A-containing vesicles forming at the cytosolic face of the plasma membrane are internalized and fuse with autophagosomes before expulsion of the autophagosomal contents by exocytosis. Early reticulocyte maturation is characterized by the selective elimination of unwanted plasma membrane proteins (CD71, CD98, and β1 integrin) through the endosome-exosome pathway. In contrast, late maturation is characterized by the generation of large glycophorin A-decorated vesicles of autophagic origin.

Diagnostic accuracy of routine antenatal determination of fetal RHD status across gestation: population based cohort study

Objectives To assess the accuracy of fetal RHD genotyping using cell-free fetal DNA in maternal plasma at different gestational ages. Design A prospective multicentre cohort study. Setting Seven maternity units in England. Participants RhD negative pregnant women who booked for antenatal care before 24 weeks’ gestation. Interventions Women who gave consent for fetal RHD genotyping had blood taken at the time of booking for antenatal care and, when possible, at other routine visits such as for Down’s syndrome screening between 11 and 21 weeks’ gestation, at the anomaly scan at 18-21 weeks, and in the third trimester when blood was taken for the routine antibody check. The results of cord blood analysis, done routinely in RhD negative pregnancies, were also obtained to confirm the fetal RHD genotyping. Main outcome measures The accuracy of fetal RHD genotyping compared with RhD status predicted by cord blood serology. Results Up to four analyses per woman were performed in 2288 women, generating 4913 assessable fetal results. Sensitivity for detection of fetal RHD positivity was 96.85% (94.95% to 98.05%), 99.83% (99.06% to 99.97%), 99.67% (98.17% to 99.94%), 99.82% (98.96% to 99.97%), and 100% (99.59% to 100%) at <11, 11-13, 14-17, 18-23, and >23 completed weeks’ gestation, respectively. Before 11 weeks’ gestation 16/865 (1.85%) babies tested were falsely predicted as RHD negative. Conclusions Mass throughput fetal RHD genotyping is sufficiently accurate for the prediction of RhD type if it is performed from 11 weeks’ gestation. Testing before this time could result in a small but significant number of babies being incorrectly classified as RHD negative. These mothers would not receive anti-RhD immunoglobulin, and there would be a risk of haemolytic disease of the newborn in subsequent pregnancies.

Guideline on the investigation and management of acute transfusion reactions Prepared by the BCSH Blood Transfusion Task Force

Although acute non‐haemolytic febrile or allergic reactions (ATRs) are a common complication of transfusion and often result in little or no morbidity, prompt recognition and management are essential. The serious hazards of transfusion haemovigilance organisation (SHOT) receives 30–40 reports of anaphylactic reactions each year. Other serious complications of transfusion, such as acute haemolysis, bacterial contamination, transfusion‐related acute lung injury (TRALI) or transfusion‐associated circulatory overload (TACO) may present with similar clinical features to ATR.

BCSH guideline for the use of anti-D immunoglobulin for the prevention of haemolytic disease of the fetus and newborn.

H. Qureshi,1 E. Massey,2 D. Kirwan,3 T. Davies,4 S. Robson,5 J. White,6 J. Jones7 & S. Allard8 1Department of Haematology, University Hospitals of Leicester, Leicester, UK, 2NHS Blood & Transplant, Bristol, UK, 3NHS Fetal Anomaly Screening Programme, UK National Screening Committee, University of Exeter, Exeter, UK, 4NHS Blood & Transplant, Manchester, UK, 5Department of Fetal Medicine, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK, 6UKNEQAS Blood Transfusion Laboratory Practice, West Hertfordshire Trust, Hertfordshire, UK, 7Welsh Blood Service, Pontyclun, UK, and 8NHS Blood and Transplant, London, UK

The ins and outs of human reticulocyte maturation: autophagy and the endosome/exosome pathway.

The maturation of reticulocytes into functional erythrocytes is a complex process requiring extensive cytoplasmic and plasma membrane remodeling, cytoskeletal rearrangements and changes to cellular architecture. Autophagy is implicated in the sequential removal of erythroid organelles during erythropoiesis, although how this is regulated during late stages of erythroid differentiation, and the potential contribution of autophagy during reticulocyte maturation, remain unclear. Using an optimized ex vivo differentiation system for human erythropoiesis, we have observed that maturing reticulocytes are characterized by the presence of one or few large vacuolar compartments. These label strongly for glycophorin A (GYPA/GPA) which is internalized from the plasma membrane; however, they also contain organellar remnants (ER, Golgi, mitochondria) and stain strongly for LC3, suggesting that they are endocytic/autophagic hybrid structures. Interestingly, we observed the release of these vacuoles by exocytosis in maturing reticulocytes, and speculate that autophagy is needed to concentrate the final remnants of the reticulocyte endomembrane system in autophagosome/endosome hybrid compartments that are primed to undergo exocytosis.

Ninety-six suspected transfusion related acute lung injury cases: investigation findings and clinical outcome.

Abstract Transfusion related acute lung injury (TRALI) is one of the complications of blood transfusion and can result in major morbidity or mortality. The diagnosis depends upon the application of strict clinical criteria defining acute lung injury (ALI) and a temporal relationship to blood transfusion. We present the clinical and immunogenetic findings of 96 suspected TRALI cases investigated between 1996 and 2004. During this time period the national haemovigilance scheme (UK) defined TRALI as a reaction occurring either during or within 24 h of blood transfusion. Using clinical, laboratory and post mortem evidence, 64/96 cases could be defined as TRALI in our series. Sensitive techniques were employed to screen for HLA class I, class II and granulocyte specific antibodies in donor serum. Donor derived antibodies were detected in 58/64 (90%) of cases. Recipient derived DNA or cells were not always available but incompatibility was confirmed by the presence of the cognate antigen on recipient leucocytes or by crossmatching in 47/64 (73%) of cases. Cases referred prior to 2001 were not tested for HLA class II antibodies. By applying strict clinical criteria and using sensitive techniques a white blood cell antibody mediated immunological pathophysiology can be implicated in the majority TRALI cases.

Neutrophil function is preserved in a pooled granulocyte component prepared from whole blood donations

Whole blood‐derived granulocytes (buffy coats) are issued as an alternative to apheresis donations, but are heavily contaminated with red cells and platelets and there is minimal in vitro data describing their functionality. We developed a purer pooled granulocyte component (PGC) from whole blood donations by pooling 10 ABO‐matched buffy coats with 400 ml of platelet additive solution (SSP+) and re‐centrifuging. The PGC was irradiated (25–50 Gy) and neutrophil viability, chemotaxis, phagocytosis and respiratory burst activity were determined by flow cytometry. Results from 13 PGC at 16–20 h following donation were compared with those obtained from 20 standard individual buffy coats and with fresh whole blood. The PGC contained similar numbers of neutrophils (approximately 0·9 × 1010) with a reduced volume and haemoglobin content when compared with 10 individual buffy coats. Neutrophils in the PGC maintained >90% viability, oxidative burst and phagocytic activity and their ability to migrate towards a chemoattractant 16–20 h following donation, which is similar to results obtained with either fresh whole blood or standard buffy coats. Therefore, neutrophil function in the PGC was preserved 16–20 h following donation, but this product had significantly lower red cell contamination compared with 10 buffy coats, which are currently transfused.