Classification of major and minor blood group antigens in the Kuwaiti Arab population.

Ethnic differences in blood group frequencies might result in clinically important mismatches for transfusions. Arab people represent a large population for which no comprehensive database of red cell genotypes is available and Kuwaitis are no exception. For instance, the Rh blood group is the most elaborate blood group system that shows a high degree of polymorphism among different ethnic groups, there has been little classification of RH alleles in Arab people. Blood samples from 917 Kuwaiti Arab donors in the Kuwaiti Bone Marrow registry were tested with a single-nucleotide polymorphism DNA array. Blood group antigen prevalence were compared to known prevalence in European populations. Multiple subjects were found to be antigen negative for certain phenotypes that is considered rare by the American Rare Donor Program; (Fy(a-,b-) and Kell). In the minor blood group antigens, the FYA allele was predicted to be low in Kuwaitis, when compared to other published accounts. The frequencies of MNS blood antigens in the study population were not significantly different from those reported for European/Caucasian populations. The predicted frequency of the Diego blood group antigen was similar to that observed in a South Asian population. The weak D 1, 2, 3 phenotypes were not prevalent in the Kuwaiti Arab population; however, other RHD variants were detected. We provided information about blood group antigens in the Kuwaiti population that is important for guiding transfusion care. Several interesting findings demonstrated clinical importance, which could be useful in developing transfusion medicine policies and approaches.

Determination of fetal RHD type in plasma of RhD negative pregnant women.

Alloimmunization against the RhD antigen is the most common cause of hemolytic disease of the fetus and newborn. Antenatal anti-D prophylaxis in addition to postnatal anti-D prophylaxis reduces the number of RhD-immunizations compared to only postnatal administration. Cell-free fetal DNA released from the apoptotic trophoblastic placental cells into the maternal circulation can be used to determine the fetal RHD type in a blood sample from an RhD negative mother. Based on this typing, antenatal anti-D prophylaxis can be recommended only to RhD negative women carrying an RhD positive fetus, since only these women are at risk of developing anti-D. The objective was to establish and validate a method for non-invasive fetal RHD typing. The fetal RHD genotype was studied in 373 samples from RhD negative pregnant women (median gestational week 24). DNA extracted from plasma was analyzed for the presence/absence of RHD exon 7 and 10 in a real-time PCR. The RHD genotype of the fetus was compared with the serological RhD type of the newborn. In 234 samples, the fetal RHD test was positive and in 127 samples negative. There was one false positive and no false negative results. In 12 samples, the fetal RHD type could not be determined, in all of them due to a maternal RHD gene. This method gives a reliable detection of fetal RHD positivity in plasma from RhD negative pregnant women. Antenatal anti-D prophylaxis based on the predicted fetal RhD type will avoid unnecessary treatment of pregnant women carrying an RhD negative fetus.

Anti-D reagents should be chosen accordingly to the prevalence of D variants in the obstetric population.

BACKGROUND: Resolving ambiguous results of D antigen typing is crucial for appropriate and rational administration of anti-D immunoprophylaxis and transfusion practice in obstetric population. The aim of the study was to establish selection criteria of anti-D reagents for our population. METHODS: A total of 12 689 samples from primiparous women in Split-Dalmatia County, Croatia, were typed for RhD antigen during the period of 5 years. Ambiguous results were submitted to additional serologic investigation and genotyping. RHD genotyping was performed by commercial genotyping kits (Ready Gene weak D ® and Ready gene CDE, Inno-Train, Kronberg, Germany). Relative frequencies and accompanying 95% confidence intervals were used to estimate the prevalence of variants. RESULTS: The prevalence of D variants was 0.42% (95% CI 0.31; 0.53). The most common partial D variant was D Va (RHD*05.05), with the prevalence of 0.08% (95% CI 0.03; 0.13). All weak D variants were weak D types 1, 2 and 3 (RHD*weak D type 1, RHD*weak D type 2, RHD*weak D type 3). Weak D samples were distinguishable from partial D in routine typing due to the difference in reactivity of partial D samples with clones D7B8 and RUM-1. Cell line RUM-1 gives weak or negative reactions with partial DVa category. CONCLUSION: The most common partial D variant in our population is DVa. It is recommended to use cell lines which do not strongly agglutinate DVa variant in routine RhD typing. The appropriate choice of reagents will enable the serology methods to recognize the cases in which RHD genotyping is required.

Evolution of the RH gene family in vertebrates revealed by brown hagfish (Eptatretus atami) genome sequences.

In vertebrates, there are four major genes in the RH (Rhesus) gene family, RH, RHAG, RHBG, and RHCG. These genes are thought to have been formed by the two rounds of whole-genome duplication (2R-WGD) in the common ancestor of all vertebrates. In our previous work, where we analyzed details of the gene duplications process of this gene family, three nucleotide sequences belonging to this family were identified in Far Eastern brook lamprey (Lethenteron reissneri), and the phylogenetic positions of the genes were determined. Lampreys, along with hagfishes, are cyclostomata (jawless fishes), which is a sister group of gnathostomata (jawed vertebrates). Although those results suggested that one gene was orthologous to the gnathostome RHCG genes, we did not identify clear orthologues for other genes. In this study, therefore, we identified three novel cDNA sequences that belong to the RH gene family using de novo transcriptome analysis of another cyclostome: the brown hagfish (Eptatretus atami). We also determined the nucleotide sequences for the RHBG and RHCG genes in a red stingray (Dasyatis akajei), which belongs to the cartilaginous fishes. The phylogenetic tree showed that two brown hagfish genes, which were probably duplicated in the cyclostome lineage, formed a cluster with the gnathostome RHAG genes, whereas another brown hagfish gene formed a cluster with the gnathostome RHCG genes. We estimated that the RH genes had a higher evolutionary rate than the RHAG, RHBG, and RHCG genes. Interestingly, in the RHBG genes, only the bird lineage showed a higher rate of nonsynonymous substitutions. It is likely that this higher rate was caused by a state of relaxed functional constraints rather than positive selection nor by pseudogenization.

[Molecular serological characteristics of weak D antigen types of the Rhesus system]. / Molekulyarno-serologicheskie kharakteristiki tipov slabogo antigena D sistemy rezus.

AIM: to estimate the spread of weak D antigen types of the Rhesus system in the citizens of the Russian Federation and a possibility of serologically identifying these types. SUBJECTS AND METHODS: The red blood cells and DNA of people with weakened expression of D antigen were investigated using erythrocyte agglutination reaction in salt medium (2 methods); agglutination reaction in the gel columns containing IgM + IgG anti-D antibodies, indirect antiglobulin test with IgG anti-D antibodies (2 methods); polymerase chain reaction to establish the type of weak D. RESULTS: A rhesus phenotype was determined in 5100 people in 2014-2015. The weakened agglutinable properties of red blood cells were detected in 102 (2%) examinees. 63 examinees underwent genotyping to identify the variants of the weak D antigen, which identified 6 weak D types. There were the most common weak D types 3 (n=31 (49.2%)) and weak D type 1 (n=18 (28.6%)), including weak D type 1.1 in one (1.6%) case. The other 4 weak D antigen types were as follows: weak D type 2 (14.3% (n=9)), weak D type 15 (4.8% (n=3)), weak D type 4.2 (DAR) (1.6% (n=1)) and weak D type 6 (1.6% (n=1)). The antiglobulin test in the gel column containing antiglobulin serum was the most sensitive serological assay to identify the weak D antigen. Only a molecular test could establish weak D type 15 in 2 samples of red blood cells with Ccdee and ccdEe phenotypes. CONCLUSION: The weak D antigen could be serologically identified in 96.8% of cases. When testing for weak D, particular attention should be given to people with the D-negative phenotype who had the C or E antigens. Our investigations conducted for the first time in Russia will be able to improve the immunological safety of red blood cell-containing medium transfusions for patients.

Distribution and clinal trends of the ABO and Rh genes in select Middle Eastern countries.

An understanding of the ABO and Rh blood group systems is important for blood transfusions and is also pertinent due to their potential association with certain morbidities and susceptibilities to infections. To investigate the diversity and differentiation of the ABO and Rh loci in Middle Eastern populations, data from twelve representative Middle Eastern populations were analyzed. Six populations were in conformity with Hardy-Weinberg equilibrium at the ABO locus. The pooled heterozygosity at both loci was calculated to be highest in the sample from Jordan and lowest in Bahrain. Heterogeneity was pronounced in the Northern compared to the Southern Middle Eastern populations. Overall, the absolute gene diversity was 0.0046 and gene differentiation was calculated to be 0.0100. Genetic diversity of the studied loci across all populations (HT) was estimated to be 0.4594, while the diversity within the populations (HS) was 0.4548. Nei's genetic distance analyses revealed highest affinities between the populations of Kuwait and Qatar, Oman and Yemen, and between Qatar and the United Arab Emirates. These results were displayed through a UGPMA dendrogram and principal component analyses, which established clustering of certain populations. Clinal trends of the allelic systems were observed by generating contour maps that allow a detailed appreciation of the distributions of alleles across the geography of the Arabian Peninsula and the Middle East. Taken together, these analyses are helpful in understanding the differentiation of blood group loci and for designing prospective studies for establishing the associations of these loci with health variables in the populations studied.

Evaluation of an automated microplate technique in the Galileo system for ABO and Rh(D) blood grouping.

BACKGROUND: A number of automated devices for pretransfusion testing have recently become available. This study evaluated the Immucor Galileo System, a fully automated device based on the microplate hemagglutination technique for ABO/Rh (D) determinations. METHODS: Routine ABO/Rh typing tests were performed on 13,045 samples using the Immucor automated instruments. Manual tube method was used to resolve ABO forward and reverse grouping discrepancies. D-negative test results were investigated and confirmed manually by the indirect antiglobulin test (IAT). RESULTS: The system rejected 70 tests for sample inadequacy. 87 samples were read as "No-type-determined" due to forward and reverse grouping discrepancies. 25 tests gave these results because of sample hemolysis. After further tests, we found 34 tests were caused by weakened RBC antibodies, 5 tests were attributable to weak A and/or B antigens, 4 tests were due to mixed-field reactions, and 8 tests had high titer cold agglutinin with blood qualifications which react only at temperatures below 34 degrees C. In the remaining 11 cases, irregular RBC antibodies were identified in 9 samples (seven anti-M and two anti-P) and two subgroups were identified in 2 samples (one A1 and one A2) by a reference laboratory. As for D typing, 2 weak D+ samples missed by automated systems gave negative results, but weak-positive reactions were observed in the IAT. CONCLUSIONS: The Immucor Galileo System is reliable and suited for ABO and D blood groups, some reasons may cause a discrepancy in ABO/D typing using a fully automated system. It is suggested that standardization of sample collection may improve the performance of the fully automated system.

Serologic and molecular investigations of DAR1 (weak D Type 4.2), DAR1.2, DAR1.3, DAR2 (DARE), and DARA.

BACKGROUND: The partial D variant DAR1 (weak D Type 4.2) is caused by three single-point mutations, 602C>G, 667T>G, and 1025T>C. Here we report a molecular study on different D variants belonging to the DAR category (DAR1, DAR1.2, DAR1.3, and DAR2) and their serologic data. STUDY DESIGN AND METHODS: A total of 42 samples belonging to the DAR category were screened for the presence of the silent mutations 744C>T and 957G>A. The samples were phenotyped for RhD and RhCE, characterized for RhD epitope expression, and sequenced for RHD exons. Flow cytometry was performed to determine RhD antigen density. RESULTS: The silent mutation 744C>T was found in all six samples previously typed as RHD*DAR2 (602C>G, 667T>G, 957G>A, 1025T>C). In addition to the three nucleotide changes originally reported for the RHD*DAR1 allele, the silent mutations 744C>T and 957G>A were found in 14 of 16 samples previously typed as RHD*DAR1. In the remaining two samples one additional silent mutation, 744C>T, was found. Serologically the DAR1.2 and DAR1.3 samples analyzed in this study showed no distinct difference in their anti-D reaction pattern compared to each other. The anti-D reaction pattern of DARA/DAR2 showed some distinct differences compared to those of DAR1.2 and DAR1.3. CONCLUSION: RHD*DARA and RHD*DAR2 are the same allele. Furthermore, the alleles RHD*DAR1.2 and RHD*DAR1.3 both exist; however, the silent mutation 957G>A (V319) showed no influence on the RhD phenotype.

D category IV: a group of clinically relevant and phylogenetically diverse partial D.

BACKGROUND: The D typing strategies in several European countries protect carriers of D category VI (DVI) from anti-D immunization but not carriers of other partial D. Besides DVI, one of the clinically most important partial D is D category IV (DIV). A detailed description and direct comparison of the different DIV types was missing. STUDY DESIGN AND METHODS: RHD nucleotide sequences were determined from genomic DNA. D epitope patterns were established with commercial monoclonal anti-D panels. RESULTS: DIV comprises several variants of the D antigen with distinct serology, molecular structures, evolutionary origins, and ethnic prevalences. The DIV phenotype is determined by 350H shared by all, but not limited to, DIV variants which are further divided into DIVa and DIVb. The DIVa phenotype is expressed by DIV Type 1.0 harboring 350H and the dispersed amino acids 62F, 137V, and 152T. The DIVb phenotype is expressed by DIV Type 3 to Type 5 representing RHD-CE-D hybrids. Four of the six postulated DIV variants were encountered among 23 DIV samples analyzed. Of 12 DIV carriers with anti-D, 10 were female and seven likely immunized by pregnancy. Two DIV-related alleles are newly described: DWN, which differs from DIV Type 4 by 350D and epitope pattern. DNT carries 152T, known to cause a large D antigen density. CONCLUSION: DIV alleles arose from at least two independent evolutionary events. DIV Type 1.0 with DIVa phenotype belongs to the oldest extant human RHD alleles. DIV Type 2 to Type 5 with DIVb phenotype arose from more recent gene conversions. Anti-D immunization, especially dreaded in pregnancies, will be avoided not only in carriers of DVI but also in carriers of other D variants like DIV, if our proposed D typing strategy is adopted.

Novel RHD alleles with weak hemagglutination and genetic Exon 9 diversity: weak D Types 45.1, 75, and 76.

BACKGROUND: Molecular variant RHD allele analysis is best complemented by detailed characterization of the associated D phenotype. STUDY DESIGN AND METHODS: Variant D types were characterized using molecular typing, RHD sequencing, extended serologic D antigen investigations, and flow cytometric D antigen quantification. RESULTS: We discovered three novel weak D types termed weak D Types 45.1, 75, and 76 with RHD nucleotide substitutions coding for amino acid exchanges in predicted intracellular RhD polypeptide stretches; antigen densities of approximately 1.990, 900, and 240 D sites per red blood cell were found, respectively. Adsorption-elution technique-supported D epitope mapping of these three weak D types demonstrated the expression of all tested D epitopes. Initial molecular typing of the three investigated samples by RHD gene exon scanning polymerase chain reaction using sequence-specific priming yielded a negative reaction for A1193 located in RHD Exon 9 and could be explained by specific mutations for weak D Types 45.1 (C818T, G1195A), 75 (G1194C), and 76 (A1215C). CONCLUSION: All novel weak D types expressed all tested D epitopes. It is of interest that for weak D Types 45.1, 75, and 76, similar alleles with a maximal divergence of one amino acid only, that is, weak D Types 45, 41, and 68, respectively, have been reported so far.

Routine testing of fetal Rhesus D status in Rhesus D negative women using cell-free fetal DNA: an investigation into the preferences and information needs of women.

OBJECTIVE: The goal of this study is to investigate women's preferences and information needs for routine implementation of fetal Rhesus D (RhD) typing using cell-free fetal DNA. METHODS: A questionnaire was developed following focus groups and interviews with both health professionals and RhD negative (RhD-) women offered fetal RhD genotyping within a research study and distributed to RhD- women attending routine antenatal appointments in four National Health Service hospitals. Current knowledge of blood types, anti-D administration, fetal RhD genotyping and future practices were explored. RESULTS: A total of 19 respondents participated in interviews and focus groups, and 270 respondents completed the questionnaires. Questionnaire respondents overwhelmingly felt that the test should be offered to all RhD- women (92.1%), and 75.9% said that they would accept this test. Most were happy to have the test even if it involved extra blood tests (89.3%) or appointments (79%). The knowledge of blood groups was poor. Although 90.7% knew that the baby could have a different blood group from themselves, only 34% knew that blood groups are inherited from both parents. More than 40% were not aware that anti-D would not be required if their baby was RhD-. CONCLUSIONS: Women would welcome the introduction of routine fetal RhD genotyping. Information leaflets and training of midwives will be essential for implementation to ensure good understanding regarding testing.

Frequencies of some human genetic markers and their association with Plasmodium falciparum malaria in the Niger Delta, Nigeria.

BACKGROUND & OBJECTIVES: There is paucity of information on the association between Plasmodium falciparum malaria and some human genetic markers in the Niger Delta region of Nigeria. Hence, a study was undertaken in children to assess the current level of subclinical malaria due to P. falciparum. METHODS: Blood groups ABO and Rhesus factor, haemoglobin electrophoretic pattern, G-6-PD deficiency status and malaria were determined among 240 apparently healthy children in a crosssectional descriptive study using standard procedures. RESULTS: The prevalence of P. falciparum malaria in this region was high (27.5%). Blood group O (51.3%) dominated the study population, followed by B (23.8%), A (21.3%), and AB (3.8%). Rhesus D positive accounted for 91.3% while Rh D negative was 8.7%. Sickle-cell trait (HbAS) prevalence was 12.5% while HbAA accounted for 87.5%. In all, 5.42% of the children were G-6- PD deficient while 94.58% had normal G-6-PD status. Chi-square analysis revealed that only blood group O and Rh D negative had a significant association with P. falciparum malaria (chi2=4.3636, p<0.05 and chi2 = 5.760, p<0.02 respectively). No significant association was found to exist between P. falciparum malaria and other genetic markers. CONCLUSION: This study has provided the current prevalence rates of some genetic markers in a malaria endemic region of Niger Delta, Nigeria. Of all the genetic markers tested, only Blood group O and Rh D negative had significant and positive associations with P. falciparum infection.

Non-invasive prenatal determination of fetal RhD genotyping from maternal plasma: a preliminary study in Pakistan.

OBJECTIVE: To determine the accuracy of the non-invasive pre-natal real-time polymerase chain reaction based fetal RhD genotyping from maternal plasma. STUDY DESIGN: Cross-sectional study. PLACE AND DURATION OF STUDY: Juma Health Sciences Research Laboratory, The Aga Khan University Hospital, Karachi, from July to December 2008. METHODOLOGY: Cell-free plasma DNA from 21 D-negative women with D-positive spouse between 20-39 weeks of gestation was tested for the presence of exon 5 region of RhD gene using real-time polymerase chain reaction. b-globin was employed as the house-keeping gene. Sensitivity and specificity of the real-time PCR-based non-invasive fetal RhD genotyping was obtained by calculating proportion of the D-positive fetuses that were D-positive at birth as well. RESULTS: Of the 21 D-negative women 13 and 8 neonates were determined to be D-positive and D-negative, respectively, by serologic studies on cord blood samples at birth. RhD status was correctly determined in 17 of 21 cases. There were three false-positive and one false-negative results. The sensitivity and specificity of the assay was 92.3% (95% CI: 62.1, 99.6) and 62.5% (95% CI: 25.9, 89.8), respectively. The positive and negative predictive value of the assay was 80% (95% CI: 51.4, 94.7) and 83.3% (36.5, 99.1), respectively. CONCLUSION: These preliminary results demonstrate the feasibility of non-invasive pre-natal diagnosis of fetal RhD status of D-negative mothers in Pakistan.

[The red blood cell antigen terminologies]. / Les nomenclatures de groupes sanguins érythrocytaires.

Since the discovery of blood groups in humans, several hundred new red blood cell antigens have been identified. Multiple terminology modes have been used to denote each new antigen identified, but without any consistent rules, nor international consensus. This was largely due to the many discoverers of these antigens, using either letters of the alphabet, numbers, part of the patient or donor's name, place of discovery or animal names. Besides, alternative terminologies for the Rh system were implemented in the middle of the twentieth century (Rosenfield, Fisher-Race, Wiener). The International Society of Blood Transfusion described for the first time in 1980 the advantages of an alphanumeric and homogeneous nomenclature, keeping with the genetic bases of blood groups, as well as a classification of all RBC antigens within several families. A variant of this new terminology, exclusively numerical, was simultaneously established, mainly designed for computer data exchange. Nearly 30 years later, 308 red blood cell antigens are described within 30 systems, 12 collections, one 700 series and one 901 series of blood groups. Any person involved in the field of immuno-haematology must master both the usual and international nomenclatures. The Wiener nomenclature used for Rh haplotypes, still largely used today, is also important to be known. The systematic use of the international nomenclature should be strongly encouraged, either in the labelling of blood products, clinical laboratory reports or blood type cards.

Automated typing of red blood cell and platelet antigens: a whole-genome sequencing study.

BACKGROUND: There are more than 300 known red blood cell (RBC) antigens and 33 platelet antigens that differ between individuals. Sensitisation to antigens is a serious complication that can occur in prenatal medicine and after blood transfusion, particularly for patients who require multiple transfusions. Although pre-transfusion compatibility testing largely relies on serological methods, reagents are not available for many antigens. Methods based on single-nucleotide polymorphism (SNP) arrays have been used, but typing for ABO and Rh-the most important blood groups-cannot be done with SNP typing alone. We aimed to develop a novel method based on whole-genome sequencing to identify RBC and platelet antigens. METHODS: This whole-genome sequencing study is a subanalysis of data from patients in the whole-genome sequencing arm of the MedSeq Project randomised controlled trial (NCT01736566) with no measured patient outcomes. We created a database of molecular changes in RBC and platelet antigens and developed an automated antigen-typing algorithm based on whole-genome sequencing (bloodTyper). This algorithm was iteratively improved to address cis-trans haplotype ambiguities and homologous gene alignments. Whole-genome sequencing data from 110 MedSeq participants (30 × depth) were used to initially validate bloodTyper through comparison with conventional serology and SNP methods for typing of 38 RBC antigens in 12 blood-group systems and 22 human platelet antigens. bloodTyper was further validated with whole-genome sequencing data from 200 INTERVAL trial participants (15 × depth) with serological comparisons. FINDINGS: We iteratively improved bloodTyper by comparing its typing results with conventional serological and SNP typing in three rounds of testing. The initial whole-genome sequencing typing algorithm was 99·5% concordant across the first 20 MedSeq genomes. Addressing discordances led to development of an improved algorithm that was 99·8% concordant for the remaining 90 MedSeq genomes. Additional modifications led to the final algorithm, which was 99·2% concordant across 200 INTERVAL genomes (or 99·9% after adjustment for the lower depth of coverage). INTERPRETATION: By enabling more precise antigen-matching of patients with blood donors, antigen typing based on whole-genome sequencing provides a novel approach to improve transfusion outcomes with the potential to transform the practice of transfusion medicine. FUNDING: National Human Genome Research Institute, Doris Duke Charitable Foundation, National Health Service Blood and Transplant, National Institute for Health Research, and Wellcome Trust.

[Identification strategy of RHCE gene variants at the National Blood Group Reference Laboratory: impact on transfusion safety]. / Stratégie d'identification des variants du gène RHCE au Centre national de référence pour les groupes sanguins: impact sur la sécurité transfusionnelle.

The RH blood group is the most polymorphic and immunogenic blood group system. The RH locus is composed of 2 highly homologous genes: the RHD gene encoding the D polypeptide; and the RHCE gene, encoding C or c together with either E or e polypeptides. Numerous variants exist for both RHD and RHCE genes. Among them we were interested in the serological and molecular definition of numerous pre-published RHCE variants encountered in different populations. The identification of these variants is crucial to ensure the transfusion safety for patients expressing these variants. Here we propose a procedure to identify some of them and discuss the adequate transfusion strategy. This procedure has enable us to identify new variants to be presented at the symposium.

Single Molecule Fluorescence Microscopy and Machine Learning for Rhesus D Antigen Classification.

In transfusion medicine, the identification of the Rhesus D type is important to prevent anti-D immunisation in Rhesus D negative recipients. In particular, the detection of the very low expressed DEL phenotype is crucial and hence constitutes the bottleneck of standard immunohaematology. The current method of choice, adsorption-elution, does not provide unambiguous results. We have developed a complementary method of high sensitivity that allows reliable identification of D antigen expression. Here, we present a workflow composed of high-resolution fluorescence microscopy, image processing, and machine learning that - for the first time - enables the identification of even small amounts of D antigen on the cellular level. The high sensitivity of our technique captures the full range of D antigen expression (including D+, weak D, DEL, D-), allows automated population analyses, and results in classification test accuracies of up to 96%, even for very low expressed phenotypes.

Section 1B: Rh flow cytometry. Coordinator's report. Rhesus index and antigen density: an analysis of the reproducibility of flow cytometric determination.

Fifty-seven IgG monoclonal anti-D antibodies were evaluated in the Rh flow cytometry section, in which 12 laboratories participated. Staining protocols and a fluorescein (FITC)-conjugated Fab fragment goat anti-human IgG (H + L) as a secondary antibody were recommended but not mandatory. A CcDEe red blood cell (RBC) sample that was shown to be homozygous for RHD by molecular methods was supplied and used as internal 'standard RBC' throughout all experiments. An RBC panel comprising two partial D and four weak D types was supplied as well. The use of standard RBC reduced the variability of the data among the laboratories and allowed the conversion of fluorescence data into epitope densities, which were compounded in an antigen density (antigen D per RBC). The highest antigen density was determined for DVI type III, followed by DVII and weak D type 3; the lowest antigen density were determined for weak D type 1 and type 2. Nine of the 12 participating laboratories discriminated three groups of aberrant RhD that had similar Rhesus indices (RI): D category VI with RI = 0; weak D type 2 and type 3 with an high RI; and D category VII and weak D type 1 with an intermediate RI. The antigen densities and the Rhesus indices obtained correlated well among the laboratories of this Workshop section despite different staining protocols, secondary antibodies and instrumentation.

Human haemoclassification by use of specific yolk antibodies obtained after immunisation of chickens against human blood group antigens.

Polyclonal antibodies, widely used in research and diagnostics, are conventionally isolated from the blood of immunised mammals, especially rabbits. The fact that antibodies can also be detected in the yolk of eggs laid by immunised hens, led to the development of yolk antibody technology (IgY-technology) as an alternative method that is less stressful to animals. This technology has become a worthwhile alternative to the blood-dependent techniques. Furthermore, because of the phylogenetic distance between birds and mammals, avian antigens have a very specific immune response to highly conserved antigens of mammals, such as human erythrocyte antigens. To evaluate the humoral immune response of hens immunised with human red erythrocyte antigens, 22 White Leghorn hens were kept in cages and immunised with total red blood cells or stroma of the human rhesus positive (Rh+) system (D antigen) by weekly intramuscular and intravenous injections, without the use of an adjuvant. The haemagglutination assay was used to evaluate the dynamics of the production of IgY antibodies against human erythrocyte antigens, and single radial immunodiffusion was used to evaluate the amount of total IgY in de-lipidated supernatants from egg yolk. The highest titres were observed four weeks after the first immunisation, and these remained stable for up to seven weeks for the intravenous route. Positive reactivity against human erythrocyte antigens A, B and O was demonstrated in de-lipidated supernatants from the egg yolks of immunised hens. The strongest reaction was observed against blood group O Rh+ (O+).

Profile of blood donors in Port Moresby.

A record analysis study was conducted on blood donors in the greater Port Moresby area to determine the trend in the number of blood donations and the profile of donors between 1985 and 1994. There was no significant change in the donation trend between 1985-1989 and 1990-1994. While there were no changes in the age distribution between these two periods, there were significant increases in female donors (from 17% to 25%) and new donors (47% to 54%) during 1990-1994. The study data show that there has been a problem in the retention of donors in the greater Port Moresby area during the 1990-1994 period.