RHCE-D-CE hybrid genes can cause false-negative DNA typing of the Rh e antigen.

BACKGROUND AND OBJECTIVES: DNA typing of the human Rh blood groups generally shows good agreement with serologically defined phenotypes. However, in the present report we describe four individuals who were declared Rh e negative by genotyping although they express the Rh e antigen. MATERIALS AND METHODS: Serotyping was performed using mono- and polyclonal Rh antisera. Fluorescent multiplex sequence-specific polymerase chain reactions (PCR-SSPs) identified RHD exons and the polymorphisms usually associated with the Rh E/e or Rh C/c/C(W) antigens. Additional PCR amplification reactions, which were carried out to reveal RHCE-D-CE hybrid genes, analysed exon 5 of the RH genes, the location of the polymorphism (676C-->G) coding for the Rh E and Rh e antigens. RESULTS: Four individuals were identified who expressed Rh e antigens but were negative by PCR-SSP typing for common Rhe-coding sequences. In one family analysed in detail, an RHCE-D5-CE hybrid gene associated with Rh e antigen expression was identified. A concomitant RHcE allele accounted for a seemingly regular typing pattern by conventional RH PCR. CONCLUSIONS: The presence of RHCE-D5-CE hybrid alleles may cause false-negative DNA-typing results for the Rh e antigen that are easily overlooked unless appropriate RH hybrid PCR-SSPs are incorporated into conventional DNA-typing protocols. These and previous data strongly caution against an uncritical interpretation of RH DNA-typing results.

PCR screening for common weak D types shows different distributions in three Central European populations.

BACKGROUND: DNA sequencing showed RHD mutations for all weak D phenotypes investigated in a study from Southwestern Germany. Molecular classification of weak D offers a more reliable basis than serotyping and is relevant for optimal D transfusion strategies. STUDY DESIGN AND METHODS: Sequence-specific primers were designed to detect weak D types 1 to 5 and the partial D phenotype HMi in a modular set for conventional PCR analysis. Alternatively, all reactions were multiplexed into a single tube, and the products were identified after automated capillary electrophoresis by their size and fluorescence. Weak D phenotype samples from 436 donors in the Tyrol (Austria) and Northern Germany were investigated by PCR. RESULTS: More than 90 percent of the weak D types identified by PCR represented type 1, 2, or 3. The distribution among the common types varied between the Tyrol and Northern Germany (p<0.0001). Three new RHD alleles were identified. CONCLUSION: A PCR method of detecting the common weak D types was validated. This PCR system introduces a simple and rapid tool for routine DNA typing of weak D samples. The results confirmed that all weak D phenotype samples identified by current serologic criteria carry altered D proteins.

Fyx is associated with two missense point mutations in its gene and can be detected by PCR-SSP.

The Duffy blood group antigens are encoded by the Duffy gene with its three major alleles: Fy*A (Fya+), Fy*B (Fyb+), and a nonexpressed Fy*Fy (Fya-b-), which is most commonly found among black people. Additionally, a fourth allele, Fyx, is found among white people and defined as weak Fyb not detectable by all anti-Fyb. Three polymerase chain reactions (PCRs) using sequence-specific priming (SSP) for detection of the major FY alleles were developed. Eighteen Fy(a-b-) samples of Tanzanian origin were correctly typed and of 300 random donors of Caucasian origin with known Fy phenotype, only four out of 59 Fy(a+b-) donors showed the discrepant DNA-type Fy(a+b+). Serologic reinvestigation by adsorption and elution techniques confirmed weakly expressed Fyb antigen in these cases and DNA sequencing of the entire Duffy gene revealed identical point mutations in all of them. Specific PCR reactions were used to reinvestigate the C265T (Arg89Cys) and G298A (Ala100Thr) substitution in the 300 samples. A298 was found to be present in both FY*X and FY*B alleles, pointing to an allelic variation among FY*B alleles. T265 was encountered exclusively in FY*X and is thought to be FY*X specific. Combining the T265 specific reaction with the three PCR-SSPs described above, we were able to correctly DNA-type all phenotypes investigated in our study.

Molecular basis of weak D phenotypes.

A Rhesus D (RhD) red blood cell phenotype with a weak expression of the D antigen occurs in 0.2% to 1% of whites and is called weak D, formerly Du. Red blood cells of weak D phenotype have a much reduced number of presumably complete D antigens that were repeatedly reported to carry the amino acid sequence of the regular RhD protein. The molecular cause of weak D was unknown. To evaluate the molecular cause of weak D, we devised a method to sequence all 10 RHD exons. Among weak D samples, we found a total of 16 different molecular weak D types plus two alleles characteristic of partial D. The amino acid substitutions of weak D types were located in intracellular and transmembraneous protein segments and clustered in four regions of the protein (amino acid positions 2 to 13, around 149, 179 to 225, and 267 to 397). Based on sequencing, polymerase chain reaction-restriction fragment length polymorphism and polymerase chain reaction using sequence-specific priming, none of 161 weak D samples investigated showed a normal RHD exon sequence. We concluded, that in contrast to the current published dogma most, if not all, weak D phenotypes carry altered RhD proteins, suggesting a causal relationship. Our results showed means to specifically detect and to classify weak D. The genotyping of weak D may guide Rhesus negative transfusion policy for such molecular weak D types that were prone to develop anti-D.

Three molecular structures cause rhesus D category VI phenotypes with distinct immunohematologic features.

Rhesus D category VI (DVI) is the clinically most important partial D. DVI red blood cells were assumed to possess very low RhD antigen density and to be caused by two RHD-CE-D hybrid alleles. Because there was no population-based work-up, we screened three populations in central Europe for DVI. Twenty-six DVI samples were detected and examined by exon-specific RHD polymerase chain reaction with sequence-specific primers (PCR-SSP). A new genotype, hereby designated D category VI type III, was characterized as a RHD-Ce(3-6)-D hybrid allele by sequencing of the cDNA, parts of intron 1, and by PCR-restriction fragment length polymorphism (PCR-RFLP) of intron 2. Rhesus introns 5 and 6 were sequenced and the 3' breakpoints of all known DVI types shown to be distinct. We differentiated the 5' breakpoints of DVI type I and DVI type II by a newly devised RHD-PCR. Thus, the DVI phenotype originated in at least three independent molecular events. Each DVI type showed distinct immunohematologic features in flow cytometry. The number of RhD proteins accessible on the red blood cells' surface of DVI type III was normal (about 12,000 antigens/cell; DVI type I, 500; DVI type II, 2,400) based on the determination of an RhD epitope density profile. DVI type II and DVI type III occurred as CDe haplotypes, and DVI type I as a cDE haplotype. The distribution of the DVI types varied significantly in three German-speaking populations. Genotyping strategies should take account of allelic variations in partial RhD. The reconsideration of previous serologic and clinical data for partial D in view of the underlying molecular structures may be worthwhile.

Manual and automated methods for the determination of leukocyte counts at extreme low levels: comparative evaluation of the Nageotte chamber and the Abbott Cell Dyn 3500 analyser.

Leukodepleted or leukocyte-poor blood products (fresh-frozen plasma, packed red cell and platelet concentrates in particular) are widely used in current clinical practice. However, because the monitoring of leukodepletion efficiency is generally carried out (if at all) using the labour-intensive and relatively inaccurate manual Nageotte chamber technique, it is clear that any increased demand for leukodepletion monitoring would be difficult, if not impossible, to meet. As the need to identify an automated alternative to the Nageotte technique is important, this study was undertaken to evaluate such a possibility. White blood cells were enumerated in a representative series of filtered and non-filtered human blood components by both microscopic counting in the Nageotte chamber, and with the Abbott CD3500 automated haematology analyser. For the Nageotte estimate, a single analysis was made in accordance with standard procedures, whereas the automated analysis was achieved by making six replicate counts and determining the mean of four replicates after excluding the highest and lowest estimates. To determine linearity limits of the manual and automated procedures, freshly isolated leukocytes were admixed with cell-free plasma-pheresis plasma. Reasonable reproducibility (mean CV 10% for cell counts exceeding 100 cells/microL) and good linearity (r > 0.9) were observed for CD3500 determinations in four separate experiments. The manual and automated measurements also correlated well (r > 0.9) with no obvious inter-method bias for cell counts up to 40 cells/microL although there was some suggestion of lower absolute CD3500 counts in the range 40-130 cells/microL. For the comparative studies with filtered and non-filtered blood products, no significant method bias was seen with 70 individual red cell concentrates, but systematically higher CD3500 white blood cell counts were observed in the series of 68 platelet concentrates (probably due to the presence of platelet clumps). This study concludes that automation of white cell counts in blood products with the CD3500 analyser is feasible for quality control in the preparation of fresh-frozen plasma and red cell concentrates but is limited for the analysis of filtered platelet concentrates.

Leuko-reduction using an integral Sepacell filter early (4 h, 24 h) or late (48 h, 120 h) after plateletpheresis does not affect platelet function.

Leuco-reduction of aliquots of single-donor platelet concentrates with an integral Sepacell PLS-5A filter was performed 4, 24, and 48 h after apheresis including a control after 120 h (not recommended by the manufacturer) to evaluate the effect of both early and late filtration on the concentrates in a paired study. Highly efficient (> 2 log10) leuko-reduction was consistently observed for filtration any time after apheresis. Various proaggregatory stimuli were tested to determine the stimulus concentration (EC50 values) required for half-maximal aggregation of platelet samples (200 platelets/nl). Neither glycoprotein IIb/IIIa-dependent (thrombin-, collagen-, and APD-induced) nor glycoprotein Ib-mediated (ristocetin-induced) platelet function were affected by filtration 4, 24, 48, or 120 h after plateletpheresis. For single-donor plateletpheresis using a closed system with an integral Sepacell filter, our in vitro results suggest that the timing of leuko-reduction does not affect the platelet-dependent hemostatic qualities of the product.

Platelet aggregation in response to collagen and thrombin reliably detects the ingestion of low-dose aspirin.

The exposure of blood donors to aspirin is not reliably excluded by pharmacokinetic measurements due to the irreversible effects of aspirin which persist even after elimination of aspirin and its metabolites from plasma. Tests of platelet functions do overcome this deficit, but are usually limited by substantial inter-individual variability of parameters of platelet function. We have evaluated platelet aggregation ex vivo in 20 aspirin-treated (100 mg single oral dose/day) patients in comparison with a control group of 20 aspirin-free donors. The results demonstrate a significant reduction in the collagen(2.5 micrograms/ml)-induced platelet aggregation by aspirin treatment, whereas thrombin(50 mumol/l TRAP-6)-induced platelet aggregation was not affected at all. Assessment of collagen-induced platelet aggregation relative to platelet responses of the same subject elicited either by thrombin or by a combination of collagen and thrombin does substantially improve the reliability of functional assays of aspirin.

Detection of malignant cells of epithelial origin in mononuclear cell fractions.

Qualitative and quantitative detection of contaminating malignant cells in stem cell apheresis products intended for supportive therapy after high-dose chemotherapy of sensitive solid tumors is a demanding task for the laboratory. Human breast cancer cells in differing concentrations among mononuclear cells were quantitated by multicolor flow cytometry and immunocytochemistry. Comparing specificity, sensitivity, and laboratory processing time, the immunocytochemical detection of the above-mentioned malignant cells of epithelial origin was demonstrated to be the superior method.

Routine rhesus-D genotyping in amniotic fluid: analysis of exon 10 versus intron 4 by polymerase chain reaction.

Both the exon 10 and the intron between exons 4 and 5 of the Rh-D gene were analyzed in DNA from amniotic cells by polymerase chain reaction (PCR) in order to determine the fetal Rh-D status. Residual (0.3-2 ml) samples of amniotic fluid obtained by diagnostic amniocentesis in Rh-D-negative women were analyzed. It is important to standardize the sampling and preanalytical storage and to optimize DNA extraction procedures as well as the detection of PCR products for maximal sensitivity. Genotyping was successful in approximately 90% (229 of 256) of amniotic fluid samples tested. Consistent Rh-D results were obtained by both methods in all 229 typed samples with a single exception. Our experience demonstrates that routine genotyping of the Rh-D blood group in small volumes of amniotic fluid (0.5-2 ml) is feasible. Its validity is currently tested by comparison of the prenatal genotype with the serotype of the newborn.

Genotyping of the human platelet antigen-1 by ELISA detection of allele-specific amplicons.

BACKGROUND AND OBJECTIVES: The purpose of the study was to establish a valid and efficient method for genotyping of the human platelet alloantigen-1 (HPA-1) in large sample numbers. MATERIALS AND METHODS: Digoxigenin- and fluorescein-labelled allele-specific primers and a biotinylated common primer were included in the hot-start PCR. Amplicons were bound to avidin-coated plates to identify the products by ELISA. RESULTS: This approach reduced the number of PCR analyses for HPA-1 typing by half. PCR products were detected with high sensitivity and good reproducibility (interassay-CV: < 8%) in the ELISA. The typing of 100 blood donors with both PCR plus gel electrophoresis and ELISA-PCR showed identical results. CONCLUSION: This method offers efficient HPA-1 genotyping in large numbers of donors and patients.

Rhesus D genotyping in amniotic fluid.

Rhesus D genotyping by PCR of amniotic fluid offers a very valuable tool for prenatal care of Rhesus D-alloimmunized women. Misleading results have been reported for the method of Bennett et al. analysing the 3'-terminal end of the RhD gene. We have observed also problems with Rhesus D genotyping of DVI-variant samples in the assay of Simsek et al. utilizing size differences of intron 4 between the Rhesus D and the Rhesus CcEe gene. We, thus, prefer to amplify two different regions including the 3'-terminal region of the Rhesus D gene to further minimize the very low risk of false-negative results. This rapid Rhesus D genotyping usually does not require more than 2 ml of amniotic fluid.

Quantitation of circulating hematopoietic progenitor cells early after transplantation.

By multicolor flow cytometry CD34 positive blood progenitor cells (BPC) were determined by standard procedures before and after reinfusion. Measurements of circulating progenitor cells after transplantation lead to cell numbers that are considered together with reconstitution potential, especially with the number of transfused apheresis platelet concentrates after transplantation. Decreased concentrations of CD34 positive cells are associated with an increased frequency of platelet transfusions after transplantation.

Unsatisfactory specificities and sensitivities of six enzyme immunoassays for antibodies to hepatitis B core antigen.

To examine the consistency and comparability of anti-hepatitis B core antigen (anti-HBcAg) assays, four blood donation centers of the Red Cross in the Federal Republic of Germany tested 4,080 unselected blood donors with six different tests in parallel. Confirmation testing of reactive samples was done in the National Reference Center for Viral Hepatitis. Depending on the test kit used, 4.1 to 9.9% of serum samples were initially positive and 2.9 to 7.5% were repeatedly positive. Sixteen percent of serum samples were positive in at least one test but only three percent were positive in all six tests. Statistical analysis of frequency distribution of optical densities for each test suggested that there should be a correction of the cutoff values. This reduced the number of false-positive results by half, but a significant proportion of discrepant results could not be resolved. The lack of specificity and consistency requires cautious interpretation of isolated anti-HBcAg results in clinical specimens. Screening of predominantly anti-HBcAg-negative populations (e.g., blood donors) by the current anti-HBcAg test kits will almost necessarily give unsatisfactory results.

Human factor H (beta 1H-globulin): linkage analysis.

Linkage data on human factor H (HF) and 22 other human genetic markers are presented. Close linkage at theta less than 0.10 can be ruled out for a series of marker systems (Rh, PGM1, ACP1, Jk, Tf, Gc, MNSs, ME2, HLA, GLO1, ORM, Gt, PI, Hp, GPT). Strong evidence for linkage was obtained for peptidase A (PEPA) with lods greater than 3.0 at theta = 0.10 in males and at theta = 0.20 for the sexes combined. From this result the HF locus can be provisionally assigned to chromosome 18.

HIV-specific antibody among voluntary blood donors in Lower Saxony (FRG).

Anti-HIV test results of the Red Cross Blood Transfusion Service of Lower Saxony from 1 June 1985 to 31 July 1986 inclusive were analysed retrospectively. Nine out of 70,936 donors who had not donated blood before 1 June 1985 (first-time donors) and 9 out of 261,231 donors who had donated blood before this date (repeating donors) were found anti-HIV confirmed positive at the time of the first blood donation during the study period. The prevalence of HIV antibody in first-time donors was significantly higher than in repeating donors (p less than 0.01). It was concluded that some members of risk groups used blood donation to obtain an anti-HIV test result. One out of 30,300 blood donations was confirmed anti-HIV positive. The results of this study justify the transfusion of blood donations that are reactive only in the initial ELISA test.