German Blood Donors Research Articles

Molecular Screening for Vel- Blood Donors in Southwestern Germany

Background: The SMIM1 protein carries the Vel blood group antigen, and homozygosity for a 17 bp deletion in the coding region of the SMIM1 gene represents the molecular basis of the Vel- blood group phenotype. We developed PCR-based methods for typing the SMIM1 17 bp (64-80del) gene deletion and performed a molecular screening for the Vel- blood type in German blood donors. Methods: For SMIM1 genotyping, TaqMan-PCR and PCR-SSP methods were developed and validated using reference samples. Both methods were used for screening of donors with blood group O from southwestern Germany. Heterozygotes and homozygotes for the SMIM1 64-80del allele were serologically typed for the Vel blood group antigen. In addition, the rs1175550 SNP in SMIM1 was typed and correlated to the results of the phenotyping. Results: Both genotyping methods, TaqMan-PCR and PCR-SSP, represent reliable methods for the detection of the SMIM1 64-80del allele. Screening of 10,598 blood group O donors revealed 5 individuals homozygous for the deletional allele. They were confirmed Vel- by serological typing. Heterozygotes for the 64-80del allele showed different antigen expressions ranging from very weak to regular positive. Conclusion: Molecular screening of blood donors for the Vel- blood type is feasible and avoids the limitations of serological typing which might show false-negative results with heterozygous individuals. The identification of Vel- blood donors significantly contributes to the adequate blood supply of patients with anti-Vel.

Characterization of mannose‐binding lectin (MBL) variants by allele‐specific sequencing of MBL2 and determination of serum MBL protein levels

Mannose-binding lectin (MBL) is a major component of the lectin pathway of complement activation. High and low MBL levels have been associated with susceptibility and severity of a variety of infectious and autoimmune diseases. Several single-nucleotide polymorphisms (SNPs) in the promoter region and exon 1 of the MBL2 gene are responsible for variations in serum MBL levels. We developed a sequence-based typing method for allele-specific MBL2 genotyping and measured serum MBL protein levels in 24 German blood donors. We identified the common MBL2 haplotypes including five promoter polymorphisms in linkage with the Q allele and correlated serum MBL levels with the respective genotypes. The genotyping method presented here could provide a basis for confirmatory studies in larger cohorts.

Donor Satisfaction with a New German Blood Donor Questionnaire and Intention of the Donor to Return for Further Donations

Background: The aim of this study was to describe donor satisfaction regarding different aspects of the new German blood donor questionnaire (BDQ) and to assess whether donor satisfaction is associated with the intention to return for further donations. Methods: A random number of 6,600 blood donors, donating at the German Red Cross Blood Service Baden-Wuerttemberg - Hessen, were asked to rate their satisfaction with four different aspects of the BDQ. Chi-square statistics was used to test for associations between satisfaction and the intention to return. Results: Most of the donors were satisfied with format and layout (72.7%) and the clarity of the questions (72.5%). However, only 39.5% of the donors were satisfied with the scope of the BDQ and 44.3% with the questions about sexual risk behavior. The lowest satisfaction seemed to be among experienced donors and among donors from small municipalities. Among experienced and very experienced donors, a significant association between the satisfaction with the different aspects and the intention to return became apparent. Conclusion: When considering the implementation of the BDQ, Blood Donor Services have to weigh up the advantages of increased deferral rates among donors with high-risk behavior against the potential drop-out of dissatisfied blood donors.

Zoonotic hepatitis E virus strains in German blood donors

Dear Editor,We have read the letters of Baylis et al. [1] and Ijaz et al.[2] on hepatitis E virus (HEV) in blood products with greatinterest. We wish to add data from a similar study onplasma samples from German blood donors collected dur-ing 2009–2010. Viral RNA from pools consisting of up to96 plasma samples was purified as described previously [3]and mixed in metapools of 20, corresponding to 93 955individual donations. A highly sensitive hemi-nestedRT-PCR assay targeting conserved sites of the HEVRNA-dependent RNA polymerase was used for virus detec-tion [4]. In total, 14 of 1185 (1AE18%) individual plasmapools were positive. Unfortunately, the scarcity of availablespecimens did not permit testing of individual donations.Still, extrapolation of HEV-positive pools to individualsamples corresponded to a minimum prevalence of 0AE015%in healthy blood donors. This was compatible withdetection rates in blood products of 0AE012% in Sweden and0AE022% in Germany [1].Positive pools were quantified using a commerciallyavailable real-time RT-PCR assay (RealStar HEV RT-PCRkit; Altona Diagnostics, Hamburg, Germany). Comparable

Novel Approach for Detection of Hepatitis E Virus Infection in German Blood Donors

The risk of transfusion-transmitted hepatitis E virus (HEV) infections by contaminated blood products remains unknown. In the present study, we evaluated and compared different nucleic acid amplification technique (NAT) methods for the detection of HEV in blood components. Minipools of a total of 16,125 individual blood donors were screened for the presence of HEV RNA using the highly sensitive RealStar HEV RT-PCR kit, revealing a minimum detection limit of 4.66 IU/ml. Thirteen donors were HEV RNA positive (0.08%), and of these donors, only three already showed reactive IgM antibody titers. The detected HEV strains all belonged to genotype 3 and were most closely related to German HEV strains from wild boars and pigs as well as from human hepatitis E cases. Furthermore, HEV RNA and HEV-specific IgM and IgG titers were determined in 136 blood donors with elevated alanine aminotransferase (ALT) levels and in 200 donors without pathological findings. HEV RNA was not detectable, but 8.08% (elevated ALT) and 0.5% (nonelevated ALT) of donors showed reactive HEV IgM titers. The overall seroprevalence rate of HEV IgG amounted to 5.94% (elevated ALT, 5.88%; nonelevated ALT, 6.0%). The clinical relevance of transfusion-associated hepatitis E infection still requires further investigation. However, in connection with raising concerns regarding blood safety, our NAT method provides a sensitive possibility for HEV testing.

The role of human leukocyte antigens as predisposing and/or protective factors in patients with idiopathic thrombotic thrombocytopenic purpura

Fifty-four adult German patients suffering from idiopathic thrombotic thrombocytopenic purpura (TTP) have been examined for HLA class II. All patients presented autoantibodies against ADAMTS13 and ADAMTS13 activity levels <5%. Blood samples have been analyzed for HLA-DRB1 and DQB1 alleles using sequence-specific primer PCR and sequence-specific oligonucleotide PCR. Reference data of German bone marrow and blood donors were obtained from www.allelefrequencies.net. The results were evaluated employing two-sided binomial tests, and p values were corrected using the Benjamini-Hochberg procedure. A significant accumulation for DQB1*02:02 (p < 0.001) and DRB1*11 (p = 0.003) was found within the patient group. Twenty percent (DQB1*02:02) or 48.1% (DRB1*11) of TTP patients were tested positive for the particular HLA antigen compared to 1.2% (DQB1*02:02) or 23.5% (DRB1*11) in the control group. A tendency for a reduced occurrence of HLA-DRB1*04 was revealed (7.4% in patients compared to 24.6% in controls). An association between the HLA antigens DQB1*02:02 and DRB1*11 and disease susceptibility for idiopathic TTP has been found. A higher risk for disease outbreak within persons carrying the mentioned alleles can be assumed. The reduced occurrence of HLA-DRB1*04 in TTP patients indicates a possible protective effect of this HLA allele in disease development.

Donor Deferral Rates after the Implementation of a New German Blood Donor Questionnaire

Background: The implementation of a new national German blood donor questionnaire was proposed to improve donor and recipient safety. Methods: We compared deferral/exclusion rates of whole blood donors before (May 2010, n = 64,735) and after (May 2011, n = 71,687) the implementation of a new blood donor questionnaire. Considering seasonal variations, analysis was performed with respect to collection site (mobile vs. fixed), sex, donor status (first-time vs. repeat), age, and the frequencies of sexual risk behavior and other reasons for deferral. Results: We observed a statistically significant increase (p < 0.001) of the overall deferral/exclusion rate from 6.2 to 8.1%, irrespective of type of collection site (fixed: from 6.0 to 8.5%; mobile: from 6.2 to 8.0%), sex (females: from 7.5 to 9.9%; males: from 5.1 to 6.6%), donor status (first-time donors: from 19.7 to 24.7%; repeat donors: from 4.6 to 6.3%) or age (18–29 years: from 9.1 to 11.7%; 60–71 years: from 5.1 to 6.6%). Confidential self-exclusion increased from 0.08 to 0.14% (p < 0.001). Besides risk behavior, various medical reasons could be identified that explain this increase. Conclusions: The new blood donor questionnaire resulted in an increased deferral/exclusion of all donor groups. Thus the impact on future blood supply must be considered carefully, and long-term studies and investigation of donor acceptance will be needed.

Human neutrophil alloantigen genotype frequencies among blood donors with Turkish and German descent

Antibodies against the human neutrophil antigens (HNA) are able to stimulate transfusion reactions, autoimmune and neonatal neutropenia. The aim of this study was to determine the HNA allele frequencies in the largest ethnic minority group in Germany in comparison with the German population for predicting the risk of alloimmunization and associated transfusion reactions, as well as the risk of developing neonatal neutropenia for the newborn of racial mixed couples. However, there exists no data about HNA genotype distribution in Turkish population. DNA was isolated from blood samples of 119 German and 118 Turkish blood donors and typed them for HNA-1, -3, -4, and -5 by using a commercial polymerase chain reaction kit with sequence-specific primers (SSP-PCR) and compared the HNA genotype distribution of both groups. In German blood donors, the gene frequencies for HNA-1a and HNA-1b were 0.391 and 0.601, for HNA-3a and -3b, 0.744 and 0.256, for HNA-4a and -4b, 0.908 and 0.092, and for HNA-5a and -5bw, 0.731 and 0.269. In Turkish blood donors, we observed 0.420/0.564, 0.737/0.263, 0.881/0.119, and 0.754/0.246 for HNA-1a/1b, -3a/3b, -4a/4b, and -5a/5bw. No statistic significant difference between genotypes in these populations was observed. This study is the first to report HNA gene frequencies in a Turkish population. It showed that there is no difference of HNA genotype in blood donors with Turkish descent in comparison with German blood donors. The alternating transfusion of blood and blood components is no increased risk for developing alloantibodies against HNA antigens. In pregnancy of mixed couples no special screening programs for HNA are necessary.

ARCHITECT rHTLV-I/II immunoassay for blood screening and diagnosis

Background Detection of HTLV infection continues to be important to control spread of human retroviruses and to protect safety of blood supply. The ARCHITECT rHTLV-I/II assay is a chemiluminescence-based immunoassay, which utilizes HTLV-I, and HTLV-II recombinant antigens and peptides in a double-antigen-sandwich assay format on the Abbott ARCHITECT automated platform. Precision, sensitivity and specificity of the ARCHITECT rHTLV-I/II assay is being presented. Methods Assay performance was evaluated on confirmed HTLVI/II positive specimens and specimens from German Blood Donors and hospitalized patients. External performance evaluation was conducted at two different sites in Portugal. Murex HTLV-I/II EIA was used as comparator assay and INNO-LIA or MP diagnostics HTLV Blot were used for confirmation. Results The ARCHITECT rHTLV-I/II assay detected all HTLVI and HTLV-II specimens (N=406) in accordance to the Murex assay. Analytical sensitivity was equivalent to Murex based on end-point-dilutions. Specificity on HTLV-I/II blood donors was 99.95% (N=5646; 95% CI 99.84-99.99%), SD to cut-off was 15.6. Specificity on a diagnostic population was 99.86% (N= 692; CI 95% 99.20-100%). Precision for samples (1-6 S/CO) was 3.984.31%. Presented data support selected cut-off multiplier-value of 0.25 for the ARCHITECT rHTLV-I/II assay. Results from the Receiver-Operating-Curve showed a 99.98% area under the curve which is indicative of high separation power between negative and positive populations. Conclusions This study demonstrated excellent sensitivity and specificity of the ARCHITECT rHTLV-I/II assay. The assay is suitable for both blood screening and diagnosis of HTLV infections and therefore reduces risk of transfusion transmitted HTLV and improves safety of blood supply.

Sequence‐based typing of major histocompatibility complex class I chain‐related gene A alleles by use of exons 2–5 information

The polymorphic MICA (major histocompatibility complex class I chain-related gene A) (Gene ID: 100507436) gene products are a ligand of the activating natural killer cell receptor, NKG2D. Their clinical importance spans from solid organ transplantation to bone marrow transplantation and disease susceptibility. Typing of MICA genes by sequencing is hampered by an exon 5 short tandem repeat, the definition of which is critical for the final allelic and functional assignment. We present a novel sequencing approach, which uses group-specific (7T/8T) exon 5 polymerase chain reactions (PCRs) and facilitates hemizygous exon 5 MICA-PCR in approximately 70% of the tested individuals. With this method we typed the International Histocompatibility Workshop Group MICA reference panel (40 cell lines) as well as 110 healthy South German blood donors. All ambiguities, with the exception of MICA*008:01/008:04 (synonymous substitution in exon 1) and MICA*009:01/049 (nonsynonymous substitution in exon 6), could be resolved with our method. Analysis of Hardy-Weinberg equilibrium for our cohort showed no significant difference between expected and observed frequencies of MICA alleles (P = 0.6142). The three most frequent alleles in our blood donor cohort were MICA*008:01/008:04 (40.5%), MICA*002:01 (13.2%), and MICA*009:01/049 (8.6%). The 7T polymorphism was observed in 67.7% and the 8T polymorphism in 32.3% of our blood donor cohort. Individuals (24.5%) tested were homozygous. The approach described in this paper is suitable for accurate sequencing of large sample numbers, including direct readout of exon 5 sequences. It is compatible with laboratory automation and commercial human leukocyte antigen analysis software tools. It may therefore be applied in large clinical trials.

High prevalence of antibodies against polyomavirus WU, polyomavirus KI, and human bocavirus in German blood donors

BackgroundDNA of the polyomaviruses WU (WUPyV) and KI (KIPyV) and of human bocavirus (HBoV) has been detected with varying frequency in respiratory tract samples of children. However, only little is known about the humoral immune response against these viruses. Our aim was to establish virus-specific serological assays and to determine the prevalence of immunoglobulin G (IgG) against these three viruses in the general population.MethodsThe capsid proteins VP1 of WUPyV and KIPyV and VP2 of HBoV were cloned into baculovirus vectors and expressed in Sf9 insect cells. IgG antibodies against WUPyV VP1, KIPyV VP1, and HBoV VP2 were determined by immunofluorescence assays in 100 plasma samples of blood donors.ResultsThe median age of the blood donors was 31 years (range 20 - 66 yrs), 52% were male. 89% of the samples were positive for WUPyV IgG (median age 31 yrs, 49.4% male), 67% were positive for KIPyV IgG (median age 32 yrs, 46.3% male), and 76% were positive for HBoV IgG (median age 32 yrs, 51.3% male). For WUPyV and HBoV, there were no significant differences of the seropositivity rates with respect to age groups or gender. For KIPyV, the seropositivity rate increased significantly from 59% in the age group 20 - 29 years to 100% in the age group > 50 years.ConclusionsHigh prevalences of antibodies against WUPyV, KIPyV, and HBoV were found in plasma samples of healthy adults. The results indicate that primary infection with these viruses occurs during childhood or youth. For KIPyV, the seropositivity appears to increase further during adulthood.

Sensitivity and specificity of Anti‐HBc screening assays – which assay is best for blood donor screening?

Compared to HIV and hepatitis C virus, the residual infectious risk of hepatitis B virus (HBV) posed by blood products is about 10 times higher. In addition to HBsAg testing, screening for anti-HBc was recommended by the German Advisory Committee Blood in March 2005. Prevalence of anti-HBc in German blood donors was investigated at five test sites located in different geographic regions. In total, 12,000 blood donors were screened for anti-HBc by PRISM HBcore, and a statistically representative number of these were tested with Abbott Murex anti-HBc total, bioMérieux Hepanostika anti-HBc uniform, Bio-Rad Monolisa anti-HBc PLUS and Dade Behring Enzygnost anti-HBc. Anti-HBc repeat reactive samples were tested for anti-HBs, anti-HBe and HBV DNA by individual donation NAT. The mean prevalence of anti-HBc was 1.75% in donors that had not been tested for anti-HBc in the past. The percentage of anti-HBs in anti-HBc repeat reactive donors was 93.7%. Samples that were additionally reactive for anti-HBe were anti-HBc reactive in all tested assays. The sample to cut-off (S/Co) values for anti-HBc were lower (competitive assays) in samples that were also positive for anti-HBe, when compared to samples that were only anti-HBc reactive. Most commercially available anti-HBc assays provide sufficient sensitivity for routine screening purposes, and lacking specificity is no longer a serious issue for most of them. Assay differences were recognized for samples that were anti-HBc only reactive. The overall loss of 1.75% of positive testing donors can be significantly reduced to 0.45% by implementation of re-entry procedures for donors with an anti-HBs titre of over 100 IU/l and negative by sensitive ID-NAT.

RhCE protein variants in Southwestern Germany detected by serologic routine testing

Variant RHCE alleles with diminished expression of C, c, E, and e antigens have been described and indicate the genetic diversity of this gene locus in several populations. In this study the molecular background of variant RhCE antigens identified by standard serologic routine testing in German blood donors and patients was determined. Samples from blood donors and patients were routinely analyzed for RhCE phenotype using the PK7200 analyzer with two sets of monoclonal anti-C, -c, -E, and -e reagents. Samples with confirmed variant RhCE antigens were analyzed by nucleotide sequencing of the 10 RHCE exons. A multiplex polymerase chain reaction with sequence-specific priming (PCR-SSP) method was established for rapid typing of the rare RHCE alleles. We identified 43 samples with serologic RhCE variants. Molecular analysis revealed variant RHCE alleles in 34 samples. Altogether 22 RHCE alleles were detected; 10 have not been published before. Twenty alleles harbored distinct single-nucleotide substitutions, 18 of which encoded amino acid changes and 2 of which occurred in noncoding regions. Two samples represented RHCE-D-CE hybrid alleles involving different segments of the RHCE Exon 5. A multiplex PCR-SSP screening for 17 RHCE alleles was negative in 1344 samples of the DNA bank GerBS. The cumulative phenotype frequency was estimated between 1 in 488 (0.20%) and 1 in 8449 (0.012%). Single-amino-acid substitutions were the molecular basis for variant RhCE antigen expression in most samples. Nucleotide substitutions in RHCE exons were excluded as possible mechanism of diminished RhCE antigen expression in one-fifth of the serologically identified samples.

Multidimensional reference regions for IGF-I, IGFBP-2 and IGFBP-3 concentrations in serum of healthy adults

Context: The insulin-like growth factor (IGF) system exerts many effects on the growth and differentiation of both normal and malignant cells. The serum concentrations of insulin-like growth factor I (S-IGF-I), insulin-like growth factor-binding protein 2 (S-IGFBP-2) and insulin-like growth factor-binding protein 3 (S-IGFBP-3) and their inter-relations may differ in certain disease states from those seen in healthy individuals. Objective: To estimate age-, gender- and body mass index (BMI)-specific univariate, bivariate and trivariate 95% reference regions for these components in healthy adults and present indices that will facilitate interpretation of patient observations in relation to these reference regions. Design: Blood samples were taken in the morning from 427 healthy, non-fasting German blood donors of both genders (age range, 18–79 years; BMI range, 16–44 kg/m 2). Reference regions were developed with multivariate regression methods. Results: Regression analyses showed that S-IGF-I and S-IGFBP-3 levels decrease with increasing age, whereas S-IGFBP-2 concentrations increase with age ( P < 0.0001). Females had significantly higher S-IGFBP-3 levels than males ( P < 0.0001) and similar S-IGF-I and S-IGFBP-2 concentrations. Increasing BMI was associated with decreasing S-IGFBP-2 ( P < 0.0001), but was not significantly associated with the concentrations of the other two analytes. Controlling for age, gender and BMI, S-IGF-I and S-IGFBP-3 were positively correlated ( r = 0.58), whereas S-IGF-I and S-IGFBP-2, and S-IGFBP-2 and S-IGFBP-3 were negatively correlated ( r = –0.11 and r = –0.18, respectively). Based on the regression models, which were controlled for age, gender and BMI, two- and three-dimensional 95% reference regions with associated patient indices were described for each pair of analytes and for the trio of analytes, respectively. Conclusions: The multivariate reference regions developed in this study should be useful in identifying patients with an abnormal IGF–IGFBP axis. It is suggested that introducing multidimensional reference regions and the described patient indices into clinical practice may aid monitoring of the safety of GH therapy. These patient indices may also be useful in the assessment of cancer risk.

Pharmacogenetics of Oral Anticoagulation - VKORC1-Haplotypes Determine the Inter-Individual and Inter-Ethnical Variability.

Purpuse: Coumarins are known for their high inter-individual and inter-ethnical variability of the dose-response association (i.e. significant higher maintenance dose in Africans and lower dose in Chinese). Recently, common SNPs of the VKORC1 gene (encoding the molecular target of coumarins, vitamin K epoxide reductase) were shown to be associated with lower warfarin dose requirement (c.-1639G>A [rs17878363], c.173+1000C>T [rs9934438]). In order to clarify whether these SNPs are part of more extended haplotypes, we established a comprehensive haplotype map of the entire VKORC1 gene locus. The association of VKORC1-haplotypes with coumarin dose requirement was analysed in different ethnical populations and patient cohorts.Methods: In 200 German blood donors the VKORC1 gene locus was analysed by direct sequencing. VKORC1 genotype data in 7 additional populations from Africa, Asia and Europe were retrieved from internet databases. Haplotype frequencies were inferred using EHplus and FAMHAP. Unselected patients receiving phenprocoumon (n=61) and patient cohorts of European origin with either increased coumarin sensitivity (n=14, weekly phenprocoumon dose <6mg) or partial coumarin resistance (n=36, weekly phenprocoumon dose >42 mg or warfarin dose>70mg) were typed for VKORC1 haplotypes.Results: In 200 controls we identified 28 SNPs within the VKORC1 gene. 6 SNPs formed 3 main haplotypes covering more than 99% of the genetic variability of VKORC1 (VKORC1*2: 42%, VKORC1*3: 38%, and VKORC1*4: 20%). SNPs associated with low warfarin dose (rs17878363, rs9934438) were in complete linkage disequilibrium with the VKORC1*2 haplotype. Haplotype frequency in Africans and Chinese differed significantly from the European sample (for VKORC1*2: Europeans 42%, Chinese 95%, Africans 14%). In 61 unselected patients receiving phenprocoumon c.-1639AA genotype patients (homozygous VKORC1*2) required less phenprocoumon (1.40 mg/d) than AG (2.12 mg/d) and GG patients (3.02 mg/d).13 of 14 patients (93%) with increased coumarin sensitivity but none of the patients with partial coumarin resistance were c.-1639AA. Vice versa, the c.-1639G allele (present in the non VKORC1*2 haplotypes) was found homozygous in 31 patients (86%) with partial coumarin resistance but in none of the patients with increased coumarin sensitivity.Conclusions: Three main haplotypes represent more than 99% of VKORC1's genetic variability in Europeans. VKORC1 haplotypes are strongly associated with the inter-individual and inter-ethnical variability of oral anticoagulation. In future studies, VKORC1-haplotype testing may provide a clinically relevant predictor of coumarin dosing and bleeding risk in oral anticoagulation.

Identification of a new HLA‐B allele, HLA‐B*4443†, in a German family

A novel human leukocyte antigen (HLA)-B allele is described. The allele was identified in a German blood donor of Caucasian origin. Because high-resolution HLA-typing using sequence-specific primers gave inconclusive results, sequence-based typing was performed. Nucleotide sequences of exons 2 and 3 most closely match with HLA-B*4417 and HLA-B*440301 (99.5% identity). The predicted protein sequence revealed a single amino acid substitution (D156L) compared with the HLA-B*4417 allele but two substitutions (Y113H, D116S) compared with the HLA-B*440301 allele. Therefore, the novel allele has been officially assigned HLA-B*4443 by the WHO Nomenclature Committee. The HLA-B*4443 allele was found with the A*2301, Cw*0401, B*4443, DRB1*0701, DRB4*0107, and DQB1*0202 haplotype.

Polymorphisms in the human surfactant protein-D (SFTPD) gene: strong evidence that serum levels of surfactant protein-D (SP-D) are genetically influenced

The collectin surfactant protein-D (SP-D) plays a significant role in innate immunity. Epidemiological studies described associations between single nucleotide polymorphisms (SNPs) of the human gene coding surfactant protein-D (SFTPD) and infectious pulmonary diseases. Studies on twins indicated very strong genetic dependence for serum levels of SP-D. The aim of this study was to determine the genetic influence of sequence variations within the SFTPD gene on the constitutional serum SP-D levels. We sequenced the 5′ untranslated region (5′UTR), the coding region and the 3′ region of the SFTPD gene of 32 randomly selected blood donors. Six validated SNPs were genotyped with sequence-specific probes (TaqMan 7000) in 290 German blood donors. Serum SP-D levels were analysed by ELISA, and the association of SFTPD haplotype estimates with the quantitative phenotype serum SP-D level was determined. One single SFTPD haplotype (allele frequency 13.53%) revealed a negative association with serum SP-D levels (P<0.0001). This was confirmed in a second prospectively collected group of blood donors (n=160, P=0.0034). The discovery of a frequent negative variant of the SFTPD gene provides a basis for genetic analysis of the function of SP-D in the resistance against pulmonary infections and inflammatory disorders in humans.

Evaluation of a standardized F1 capsular antigen capture ELISA test kit for the rapid diagnosis of plague

Rapid detection of soluble F1 capsular antigen in serum, bubo fluid or urine of patients proved to be a valuable tool in the presumptive diagnosis of plague. We evaluated a F1 capsular antigen capture ELISA resembling a commercially available test kit. The minimal detectable concentration was 4 ng/ml. The specificity was 100% when investigating 47 sera from healthy Malagasy subjects and 98.4% when 365 sera from German blood donors were studied. Sensitivity was determined on sera ( n=11) and buboes ( n=18) from bacteriologically confirmed Malagasy plague patients. Sensitivity was 90.1% for serum and 100% for buboes. A standardized F1 capsular antigen capture ELISA test kit might be well suited for the early detection of plague particularly in non-endemic areas where clinical microbiological laboratories have only limited access to alternative techniques for rapid identification of Yersinia pestis.

Gene and haplotype frequencies for the loci hLA-A, hLA-B, and hLA-DR based on over 13,000 german blood donors

Numerous applications in clinical medicine and forensic sciences depend on reliable data concerning the frequencies of human leukocyte antigen (HLA) genes and haplotypes. Assuming a Hardy-Weinberg equilibrium of the underlying population, these frequencies can be estimated from phenotype data using an expectation-maximization-algorithm also known under the name “gene counting.” We have refined this algorithm in order to cope with the heterogeneous resolution of HLA phenotypes frequently occurring in large datasets due to the structure of the HLA nomenclature. This was a prerequisite to analyze a set of 13,386 blood donors contributed by over 40 blood banks who were tested for HLA-DR when they volunteered to become marrow donors. This data set is still unique in the German national donor registry because their HLA-DR-typing was not biased by patient oriented searches or other strategies for selective typing. As a consequence of the size of the sample, the frequency estimates for the genes and the two- and three-locus haplotypes of HLA-A, HLA-B, and HLA-DR are of unprecedented precision and allow interesting projections concerning the efficiency and economic aspects of the development of a large donor registry in Germany.

Immunity against diphtheria and tetanus in German blood donors.

After the recent diphtheria epidemics in Eastern Europe in the early 1990s, we re-evaluated the diphtheria and tetanus immunity of 321 German blood donors (192 men and 129 women). The mean antitoxin levels of all blood donors in this study, measured by commercial ELISA, revealed a questionable protection (0.1-1.0 IU/ml) against diphtheria. In 1994, 66.4% were without immunity against diphtheria (55.0% in 1997/98), 32.1% (41.5% in 1997/98) showed questionable protection and only 1.5% (3.5% in 1997/98) had protective antitoxin levels. The evaluation of tetanus immunity revealed only 0.5% (1.1% in 1997/98) of the subjects with no protection and 9.1% (8.5% in 1997/98) with questionable protection. For this reason, we conclude that the diphtheria epidemics only lead to an insufficient improvement of the immunization status in a healthy German population.