An internal positive control for the enumeration of CD45(+) and CD34(+) cells by flow cytometry allows monitoring of reagent and operator performance.

BACKGROUND: Three-color flow cytometry assays are used to determine CD34(+) cell doses prior to stem cell transplantation. These assays require high-quality reagents that are dispensed accurately to ensure reproducible results. We have developed a flow cytometry assay for CD34(+) cells with an integral positive control (KG1a cells) for monitoring reagent and operator performance. METHODS: The method was validated using samples from 127 allogeneic donations (42 BM, 85 PBSC) from healthy donors and 195 autologous donations (46 BM, 149 PBSC) from patients in remission from hematologic malignancies. The mean, SD and range of CD45(+) and CD34(+)cell counts were determined for each donation type. An internal control was used to assess performance of reagents and operators by comparison with a predetermined target value and an experienced operator. RESULTS: Replicate studies showed the method to be accurate and precise, with KG1a cells at 97.7+/-3.9% of the target value and a CV of 4.0%. In routine use over 322 samples, the accuracy was 91.7+/-17.7% of the target value, with a CV of 19.3%. Investigations into the cause of the reduced precision showed that reagents performed consistently well but operator performance was variable, with two of six operators significantly under-dispensing KG1a cells. DISCUSSION: This study validates our three-color flow cytometry assay and demonstrates that KG1a cells may be used to monitor test performance in the routine working environment. In addition to monitoring performance within a single laboratory, its wider use in multicenter studies may be helpful regarding standardization of methods.

Factors affecting volume reduction and red blood cell depletion of bone marrow on the COBE Spectra cell separator before haematopoietic stem cell transplantation.

The COBE Spectra is used to volume/red blood cell (RBC) deplete BM before transplantation or cryopreservation. We have audited our results to identify the effect of transit time, refrigerated storage, age and cellular composition on mononuclear cells (MNC) and CD34+ cell recoveries, volume/RBC depletion and neutrophil engraftment. In total, 88 consecutive collections from autologous (n = 25) and allogeneic (n = 63) donors were included. The mean collection volume was 1250 +/- 398 ml with RBC content of 341 +/- 113 ml. The MNC and CD34+ cell recoveries were 83.3 +/- 18.5 and 88.1 +/- 18.9%, respectively, volume depletion was 88.2+/-4.4% and RBC depletion 98.3 +/- 1.8%. Neutrophil engraftment was achieved in 20.1 +/- 6.4 days. Factors affecting MNC and CD34+ cell recoveries were transit time (P = 0.0060), overall age (P < 0.0210) and MNC/CD34+ cell concentrations (P < 0.0313). The presence of crenated RBC also reduced CD34+ cell recovery (P = 0.0028). Refrigerated storage did not adversely affect cell recovery (P > 0.8161) or neutrophil engraftment (P = 0.8959). This study demonstrates that time in transit, overall age, MNC and CD34+ cell concentrations and RBC condition were important factors affecting processing. RBCs show artefacts soon after collection at ambient temperatures and these may interfere with the separation and collection of MNC/CD34+ cells. Refrigeration at 4-6 degrees C during transit and storage may reduce formation of RBC artefacts and maximize MNC and CD34+ cell recoveries.

Five-locus HLA typing of hematopoietic stem cell donor volunteers using PCR sequence specific primers.

We have developed a strategy for five-locus human leukocyte antigen (HLA) typing of hematopoeitic stem cell (HSC) donors using the polymerase chain reaction with sequence-specific primers (PCR-SSP). The PCR-SSP method is robust, reproducible, and accurate. New PCR-SSP mixtures can be added as required and all reactions are carried out under the same conditions, which can easily be applied to the typing of other loci, e.g., ABO blood groups. Initially, 127 PCR-SSP reactions were used to detect simultaneously HLA-A, -B, -C, -DRB1/3/4/5, and DQB1 alleles, differentiated generally to the level of the first two digits of the allele name, essentially equivalent to the serological split specificity. Approximately 40% of subjects were tested against a further 29 HLA-A, -B SSP mixtures to exclude rare alleles and unambiguously assign a two-digit HLA allele family. This gave an overall typing resolution equivalent to or greater than the split specificity level and covered all HLA-A, -B, -C, -DRBland DQB1 alleles listed in the WHO's Nomenclature for Factors of the HLA System, 2000. The Welsh Bone Marrow Donor Registry has used this strategy to HLA type over 35,000 HSC donors over 9 years. Comprehensive and accurate five-locus HLA typing allows confident and rapid identification of potential matched HSC donors for patients requiring stem cell transplantation generally without the need for typing additional loci. This allows resources to be focused directly on allele level typing of DRB1 and other loci. This strategy decreases overall donor work-up time, which is a major benefit to patients.

HFE mutations, iron deficiency and overload in 10,500 blood donors.

People with genetic haemochromatosis (GH) accumulate iron from excessive dietary absorption. In populations of northern European origin, over 90% of patients are homozygous for the C282Y mutation of the HFE gene. While about 1 in 200 people in the general population have this genotype the proportion who develop clinical haemochromatosis is not known. The influence of HFE genotype on iron status was investigated in 10 556 blood donors. The allele frequencies of the C282Y and H63D mutations were 8.23% and 15.3% respectively. Heterozygosity for C282Y occurred in 1 in 7.9 donors, for H63D in 1 in 4.2 donors, and 1 in 42 were compound heterozygotes. Homozygosity for H63D occurred in 1 in 42 donors and 1 in 147 (72) were homozygous for C282Y. Mean values increased for transferrin saturation (TS) and serum ferritin (sFn), and decreased for unsaturated iron binding capacity (UIBC) in the order: donors lacking the mutations, H63D heterozygotes, C282Y heterozygotes, H63D homozygotes, compound heterozygotes and C282Y homozygotes, but serum ferritin (sFn) concentrations were no higher in H63D heterozygotes and C282Y heterozygous women than in donors lacking mutations. The percentage of donors failing the screening test for anaemia or of those with sFn < 15 microg/l did not differ among the genotype groups. C282Y and H63D heterozygotes and donors homozygous for H63D were at no greater risk of iron accumulation than donors lacking mutations, of whom 1 in 1200 had both a raised TS and sFn. The risk was higher for compound heterozygotes (1 in 80, P = 0.003) and for C282Y homozygotes (1 in 5, P < 0.0001). There was no correlation between sFn and either age or donation frequency in C282Y homozygotes. None of the 63 C282Y homozygous donors interviewed showed physical signs of overload or were aware of relatives with haemochromatosis. The Welsh Blood Service collects blood from about 140 000 people each year including an estimated 950 who are homozygous for HFE C282Y. They are probably healthy and unaware of any family history of iron overload.

Identification and nucleotide sequence of HLA-A*02015.

A novel HLA-A*02 allele, A*02015, is described that possess a unique non-coding substitution of G to A at position 113 in exon 3.

Sequence, genetics and serology of a new HLA-B allele: B*4903.

A new HLA-B allele - B*4903 - was detected by the polymerase chain reaction using sequence-specific priming (PCR-SSP), in a Caucasoid bone marrow panel donor, that differs from B*4901 by 8 nucleotides at positions 141, 142, 144, 165, 167, 193, 206 and 213 in exon 2. These substitutions all occur in HLA-B*51 and B*52 alleles and encode 4 amino acid substitutions at positions 24 (Thr to Ala), 32 (Leu to Gln), 41 (Thr to Ala) and 45 (Lys to Thr). This suggests that B*4903 occurred following a gene conversion-like event involving B*4901 and probably a B*51 allele. HLA-B*4903 was identified on a haplotype with: HLA-A*0201; Cw*07; DRB1*1302/34; DRB3*0301; DQA1*0102; DQB1*0604; BfS; C4A3; C4BQ0 and encodes a unique serological specificity which was characterised by the reactivity of 55 antisera directed towards at least four predicted epitopes. No further examples of B*4903 were found in 15,796 consecutive HLA PCR-SSP typed donors from the Welsh Bone Marrow Donor Registry, indicating that this allele has a phenotype frequency of <0.01% and a gene frequency of <0.00004.

Identification and nucleotide sequence of HLA-A*03013.

We have identified a further HLA-A*03 allele, A*03013, in three unrelated Caucasoid individuals. The allele was initially detected by the polymerase chain reaction using sequence-specific primers (PCR-SSP) and was shown to differ from HLA-A*03011 by a single non-coding substitution of G to T at position 167 in exon 2 by DNA sequencing. The A*03013 bearing haplotype in one of the three individuals, was HLA-A*03013, B*07, Cw*0702, DRB1*1101, DRB3*0202, DQA1*05, DQB1*0301. The estimated phenotype and gene frequencies for A*03013 was <0.02% and <0.00007, respectively.

Hypothyroidism and aging: the Rosses' survey.

An earlier impression of a high prevalence of hypothyroidism in a general practice (4,190 patients including 1,544 adult females aged 18 years or more with 544 aged 50 years or more) in the Rosses, a coastal area in the northwest of Ireland was confirmed by this study. The accumulated prevalence of overt spontaneous primary hypothyroidism was 8.6% in 544 females aged 50 years or more but only 0.9% in the 1,000 females between 18 and 50 years of age. This prevalence was approximately twice that of an Irish National general practice population sample of 4,314 females aged 50 years or more (8.6% vs. 4.6%) p < 0.001. The reasons for this difference are unclear but may reflect the high level of opportunistic screening carried out in West Donegal. Thyroid peroxidase antibodies measured by radioimmunoassay were found in 75.6% of hypothyroid patients compared to 18.6% of practice controls (p < 0.01). Neither HLA-DRB1, DQA1, and DQB1 phenotype frequencies nor dietary iodine intake (median urinary iodine excretion 104 microg/L) appeared to be contributory factors. The finding of an 8.6% accumulated prevalence of hypothyroidism in females greater than 50 years of age when a population is aggressively investigated demonstrates the relative importance of its contribution to total morbidity and suggests that the disorder may be underdiagnosed, thus supporting the concept of targeted screening in this age group.

Population screening for hemochromatosis by PCR using sequence-specific primers.

Over 90% of patients with hemochromatosis in the United Kingdom are homozygous for the C282Y mutation on the HFE gene. The Centers for Disease Control (CDC) in the United States has recommended that adults should be screened for HFE mutations to identify susceptible individuals before onset of disease. The aim of this study was to evaluate the polymerase chain reaction using sequence-specific primers (PCR-SSP) as a method of large-scale population screening for the common HFE gene mutations, H63D and C282Y. A total of 10,583 consenting blood donors were tested using nonautomated procedures. Three alleles, termed HFE-1, -2, and -3, were detected with phenotype frequencies of 94.56%, 28.33%, and 15.79%, respectively, and gene frequencies of 0.76421, 0.15342, and 0.08237, respectively. All donors identified as homozygous for the C282Y mutation or heterozygous for both the H63D and C282Y mutations were confirmed by heterduplex analysis and/or PCR-SSP. The number of technical failures that affected the identification of donors homozygous for the C282Y mutation was 390 giving an overall repeat rate 3.7%, although this fell to 1% over the last quarter of the study. This study demonstrates that PCR-SSP may be used for large-scale population screening for the C282Y genotype associated with hemochromatosis.

Molecular, serological and genetic studies on two new HLA-DRB1 alleles--HLA-DRB1*0704 and HLA-DRB1*1507.

Two new HLA-DRB1 alleles (DRB1*0704 and DRB1*1507) were detected during routine polymerase chain reaction (PCR)-based typing of two Caucasoid bone marrow panel donors due to apparent DRB1* "blanks" being associated with unexpected DRB4, DRB5 and DQB1 alleles. HLA-DRB1*0704 differed from DRB1*0701 by five consecutive nucleotides at positions 217 to 221 of exon 2 encoding two amino acids substitutions of tyrosine to asparagine at codons 77 and valine to tyrosine at codon 78. DRB1*1507 differed from DRB1*1501 by a single nucleotide at position 127 encoding an amino acid substitution of phenylalanine to tyrosine at codon 47. Their specificities were unequivocally assigned by serology as HLA-DR7 and DR15, respectively, and family and population studies allowed their likely HLA-A,B,C,DR,DQ and complement (Bf, C4A, C4B) bearing haplotypes to be identified. No further examples were found in 19,113 HLA-DR,DQ typed donors from the Welsh Bone Marrow Donor Registry indicating that both these alleles have a phenotype frequency of <0.01% and a gene frequency of <0.00003.

Human platelet antigens: typing by PCR using sequence-specific primers and their distribution in blood donors resident in Wales.

We developed and validated HPA-1 to HPA-6 typing by PCR-SSP using a combination of established, modified and newly designed sequence-specific primers. We confirmed that the PCR primer mixtures functioned under the same PCR conditions as our standard HLA-A, -B, -C, -DR, -DQ PCR-SSP typing system. This allows concurrent testing for both HPA and HLA specificities and is therefore the system of choice for both clinical and large-scale blood donor panel HPA and HLA typing by PCR-SSP. Test validation included typing a population of blood donors living in Wales. These HPA frequencies were consistent with those of other European Caucasoid populations. HPA-4b and -6b were absent and HPA-5b, which shows some frequency variation, had a phenotype frequency of 18.9% (allele frequency 0. 0973), being close to that of the Dutch (19.7%) and Austrian (20.4%) populations and almost twice that found in Finns (10.0%). HPA genotype frequencies showed a good fit to Hardy-Weinberg equilibrium, further supporting the validity of our typing method.

HLA-B*4703: sequence confirmation, serology and distribution.

This study has confirmed the nucleotide sequence of exons 2 and 3 of the B*4703 allele, discovered by an unusual HLA-B47 and Bw6 serological pattern, in two subjects of Black/Japanese and Caribbean Black descent. Titration studies on 25 HLA-B47 cross-reactive sera, stimulated by B13, B27, B44 and B60, and nine Bw6 antisera/monoclonal antibodies, showed that the B*4703 product can be distinguished from the established HLA-B47 specificity. The phenotypes of these donors and an International Cell Exchange donor suggests an association between B*4703 and Cw*0701/ 06 in Black subjects. No examples of B*4703 (or B*4702) were found in 10,194 PCR-SSP HLA-A,B typed Welsh Bone Marrow Donor Registry panel members indicating a phenotype frequency of <0.0098% in this primarily Northern European Caucasoid population.

Molecular, serological and population studies of the alleles and products of HLA-B*41.

The HLA-B41 specificity was first identified over 25 years ago and, although both serological and biochemical studies have suggested its subdivision, it is only recently that two HLA-B*41 alleles (B*4101 and B*4102) have been identified and sequenced. We designed three oligonucleotide primers, combined in two mixtures to define these alleles by PCR using sequence-specific primers (PCR-SSP) in a random normal population of 9,464 HLA-A, B, DR, DQ typed Northern European Caucasoid donors from the Welsh Bone Marrow Donor Registry. The HLA-B41 phenotype frequency was 0.835%, and of the 79 HLA-B41 subjects 22 (27.85%) were B*4101 and 57 (72.15%) were B*4102. The phenotype frequencies of B*4101 and B*4102 were 0.232 and 0.602%, respectively, and the gene frequencies were 0.00116 and 0.00301, respectively. Formal two-locus linkage disequilibrium (LD) analysis demonstrated significant associations between B*4101 and A30 and DR11, and between B*4102 and A66 and DR13. LD analysis of three loci showed significant associations between B*4101, DR7, DQ2 and B*4101, DR11, DQ7 (DQB1*0301/0304) and between HLA-A3, B*4102, DR13; A66, B*4102, DR7; A66, B*4102, DR13; B*4102, DR13, DQ6 and B*4102, DR13, DQ7. Examination of the HLA phenotypes (including HLA-C) of the B*41 subjects, together with the LD analysis findings, suggested four different HLA haplotypes bearing B*4101 and five B*4102 haplotypes. The most frequent B*4101 haplotype was: HLA- A30 or other A allele, Cw*1701, B*4101, DRB1*1102, DQB1*0301 and the most freqent B*4102 haplotype was: A*6601 or A3 or other A allele, Cw*1701, B*4102, DRB1*1303, DQB1*0301. In addition, the well-known association of A66 with B41 was between A*6601 and B*4102, and although both B*41 alleles were commonly in association with Cw*1701, B*4101 was found with Cw*07. One-dimensional isoelectric focusing (1D-IEF) analysis of HLA-B proteins from 2 B*4101 and 2 B*4102 subjects clearly showed that the B41.1 and B41.2 1D-IEF variants, identified in the 10th International Histocompatibility Workshop, corresponded to B*4101 and B*4102 products, respectively. Serological titration studies, with 59 lymphocytotoxic pregnancy antisera, containing an HLA-B41 component and stimulated by up to five different HLA-B specificities, were unable to differentiate the two groups of B*41 subjects. This suggests that partition of the HLA-B41 specificity will not normally be achieved by classical serological methods. It is suggested that the previous alleged serological subdivision of HLA-B41 was founded on the unwitting use of antisera detecting the HLA-Cw*17 products.

Identification, sequencing and serology of HLA-B*3527.

We describe a variant HLA-B*35 allele (B*3527) that was detected by the polymerase chain reaction using sequence-specific primers (PCR-SSP) in an individual on the Welsh Bone Marrow Donor registry. B*3527 differs from B*3501 by a single base (G/A) at position 302 that encodes an amino acid change of Ser to Asn at position 77 in the Bw6 epitope. Serological studies using 38 B35-, 4 Bw6- and 7 Bw4-reactive sera indicated that the B*3527 product was indistinguishable from the B35 specificity, and that the substitution did not significantly affect the Bw6 epitope. A family study determined the B*3527 bearing haplotype as: HLA-A*29, B*3527, Cw*0401, DRB1*0404, DRB4*0101/0103, DQA1*03, DQB1*0302, BFS, C4A3, C4B1. The phenotype and gene frequencies in the local Welsh population were<0.01% and <0.00005%, respectively.

Identification of HLA-A*0224: implications for PCR-SSP HLA typing.

We describe a novel HLA-A*02 allele, A*0224, that was identified after a comparison of DNA and serological typing revealed a discrepancy in the HLA-A types: HLA-A2 was defined by serology but was not detected by the polymerase chain reaction using sequence-specific primers (PCR-SSP). DNA sequencing indicated the presence of a variant HLA-A*02 allele that differed from A*0201 by a single base (C/A) at position 453. This base substitution corresponded to the annealing site of a primer common to the two A*02-amplifying PCR-SSP mixtures used in the method. This provides an explanation for the results and highlights a limitation of PCR-SSP methods even where two PCR mixtures are used to detect alleles. Serological titration studies suggested that A*0201, A*0205 and A*0224 are unlikely to be differentiated during routine serological typing.

Rapid detection of genetic mutations associated with haemochromatosis.

BACKGROUND AND OBJECTIVES: The purpose of this study was to establish a rapid method suitable for large-scale population screening, including blood donors, for the detection of two genetic mutations at codons 63 and 282 on the HFE gene that are associated with haemochromatosis. MATERIALS AND METHODS: A method using the polymerase chain reaction with sequence-specific primers (PCR-SSP) was designed and tested using a panel of 185 individuals previously typed for HFE mutations by PCR-RFLP. RESULTS: The PCR-SSP method detected the two mutations showing complete agreement with the genotypes obtained by PCR-RFLP. Three HFE alleles, termed HFE-1, -2, and -3, were identified. CONCLUSIONS: This PCR-SSP method allows efficient HFE genotyping for large numbers of individuals.

HLA class I (A, B) and II (DR, DQ) gene and haplotype frequencies in blood donors from Wales.

Accurate estimates of HLA-A, B, DR and DQ phenotype, gene and haplotype frequencies (HF) in the normal population are of importance in, for example, disease susceptibility studies, platelet transfusion support and transplantation. HLA population genetics studies have been performed on numerous groups, however, no major studies have been carried out on the population of Wales. As part of the validation process for our routine HLA-A and B typing by PCR using sequence-specific primers (PCR-SSP) we examined 1,798 normal, unrelated Caucasoid blood donors living in Wales and recruited onto the Welsh Bone Marrow Donor Registry (WBMDR). Typing was performed by serology (HLA-A, B) and PCR-SSP at low resolution (HLA-A, B, DR, DQ) resulting in a particularly rigorous level of HLA specificity assignment. Four discrepancies were found between the HLA-A and B serological and PCR-SSP specificity assignments: (1) two instances of HLA-A2 by serology were undetected by PCR-SSP and were a new HLA-A2 allele - A*0224; (2) one example of HLA-B*15 by PCR-SSP failed to react by serology, and remained undetectable by serology in subsequent samples, and (3) one example of HLA-B45 by serology was identified as HLA-B*5002 by PCR-SSP. Hardy-Weinberg and homozygosity analysis showed that the goodness-of-fit was excellent (p > 0.05), for both phenotype distribution and the number of homozygotes identified, for all four loci. The phenotype and gene frequencies for the 18 HLA-A, 34 -B, 15 -DR and 8 -DQ specificities identified and two- and three-locus HF, linkage disequilibrium and related values for HLA-A/B, B/DR, DR/DQ and HLA-A/B/DR and B/DR/DQ were essentially typical of a northern European population. HLA-A2, B44, DR4 and DQ2 were the highest frequency phenotypes and HLA-A2403, A34, A74, B42, B75, B2708, B48, B67 and B703 occurred once only. There were no examples of: A36, A43, A69, A80, B46, B54, B59, B73, B76, B77, B7801, B8101 or DR18 specificities. DR17, DQ2 and A1, B8, DR17 were the highest frequency two- and three-locus haplotypes identified. Diverse HLA-A, B, DR phenotypes were identified in 87.0% (1,564) of subjects. When HLA-DQ was also considered, different four locus phenotypes were identified in 89.1% (1,602) of subjects. This frequency information will be beneficial as a high-quality reference control for disease susceptibility studies and in calculating the chances of identifying a bone marrow donor in a patient's extended family. This process was successful for the validation of our HLA-A and -B PCR-SSP typing procedure and the findings suggest an accurate level of specificity assignment of WBMDR panel donors who had previously been typed by serology alone.

Molecular, serological and population studies on a novel HLA-B allele--HLA-B*5002.

A novel HLA-B allele (B*5002), detected as a discrepancy between serological and PCR-SSP HLA-A and B phenotyping of bone marrow panel donors, was identified by nucleotide sequencing of exons 2 and 3. Titration studies on 39 HLA-B12/B21 cross-reactive antisera showed that the serological specificity of HLA-B*5002 was HLA-B45. PCR-SSP testing of 287 serologically defined HLA-B45-positive subjects from a panel of 12,411 donors, together with HLA-B*45 and B*5002 frequency data on 4,342 PCR-SSP typed subjects, indicated that 4.53% of serologically defined HLA-B45-positive subjects possess HLA-B*5002 and not HLA-B*4501. The phenotype frequency of HLA-B*5002 was 0.08954%; gene frequency was 0.00045 (n=16,753). In 73.3% of instances B*5002 appeared to be present on a haplotype with DRB1*0406 and DQB1*0402, 54.6% of which possessed A*2301. The B*5002, DRB1*0406, DQB1*0402 haplotype represents 52.4% of all haplotypes with DRB1*04 and DQB1*04 and 78.6% of haplotypes possessing DRB1*0406 and DQB1*0402.

Serological and molecular identification of an HLA-B8 variant, HLA-B8Jon (B*0802).

The serological and molecular characteristics of the first HLA-B8 variant (B8Jon), located on a haplotype bearing HLA-A1, -B8Jon, -Cw*07, DRB1*0301, DQA1*05, DQB1*0201, BfS, C4AQ0, C4B1 and the microsatellite alleles D6S265-3, 258-11, 105-8, 299-1 and 202-2 are identified and described. This new HLA-B antigen was distinguished from the usual B8 by its lack of reactivity with Bw6 and most Bw4 antisera, together with its failure to react with most antisera cross-reactive with HLA-B8. B8Jon gives a 'weaker' response than HLA-B8 in titration studies with B8 antisera. It cannot be differentiated from the usual B8 by one-dimensional iso-electric focusing. Time course studies, absorption analyses and serological tests on 17 Bw4 antibodies suggest that B8Jon possesses an unusually 'weak' Bw4 epitope. Studies on the Bw4 sequence motif by PCR, using sequence-specific primers, indicate that B8Jon has a Bw4 sequence in common with other HLA-B alleles, including B*4402-B*4405, rather than the Bw6 motif found on the familiar HLA-B8 molecule.