Preliminary report of HLA (DNA) typing of Nigerians using sequence-specific primer technique.

AIMS AND OBJECTIVES: This communication is an attempt to present the experience and a preliminary report of results over a one-year period. PATIENTS AND METHODS: From December 2011 to December 2012, a prospective determination of the HLA types of 20 individuals referred to the Tissue Typing Laboratory of the Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC), Ile-Ife was done. These consisted of prospective transplant recipients, their donors, and a migrant pair for kinship determination. DNA was extracted from the client's peripheral blood sample, using the QIAmp Blood DNA Mini kit, (Qiagen). PCR was done using OlerupR low-resolution PCR-SSP typing kit. The PCR product was resolved in 2% agarose gel, and the bands visualised under UV light. The HLA types were determined using provided tables and/or Helmberg software. Data were presented using descriptive statistics whileHLA antigen frequency (AF) was expressed in percentage and gene frequency (GF) was determined using square root method (1-(1-AF)1/2). RESULTS: A total of 20 individuals (13males and 7females) consisting of seven renal transplant recipients and seven prospective donors; a stem cell recipient and three donors and a migrant pair for kinship determination were typed. Age ranged from 4-65 years. 44 HLA alleles were detected, while HLA-A, B, C, DRB1 and DQB1 were 7, 10, 11, 8, 8 alleles respectively. The alleles were heterogeneous in distribution while 6 antigens (HLA-A*02, B*30, C*15, DRB1*03, DRB1*08 and DQB1*06) were having frequencies e"25%. CONCLUSION: This report confirms that DNA-based HLA typing is feasible locally, andit was observed that renal transplantation procedure is the most frequent indication. The HLA antigens observed to have very high frequencies (e"25% frequency) in this population were HLA-A*02, B*30, C*15, DRB1*03, DRB1*08 and DQB1*06. There is a strong need to develop a broad-based HLA data bank for Nigeria to further strengthening her transplantation programmes.

Resolution of HLA class I sequence-based typing ambiguities by group-specific sequencing primers.

The increasing demand for allele-level human leukocyte antigen (HLA) typing has led the sequence-based typing (SBT) to become the preferred method. In turn, the steady increase in the number of HLA alleles driven by the adoption of SBT as the ultimate typing method leads to the ever increasing number of cis/trans ambiguities. Over the last few years, additional sequencing with the commercially available group-specific sequencing primers (GSSPs) has replaced sequence-specific primer-polymerase chain reaction and group-specific amplification as the means of resolving cis/trans ambiguities in many laboratories. Here we summarize our 3-year experience in designing and utilizing GSSPs for resolution of HLA class I ambiguities. The panel of GSSPs used in our laboratory includes 14 primers for HLA-A, 18 for HLA-B, and 13 primers for HLA-C. The panel resolves 99.9% of all ambiguities.

Quantitation of residual white cells in filtered blood components by polymerase chain reaction amplification of HLA DQ-A DNA.

BACKGROUND: Over the past several years, blood filtration technology has improved dramatically, such that currently available experimental filters are capable of reducing white cells (WBCs) in blood components to less than 0.1 WBC per microL. These residual WBC concentrations are below the sensitivity of automated cell counters, as well as of large-volume (Nageotte) hemocytometers. STUDY DESIGN AND METHODS: A quantitative polymerase chain reaction (PCR) amplification assay directed at HLA DQ-A DNA sequences has been developed for the enumeration of WBCs in filtered blood. To ensure quantitative recovery of WBCs at very low residual cell concentrations, a direct red cell lysis and WBC concentration protocol using 0.5 mL of filtered blood was perfected. Amplified product is detected by oligomer hybridization using 32P-labeled probes, with quantitation by image analysis of autoradiographic signals relative to a standardized dilution series processed in parallel. RESULTS: Recovery of residual WBCs in filtrates was shown to be enhanced by the addition of xenogeneic WBCs or polystyrene beads, which served as "carrier" particles during red cell lysis and wash steps. A contribution of nuclear fragments in filtered blood to PCR signal in the range of 0.01 to 0.5 WBCs per microL was observed; a modified protocol was developed to minimize this effect. Parallel analysis of spiked dilution series and evaluations of 39 red cell components filtered through commercial filters indicated good correlation between PCR and standard Nageotte counts in the range of 0.1 to 10 WBCs per microL (r2 = 0.94); only PCR was able to detect residual WBCs in filtrates from prototype 6 log10 WBC-reduction filters. CONCLUSION: This assay should prove useful for the development and quality assurance of increasingly efficient WBC-reduction filters.

Benign muscular dystrophy: risk calculation in families with consanguinity.

This report concerns two families in which the index patients are sporadic cases of a benign form of muscular dystrophy. In both families the sisters of the patients have married a close relative. The respective risks for a child of these consanguineous marriages being affected with either X linked Becker muscular dystrophy or autosomal recessive limb girdle muscular dystrophy is calculated using pedigree information, results of serum creatine kinase determinations, and also, in one family, results of DNA typing using RFLPs from the short arm of the X chromosome.