Genotyping of Blood Group Systems among Multi-Transfused Thalassemia Patients in Palestine for Safe Blood Transfusion

Abstract

Study DesignThis cross-sectional study was conducted in Palestine among multi-transfused thalassemia patients during 2021 (n=100). Demographic and medical data were collected from medical files of patients. In addition, blood samples were collected from patients for DNA extraction, antibody screening and antibody identification, and assessment of the biochemical, hormonal, and hematological parameters of the patients.Antibody Screening and IdentificationThree cell antigen panels were used for antibody screening. In the case of positive antibody screening, antibody identification that includes the most common antigens such as D, C, c, E, e, M, N, S, s, K, k, Lea, Leb, Fya, Fyb, Jkb and P was performed by solid phase method using NEO Iris Analyzer (Immucor, USA).Laboratory Assessment of Biochemical, Hormonal, and Hematological ParametersFor the assessment of the biochemical, hormonal, and hematological parameters of the patients, the following parameters were tested blood urea nitrogen (BUN), serum creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), Calcium (Ca), serum ferritin (SF), hemoglobin (Hb), 25-hydroxy vitamin D (vit-D), growth hormone (GH), parathyroid hormone (PTH), total triiodothyronine (TT3), free thyroxine (FT4), fasting blood sugar, and thyroid stimulating hormone (TSH).BUN, serum creatinine, Ca, AST, ALT, and ALP were performed by the photometric enzymatic methods. All measurements were performed using Roche kits and Cobas c 311 analyzer (Roche, Switzerland) following manufacturers’ instructions. TT3, FT4, TSH, and PTH, in addition to Vitamin D and SF, were measured using the Abbott ARCHITECT I1000SR Immunoassay Analyzer (Abbott, USA) following the manufacturer’s instructions using Abbot Kits. In addition, Hb levels were measured using the automatic blood cell counter MEK-6510 (Nihon Kohden, Japan). GH was measured using Maglumi 800 (Snibe Co. Ltd., China).DNA ExtractionGenomic DNA was obtained from peripheral venous blood samples collected in EDTA blood tubes. The DNA was extracted using Promega Wizard Genomic DNA Purification Kit (Promega, USA) according to manufacturer instructions. The DNA concentration and purity of each sample were determined by the measurement of optical density For each sample, a total of 9 μL of 10–30 ng/μL DNA were sent to the laboratory at the University of Gothenburg for genotyping.Molecular GenotypingERY Q Kits (BAG Diagnostics, Germany) were used for the genotyping of blood groups. These kits are intended for second-line determination of blood group characteristics. The molecular genetic typing was carried out using the sequence-specific primers (SSP)-PCR technique and real-time PCR (RT-PCR).In the first stage, DNA samples were tested using three kits: ERY Q® RH (REF 728405), ERY Q® KKD/MNS (REF 728407), and ERY Q® Rare (REF 728408). The combination of primers and probes chosen for the particular ERY Q® Kit allows the detection of clinically relevant alleles in the targeted blood groups which include Rhesus, Duffy, Kell, Kidd, Colton, Knops, Lewis, Luth, Dombrock, MNS, Diego, Cartwright (Yt), and Vel. Five samples did not show clear results for the RHD using the ERY Q® RH (REF 728405) and therefore were tested using the ERY Q® Partial D (REF 728403). Test procedures and evaluation of data were performed according to the described procedures in the Instructions Manual. In addition, the specificities of the primers and probes chosen for each particular ERY Q® Kit are also described in the Manual.</p