Variant analyzer

Worldwide, a number of genotypes manifest phenotypically as SCD. The principal genotypes include homozygosity for the β^s mutation of the HBB gene (HbSS), sickle cell-hemoglobin C disease (HbSC) and sickle cell-β-thalassemia. HbSS is the only significant cause of SCD in Kenya [12 (link)]. Results of blood tests for HbS typing, conducted by either cellulose acetate hemoglobin electrophoresis (Helena Laboratories, Beaumont, TX, USA) or by high performance liquid chromatography (Variant Analyzer, BioRad, Hercules, CA, USA), were available for a subset of children who were either tested during the course of their admission to KDH or who were members of two prospective cohort studies: (i) the Kilifi Sickle Cell Disease (KSCD) study [13 (link)] or the Kilifi Genetic Birth Cohort (KGBC) study [14 (link)]. We quantified the contribution of SCD to childhood mortality according to VA, and validated the results in the subset of deaths in children who had been involved in any of these studies, and for whom there was therefore laboratory data that confirmed or refuted a diagnosis of SCD HbSS.

Hb phenotype was determined by alkaline Hb electrophoresis (Helena; Sunderland, United Kingdom) and by quantification of Hb fractions, including HbF, by high-performance liquid chromatography (β-Thalassemia Short Program, VARIANT analyzer; BioRad, Hercules, CA). In subjects with SCD, the most recent steady-state Hb, total bilirubin, conjugated bilirubin, and lactate dehydrogenase levels were obtained from MSC records (steady state defined as absence of pain or fever, malaria rapid test negative, and with no recorded hospital admission or blood transfusion within 30 days). Full blood counts were performed using an automated cell counter (Pentra 60; Horiba ABX, Kyoto, Japan). Biochemical tests were performed using an automated chemistry analyzer (Cobas Mira [Roche, New York, NY] or Abbott Architect [Abbott Diagnostics, New York, NY]).

We measured fasting plasma glucose (FPG)—with enzymatic method using BIOSEN 5040 analyzer (EKF-Diagnostic GmbH, Germany), glycated hemoglobin (HbA1c)—with high-performance liquid chromatography (HPLC) method using Variant analyzer (Bio-Rad Laboratories Inc, USA), insulin—with radioimmunoassay IMMULITE 2000 Insulin (Siemens Healthcare Diagnostics, USA). Insulin resistance index (HOMA-IR) was calculated (HOMA-IR = fasting insulin (mU/l) × fasting glucose (mmol/l)/22.5), and oral glucose tolerance test (OGTT) with 75 g of glucose was performed according to World Health Organization (WHO) procedures (in groups DC and D).

Fasting plasma glucose (glucose oxidase-peroxidase method), serum cholesterol (cholesterol oxidase-peroxidase-amidopyrine method), serum triglycerides (glycerol phosphate oxidase-peroxidase-amidopyrine method) and HDL cholesterol (direct method-polyethylene glycol-pretreated enzymes) were measured using Hitachi-912 Autoanalyser (Hitachi, Mannheim, Germany). The intra and inter assay co-efficient of variation for the biochemical assays were <5%. Low-density lipoprotein (LDL) cholesterol was calculated using Friedewald formula. Glycated hemoglobin (HbAlc) was estimated by high-pressure liquid chromatography using the variant analyzer (Bio-Rad, Hercules, Calif., USA). Serum Insulin was estimated using enzyme-linked immunosorbent assay (Calbiotech, CA). The intra-assay and the inter-assay coefficients of variation for insulin assay was <10%. Insulin resistance was calculated using the homeostasis assessment model (HOMA-IR) using the formula: {fasting insulin (μIU/mL) x fasting glucose (mmol/L)} / 22.5.

Fasting plasma glucose (glucose oxidase-peroxidase method), serum cholesterol (cholesterol oxidase-peroxidase-amidopyrine method), serum triglycerides (glycerol phosphate oxidase-peroxidase-amidopyrine method) and HDL cholesterol (direct method-polyethylene glycol-pretreated enzymes) were measured using Hitachi-912 Autoanalyser (Hitachi, Mannheim, Germany). The intra- and inter-assay co-efficient of variation for the biochemical assays were <5%. Low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald formula [16 (link)]. Glycated hemoglobin (HbAlc) was estimated by high-pressure liquid chromatography using the variant analyzer (Bio-Rad, Hercules, Calif., USA). Serum insulin was estimated using enzyme-linked immunosorbent assay (Calbiotech, CA). The intra-assay and the inter-assay coefficients of variation for insulin assay was <10%. Insulin resistance was calculated using the homeostasis assessment model (HOMA-IR) using the formula: fasting insulin (μIU/mL)*fasting glucose (mmol/L) / 22.5.