The Use of HPLC as a Tool for Neonatal Cord Blood Screening of haemoglobinopathy: A Validation Study.

BACKGROUND: Newborn cord blood screening identifies infants with underlying haemoglobinopathies before they develop the characteristic symptoms or sequelae. AIMS: This study was performed to validate the interpretation high-performance chromatography (HPLC) along with complete blood count (CBC) results as a tool for universal neonatal screening of hemoglobin disorders in Oman. METHODS: HPLC and CBC data on subjects who participated in the National Neonatal screening program at birth were obtained from archival records. The results recorded at birth were compared with a second study performed on the same subjects, after approval from the local medical research and ethics committee. RESULTS: Only 290 subjects from amongst the original cohort of 3740 newborns could be recalled between April 2010 to March 2011, to repeat HPLC and CBC, as well as perform confirmatory DNA studies, wherever necessary. All these subjects had been documented to show an initial abnormal result. 31 cases who had no HbA at birth on HPLC were confirmed as either homozygous ß-thalassaemia major (n=5 subjects) or homozygous sickle cell anemia (n=26 subjects) by appropriate DNA analysis. Additionally, amongst 151 subjects, 72 subjects were studied in the initial study by Hb Bart's quantitation using the alpha thalassaemia short program at birth. In this cohort, 42 subjects with Hb Bart's >1% at birth could be confirmed as having either deletional or non-deletional thalassaemia by GAP PCR studies. No case of HbH was detected in this cohort. Further, carrier status for structural hemoglobin variants (HbS, HbC, HbD, HbE) (n=67) and beta thalassaemia allele with low HbA at birth (n=29 out of 41) were confirmed by relevant molecular studies. CONCLUSIONS: The study validated the earlier observation by 100% concordance with the results of CBC and HPLC. Presence of Hb Bart's at birth does not always mean the presence of alpha thalassemia, as subjects with Hb Bart's below 1% by quantitation, were shown to be normal by molecular studies.

Correlation between flow cytometry and molecular findings in autosomal recessive chronic granulomatous disease: A cohort study from Oman.

BACKGROUND: Chronic granulomatous disease (CGD) is an X-linked (XL) or autosomal recessive (AR) primary immunodeficiency disease. Respiratory burst assessment by flow cytometry is a rapid test of granulocyte stimulation, and results predict the underlying genotype. This study aims to describe the immune-phenotypic profile of patients with CGD diagnosed in our center and correlate that with underlying genetic mutations. METHODS: Immuno-phenotypic and genetic data on all patients with CGD diagnosed at Sultan Qaboos University Hospital (SQUH) were reviewed. RESULTS: A total of 32 patients were diagnosed with CGD using molecular studies. Genetically confirmed individuals included 1 patient with XL-CGD (a large deletion involving the CYBB and XK genes resulting in a McLeod phenotype), 27 patients with AR-CGD with a c.579G>A (p.Trp193X) mutation at the NCF1 gene, and 4 patients with AR-CGD with a c.784G>A (p.Gly262Ser) mutation at the NCF1 gene. Flow cytometry and molecular results were available for comparison in 26 patients with AR-CGD. The patients with AR-CGD had a range of flow cytometry-generated fluorescent patterns as follows: reduced neutrophil stimulation with a sharp peak (12/26), reduced neutrophil stimulation with a broad peak (11/26), and a complete lack of neutrophil stimulation (3/26). No consistent flow cytometry-generated fluorescent pattern was observed in either of the 2 AR mutations identified in our patients. CONCLUSION: Flow cytometry is a robust test of CGD diagnosis. However, results should be interpreted with caution when predicting the underlying probable genotype, and results need to be complemented with definitive molecular studies.

Clinical and molecular findings of chronic granulomatous disease in Oman: family studies.

Chronic granulomatous disease (CGD), a rare inherited disorder of the innate immune system, results from mutations in any one of the five genes encoding the subunits of the nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidase enzyme, and is characterized by recurrent life-threatening bacterial and fungal infections. Molecular analysis of 14 Omani CGD patients from 10 families, diagnosed to have CGD on clinical (recurrent infections) and biochemical grounds (positive for both the nitroblue tetrazolium (NBT) test and the dihydrorhodamine (DHR-1,2,3 assay), revealed that only one patient had X-linked CGD, with a large deletion involving both the gp91-phox gene (CYBB) and the McLeod gene (XK). The remaining 13 patients were all homozygotes from a previously described c.579G>A (p.Trp193X) mutation in the NCF1 gene on chromosome 7, responsible for autosomal recessive CGD (AR-CGD). Although X-linked CGD is the most common type of CGD disorder in most population groups, AR-CGD is the most prevalent type in Oman.

Potential pitfalls in the diagnosis of Hb Handsworth in areas with high prevalence of HbS.

Hb Handsworth is a rare α-globin structural variant caused by a missense mutation either on the α2 or α1-globin gene (HBA2 or HBA1: c.55G>C, p.Gly18Arg). This variant might be erroneously diagnosed as HbS unless secondary confirmative tests are carried out. We encountered a child with a prominent peak eluting in the 'S' window on high-performance liquid chromatography (HPLC). Sickle solubility test, gel electrophoresis, and selective direct nucleotide sequencing of α1, α2, and ß globin genes were performed on the patient's sample. In addition, previous HPLC results on a cord blood sample were retrieved. Sickle solubility test was negative. Gel electrophoresis revealed a band migrating at the S region with an extra faint band seen on acid gel electrophoresis. Molecular analysis of α2 globin gene revealed heterozygous state of Hb Handsworth. Hb Handsworth is a rare variant that can mimic HbS on HPLC. Failure to recognize this rare variant in regions where HbS is highly prevalent may result in serious misdiagnosis and subsequent incorrect genetic counseling.

Hematopoietic stem cell transplantation in Oman.

Hematopoietic SCT (HSCT) is an integral part of the management of patients with hematologic disorders. The Sultanate of Oman, with a population of 2.3 million, has an HSCT program based in the Sultan Qaboos University (SQU) hospital. Initiated in 1995, this two-bed unit continues to be the only program in the country. Between June 1995 and August 2006, a total of 128 patients underwent HSCT in this center, averaging about 10-12 transplants per year. The median age of these patients was 11 years (2 months to 45 years). Hematologic malignancies (49%) and inherited disorders (42%) constituted the major transplant indications, whereas BM failure accounted for the remaining. The majority of transplants carried out so far have been HLA-matched sibling-donor allogeneic HSCTs. Among the inherited disorders, homozygous beta-thalassemia and primary immunodeficiency are important transplant indications in this center. The approximate cost of an uncomplicated transplant in this center is US$50,000. The success of this program has now led to the initiation of a new and larger HSCT complex to provide the opportunity for more patients to benefit from this treatment modality within the country.

Effects of age on composition and quality of muscle Longissimus thoracis of the Omani Arabian camel (Camelus dromedaries).

The aim of this study was to determine the effects of age on chemical composition and quality characteristics of the Arabian one-humped camel's meat. Samples of longissimus thoracis (between the 10th and the 13th rib of the left side) were randomly collected from 21 Omani intact male camels of three different age groups: group 1 (1-3 years), group 2 (3-5 years) and group 3 (6-8 years). Samples were chilled (1-3°C) for 48h. Moisture, crude protein, fat and ash were determined on freeze dried ground muscle. Mineral contents were determined using an Inductively coupled plasma emission spectrometer (ICP). Meat quality including ultimate muscle pH, Warner-Bratzler shear force, sarcomere length, myofibrillar fragmentation index, expressed juice, cooking loss percent, and colour L(∗), a(∗), b(∗) were measured using standard methods. The moisture, protein, fat and ash ranged from 64.4% to 76.7%; 18.6% to 25.0%, 1.1% to 10.5% and 1.0% to 1.4% on dry matter basis, respectively. The Ca, Mg, Na, K, P, Cad, Cr, Ni, Pb, Co, Mo, Be and V ranged from, 9.2 to 46.6, 24.7 to 57.3, 104.7 to 257.0, 471.4 to 1053.0, 249.9 to 584.0, 0.005 to 0.024, 0.020 to 0.410, 0.016 to 0.187, 0.010 to 0.299, 0.010 to 0.018, 0.050 to 0.470, 0.005 to 0.030 and 0.013 to 0.141mg/100g on dry matter basis, respectively. The percentage of protein decreased and that of fat increased with increasing camel age. The ultimate pH, shear force, sarcomere length, fragmentation index, expressed juice, cooking loss, lightness (L(∗)), redness (a(∗)) and yellowness (b(∗)) ranged from 5.46 to 6.64, 4.25 to 17.82, 0.96 to 2.50, 55.91 to 94.81,19.50 to 33.63, 13.18 to 29.88, 27.86 to 43.21, 10.46 to 22.81, and 4.63 to 10.11, respectively. Muscles of younger camels (group 1) had significantly (P<0.05) lower shear force value, ultimate pH and higher sarcomere length, fragmentation index, expressed juice, cooking loss, and lightness color (L(∗)) by 48%, 3.4%, 43%, 25%, 28%, 14%, and 16% than those collected from older camels (group 3), respectively. Values of middle age camels (group 2) camels were in-between. This study confirmed that camel meat is healthy and nutritious as it contains low fat as well as being a good source of minerals. Age is an important factor in determining meat quality and composition.