Comparative analysis of cellulose acetate hemoglobin electrophoresis and high performance liquid chromatography for quantitative determination of hemoglobin A2

BACKGROUND: The present study is designed to evaluate the reliability and cost effectiveness of cellulose acetate Hb electrophoresis and high performance liquid chromatography (HPLC) in the determination of HbA2 levels. METHODS: The test population comprised 160 individuals divided into four groups: normal individuals, beta-thalassemia trait (BTT) patients, iron deficiency anemia (IDA) patients, and co-morbid patients (BTT with IDA). HbA2 levels determined using cellulose acetate Hb electrophoresis and HPLC were compared. RESULTS: HbA2 levels were found to be diagnostic for classical BTT using either method. In co-morbid cases, both techniques failed to diagnose all cases of BTT. The sensitivity, specificity, and Youden's index for detection of the co-morbid condition was 69% and 66% for HPLC and cellulose acetate Hb electrophoresis, respectively. CONCLUSION: This study revealed that semi-automated cellulose acetate Hb electrophoresis is more suitable for use in beta-thalassemia prevention programs in low-income countries like Pakistan. This technique is easily available, simple and cost effective.

Chronic myeloid leukemia with rare e1a3 BCR-ABL transcript

Chronic myelogenous leukemia [CML] results from neoplastic transformation of a hematopoietic stem cell. It is cytogenetically characterized by the presence of Philadelphia chromosome which results from reciprocal translocation t[9;22] that juxtaposes the ABL gene on chromosome 9 with breakpoint cluster region [BCR] on chromosome 22 generating BCR-ABL oncogene. All BCR-ABL fusion proteins display activated tyrosine kinase activity. Their different types are associated with different clinical course and prognosis. We report a rare case of e1a3 BCR-ABL transcript. So far only 4 cases in patients with CML have been reported

Heterogeneity of breakpoint cluster region-abelson [BCR-ABL] rearrangement in patients with chronic myeloid leukemia

Breakpoint cluster region-Abelson [BCR-ABL] rearrangement or Philadelphia [Ph] chromosome in Chronic Myeloid Leukemia [CML] is derived from a reciprocal chromosomal translocation between ABL gene on chromosome 9 and BCR gene on chromosome 22. This chimeric protein has various sizes and therefore different clinical behaviour. The purpose of this study was to determine the heterogeneity of BCR-ABL rearrangement in patients with Ph+CML in Pakistan. The study was conducted at Civil Hospital and Baqai Institute of Hematology [BIH] Karachi. Blood samples from 25 patients with CML were collected. Multiplex reverse transcription polymerase chain reaction [RT-PCR] was performed to identify various BCR-ABL transcripts. All 25 samples showed BCR-ABL rearrangements. Out of these, 24 [96%] patients expressed p210 BCR-ABL rearrangements i.e. 60% [n=15] had b3a2 and 32% [n=8] had b2a2 rearrangements. Co-expression of b3a2 /b2a2 rearrangement and p190 [e1a3] rearrangement was also identified in two patients. It is apparent that majority of the patients had p210 BCR-ABL rearrangements. Frequency of co-expression and rare fusion transcripts was very low

Frequency of JAK2 V617F mutation in patients with Philadelphia positive Chronic Myeloid Leukemia in Pakistan

Co-existence of myeloproliferative disorders [MPD] and Janus associated kinase 2 mutation [JAK2 V617F] is a well-established fact. Only few case reports are available showing presence of JAK2 V617F mutation in chronic myeloid leukemia [CML]. Purpose of this study was to determine the frequency of JAK2 V617F mutation in Philadelphia Chromosome positive [Ph [+]] CML patients in Pakistan. The study was conducted from August 2009 to July 2010 at Civil Hospital and Baqai Institute of Hematology [BIH] Karachi. Blood samples from 25 patients with CML were collected. Multiplex reverse transcription polymerase chain reaction [RT-PCR] was performed for Breakpoint Cluster Region - Abelson [BCR-ABL] rearrangement. Conventional PCR was performed for JAK2 V617F mutation on BCR-ABL positive samples. All 25 samples showed BCR-ABL rearrangement. Out of these 11 samples [44%] had JAK2 V617F mutation; the remaining 14 [56%] cases showed JAK2 617V wild type. It is concluded that the co-existence of Ph [+]CML and JAK2 V617F mutation is possible

New horizons in platelets flow cytometry

Platelet flow cytometry is an emerging tool in diagnostic and therapeutic hematology. It is eminently suited to study the expression of platelet surface receptors both qualitatively as well as quantitatively. It can serve as a useful marker for the documentation of in vivo platelet activation, and thus, fore-warn the risk of thromboembolism in patients with diabetes mellitus, coronary syndromes, peripheral vascular diseases, and pre-eclampsia. This technique can also be extended to study and compare the effect of various antiplatelet drugs on the level of activation of platelets and to establish any dose-effect relationship of these drugs. Topographical localization of platelet granules and study of platelet-platelet and platelet-leukocyte interaction is also possible by this procedure. All these parameters serve as pointers towards the presence of activated platelets in the circulation with its thromboembolic consequences. This is a simple reliable and cost effective technique which has a wide application in the diagnosis of various inherited and acquired platelet disorders. Study of platelet cluster of differentiation (CD) markers in various inherited disorders i.e. Bernard Soulier’s disease, von Willebrand disease, Glanzman’s disease, and Grey platelet syndrome may help categories the molecular lesions in these oft under-studied disorders.

Role of iron deficiency anemia in the propagation of beta thalssemia gene / 대한혈액학회지

BACKGROUND: The diagnostic criterion for beta thalassemia trait (BTT) is elevated Hb-A2 levels. Iron deficiency anemia (IDA) reduces the synthesis of Hb-A2, resulting in reduced Hb-A2 levels, so patients with co-pathological conditions BTT with IDA, may have a normal level of Hb-A2. Many socio-economic factors like unawareness, poor diagnostic facilities, and cost of molecular diagnosis (for screening purposes) result in interpretation of these subjects as normal. METHODS: Venous blood samples from 200 unmarried females having a family history of thalassemia were collected, and basic hematological parameters, hemoglobin electrophoresis, and molecular analysis for beta thalassemia were done. Patients with IDA and patients with co-pathological conditions BTT and IDA were treated with oral iron. These subjects were then followed for a period of 20 weeks. RESULTS: Of the 200 females, 34 were found to be anemic. Hemoglobin electrophoresis identified 16 of these patients as BTT. Molecular analysis of all patients confirmed this diagnosis, but identified 8 additional patients with BTT. Eight patients that were not detected with hemoglobin electrophoresis were found to have co-pathology of BTT with IDA. CONCLUSION: Patients with the co-pathological condition BTT with IDA may be interpreted as being normal, as they have normal Hb-A2 levels. These misdiagnosed subjects when marry with BTT have the potential to produce beta thalassemia major in offspring. This is one of the factors playing a major role in the propagation of beta thalassemia gene in Pakistani population, and become a serious hindrance for the thalassemia prevention program in Pakistan.

Screening of five common beta thalassemia mutations in the Pakistani population: a basis for prenatal diagnosis

Thalassemia is one of the most common autosomal single-gene disorder worldwide. The highest prevalence of the disease is in the "thalassemia belt" which includes the Mediterranean region, parts of the Middle East, the Indian subcontinent, the southern parts of the Far East, Pakistan and South-East Asia. This study aimed to detect the common molecular abnormalities of the beta thalassemia syndrome in Pakistan. The study was conducted at the Institute of Hematology, Baqai Medical University, Karachi, Pakistan from August 2004 to November 2007. Blood samples of patients with beta thalassemia major [n=400] were collected from hospital transfusion centres and diagnostic laboratories in different districts of Karachi representing five major ethnic groups including Punjabi, Pathan, Sindhi, Baluchi and Urdu speaking. All the samples were analysed for five common mutations by using the polymerase chain reaction technique ARMS [amplification of refractory mutation system]. Results: The data revealed five common mutations including IVS 1-5[G-C], Fr 41/42[-CTTT], Fr 8/9 [+G], IVS 1-1 and Del 619. These accounted for 90% of the total beta thalassemia genes in Pakistan. The IVS 1-5[G-C] was found to be the most common beta thalassemia gene in the Pakistani population with a frequency of 44.4% present in all major ethnic groups. The results of this study will be helpful in the establishment of a large scale prenatal diagnosis programme in Pakistan