Densitometry of STR-PAGE for donor chimerism in acute leukemia's: A simple method for routine use.

Objective: To evaluate a PCR based method of polyacrylamide gel electrophoresis of short tandem repeats and its quantification for detecting donor chimerism after haematopoietic stem cell transplantation in acute leukaemias. Methods: The descriptive study was conducted at Genetic Resource Centre (GRC) Lab Rawalpindi from Feb 2018 - Nov 2020. A total of twenty patients with acute leukaemias having undergone HSCT were selected and assessed for the analysis of chimerism status. DNA extraction from the whole blood was done by chelex method and short tandem repeats were amplified by using conventional STR- PCR assay. Electrophoresis was carried out and 6% polyacrylamide gels were used for the resultant amplified DNA products and then followed by their densitometry. These patients had undergone HSCT from Pakistan Institute of Medical Science and Armed Forces Bone Marrow Transplant Centre. Results: The peaks in the PAGE densitometry represented the donor chimerism in all post transplant samples of the patients. Conclusion: Our study showed that densitometry of STR PCR PAGE is a useful and cheaper method for demonstration of donor chimerism in acute leukaemia patients having undergone HSCT. Hence this method can be a valuable option in the monitoring of chimerism status in these patients and therefore helps in preventing graft failure by fast and early treatment strategies for these patients.

Bilateral versus single internal mammary artery in diabetic patients: systematic review and meta-analysis.

OBJECTIVES: Data on bilateral internal mammary artery (BIMA) versus single internal mammary artery (SIMA) on diabetics were analyzed; This is the only meta-analysis, the last 7 years. METHODS: Medline through PubMed/EMBASE/CINHAL and the Cochrane Central Register of Controlled Trials; 179 articles were studied; 19 studies deemed suitable and were included in the analysis. RESULTS: The mortality was 2.41% for BIMA versus 1.71% for SIMA (odds ratio [OR] = 0.95; 95% confidence interval [CI]: 0.74-1.22). Postoperative reopening for bleeding was higher at 3.75% for BIMA versus 2.91% for SIMA (OR = 1.49; 95% CI: 1.15-1.93). The incidence of MI was 0.87% for BIMA versus 0.83% for SIMA (OR = 0.73; 95% CI: 0.37-1.44). Deep sternal wound infection was 3.02% for BIMA and 1.95% for SIMA (OR = 1.57; 95% CI: 1.26-1.95). When skeletonized, the incidence of DSWI was 2.5% for BIMA versus 2.41% for SIMA. There was a significant difference at 5-year survival favoring the BIMA, 85.15% BIMA versus 80.77% SIMA (OR = 1.79; 95% CI: 1.60-2.01). The 10-year overall survival was 74.04% BIMA versus 61.57% SIMA (OR = 1.79; 95% CI: 1.61-1.98). The 15-year survival was 47.08% for BIMA versus 37.06% for SIMA (OR = 1.69; 95% CI: 1.52-1.88). CONCLUSIONS: Postoperative bleeding was higher in BIMA group. Bilateral internal mammary artery in diabetic patients should be carried out in a skeletonize fashion, to reduce DSWI. There is a survival benefit of using BIMA in diabetics within 5 years of surgery; it remains significant up to 15 years.

Noninvasive prenatal testing of beta-thalassemia for common Pakistani mutations: a comparative study using cell-free fetal DNA from maternal plasma and chorionic villus sampling.

BACKGROUND: The discovery of circulating cell-free fetal DNA (cff-DNA) in maternal plasma has inspired the noninvasive prenatal testing (NIPT) approaches for various genetic fetal screening including rhesus D typing, sex determination, aneuploidies, and single-gene disorders. OBJECTIVE: Noninvasive determination of paternally inherited beta-thalassemia mutations in maternal total cell-free DNA (cf-DNA) by using allele-specific amplification refractory mutation system (ARMS) real-time PCR (RT-PCR) in concordance with the conventional invasive method. METHODS: An observational study was conducted at the Armed Forces Institute of Blood Transfusion in collaboration with the genetics resource center from March 2021 to August 2021. A total number of 26 couples were selected having a history of previously affected children with beta-thalassemia. A routine chorionic villus sampling (CVS) invasive procedure was carried out, and the mutation analysis was done using conventional PCR. To assess NIPT, a total cf-DNA was also extracted from maternal plasma and analyzed using allele-specific ARMS RT-PCR. RESULTS: Based on conventional PCR testing, 13 of 26 couples were found having beta-thalassemia carriers with homozygous mutation, and 13 couples were carriers with heterozygous mutations. Further to assess NIPT, the cf-DNA of 13 pregnant females among the couples with different mutational patterns was analyzed by allele-specific ARMS RT-PCR to detect paternally inherited mutations. In comparison with conventional PCR, 11 cases (84.6%) were matched successfully, while two cases (15.4%) had no concordance with conventional invasive prenatal testing (IPT). CONCLUSION: NIPT using maternal cf-DNA by allele-specific ARMS RT-PCR can be feasible to screen paternal inherited mutant alleles to rule out pregnant women from invasive procedures where the test would be negative for paternal inheritance. However, a low amount of fetal DNA in maternal plasma is a limiting factor and required further improvement to enrich fetal cf-DNA for complete concordance with conventional IPT.

Molecular Monitoring of BCR-ABL Fusion Transcripts in Patients with Chronic Myeloid Leukemia During Treatment Using the Endpoint Fluorescence Method.

OBJECTIVE: The purpose of the study was to compare results and evaluate the agreement between the endpoint fluorescence (EPF) method and quantitative real-time polymerase chain reaction (QPCR) during molecular monitoring of patients with chronic myeloid leukemia (CML) receiving treatment. MATERIALS AND METHODS: The study was conducted at Molecular Lab of Riphah International University, Islamabad, Pakistan, from January 2017 to December 2018. A total of 150 blood specimens from 30 patients with CML were analyzed at regular intervals during therapy. The detection/quantification of transcript mRNA was done simultaneously using QPCR and the EPF method. RESULTS: Out of a total of 150 RNA specimens analyzed, 117 (78%) specimens were positive, whereas 33 (22%) were negative for the transcript using both methods at various stages of treatment. Strong linear negative correlations between the cycle threshold and relative fluorescence unit values were observed with P <.0001 at 0, 3, 6, 9, and 12 months of treatment. No significant difference (P >.05) between the means of the BCR-ABL percentage was observed in either method at all stages of treatment. The bias between the 2 methods was calculated as 0.069 ± 3.50, and 95% limits of agreement were 6.92% to -6.79%. CONCLUSION: We found that EPF is s simple method to detect/quantify BCR-ABL mRNA expression during treatment with comparable results to QPCR.

Donor Chimerism Study by Single Nucleotide Polymorphism using SYBR green based Real Time PCR.

OBJECTIVE: To optimize and evaluate a real time PCR of Single Nucleotide Polymorphism by SYBR Green method for detection of donor chimerism after haematopoietic stem cell transplantation. METHODS: This descriptive study was conducted at Genetic Resource Centre (GRC) Lab Rawalpindi from Oct 2017 - Dec 2019. A total of twenty patients of post haematopoietic stem cell transplant with various haematological disorders were studied to see the status of donor chimerism by using SNP real time PCR using SYBR Green method and short tandem repeat PCR. These patients had undergone allogeneic HSCT from HLA-matched sibling donors at Pakistan Institute of Medical Science and Armed Forces Bone Marrow Transplant Centre. RESULTS: Real time PCR using SYBR Green was able to detect significant amount of chimerism in all 20 patients having undergone HSCT. Regarding precision of the real time PCR assay the mean value of donor chimerism was 94.1% (SD 3.96) and by STR PCR it was 95.1% (SD 1.41). The assay was found to be sensitive with a detection limit of <1%. CONCLUSION: Our results demonstrate that SNP analysis by SYBR Green real time PCR may be used for the evaluation of chimerism status in patients having undergone HSCT with a sensitivity of <1%. Hence donor chimerism by this sensitive method can be used in monitoring of chimerism in post-transplant patients with various haematological disorders.

Characteristics of BCR-ABL gene variants in patients of chronic myeloid leukemia.

BACKGROUND: Depending on breakpoints of rearrangement different types of BCR-ABL fusion protein can be generated in patients of chronic myeloid leukemia (CML). The aim of this study is to observe frequencies of major transcripts in CML patients by reverse transcriptase polymerase chain reaction (RT-PCR) and their hematological features at the time of presentation. MATERIALS AND METHODS: This cross sectional study was performed at Molecular Lab of Riphah International University, Islamabad from January to June 2019. Consecutive peripheral blood samples of 70 newly diagnosed CML patients in chronic phase were analyzed by RT-PCR to detect different BCR-ABL transcripts. Routine blood cell counts were assessed by an automated hematology analyzer. RESULTS: All samples expressed typical BCR-ABL rearrangement. Expression of either e14a2 or e13a2 transcript was detected in 38 (54%) and 30 (43%) patients, respectively. Coexpression of e13a2 + e14a2 was found in 2 (3%) patients. The mean total leukocyte count was higher in group expressing e13a2 (P = 0.01). Higher mean platelet count was noted in patients with e14a2 transcript, but this difference was statistically insignificant (P = 0.1). The association of male gender was observed with the group exhibiting e14a2 (P = 0.01). There was no statistically significant association between transcript type and different ranges of age, hemoglobin levels, and platelet and total leukocyte counts (P > 0.05). CONCLUSION: e14a2 transcript was most common transcript in CML patients. Patients exhibiting e13a2 subgroup presented with significantly higher mean white blood cell count at the time of presentation. Significantly higher proportion of male patients was found to express e14a2 transcript over e13a2.

Genotype frequency and use of single nucleotide polymorphisms for detection of informative allele by polymerase chain reaction.

OBJECTIVE: To determine genotype frequency of biallelic single nucleotide polymorphisms and its use in detection of informative allele in donor/recipient pairs (sibling pairs) having undergone haematopoietic stem cell transplantation with various haematological disorders using a PCR based method. METHODS: This descriptive study was conducted at GRC Lab Rawalpindi from Jan 2018- Oct 2019.A total of twenty donor/ recipient pairs (sibling pairs) were studied for genotype frequency and informativeness of single nucleotide polymorphisms. Genomic DNA was extracted from the peripheral blood and amplification of single nucleotide polymorphisms was done by PCR based method. The amplified DNA was seen by electrophoresis on 6% polyacrylamide gel. RESULTS: A sharp band of DNA on the polyacrylamide gel indicated a positive reaction. At least two or more informative SNP markers were found in every sibling pair. CONCLUSION: Our results demonstrate that PCR amplification of polyacrylamide gel electrophoresis using single nucleotide polymorphism has allowed the successful screening and detection of informative allele in all the donor/recipient pairs. (Sibling pairs). This PCR based assay using SNPs appears to be a quick, simple, reliable and technically feasible method for a use in a Pakistani setting.

Microwave-assisted synthesis and in vitro osteogenic analysis of novel bioactive glass fibers for biomedical and dental applications.

Glass fiber-based materials have gained interest for use in biomedical and dental applications. The aim of this study was to make E-glass fiber bioactive by a novel method using the microwave irradiation technique. Industrial E-glass fibers were used after surface activation with the hydrolysis method. The ratio of calcium and phosphorous precursors was set at 1.67. After maintaining the pH of the calcium solution, E-glass fibers in two ratios, i.e. 30% (nHA/E30) and 50% (nHA/E50) wt/wt, were added. The phosphorous precursor was added later and the solution was irradiated in a microwave to obtain nano-hydroxyapatite (nHA) particles on E-glass fibers. The structural, physical and in vitro biocompatibility analyses of the resulting materials were conducted. The expression of osteopontin (OPN) and collagen (Col) type 1 was measured by reverse transcription polymerase chain reaction (RT-PCR) and comparison was made between all the groups. Fourier transform infrared spectroscopy and x-ray diffraction showed characteristic peaks of nHA, and a change in the peak intensities was observed with an increase in the concentration of E-glass fibers. Scanning electron microscopic (SEM) images confirmed the homogenous adhesion of nHA spherical particles all over the fibers. Cell viability with mesenchymal stem cells showed growth, proliferation, and adhesion. All the materials were able to upregulate the expression of the OPN and Col, where gene expression was highest in nHA followed by nHA/E30 and nHA/E50. The bioactive glass fibers were synthesized in the shortest time and showed osteogenic properties. These materials have the potential for use in bone tissue engineering, dental prosthesis, and tooth restoration.

Effect of Iron Deficiency on the Phenotype of β-Thalassaemia Trait.

The objective of this study was to determine the effect of iron deficiency on Hb-A2 level in ß-thalassaemia trait and to determine the frequency of individuals with ß-thalassaemia trait who could be missed due to concomitant iron deficiency. A total of 120 patients were studied, out of which 23 were iron deficient (serum ferritin < 20 ng/ml). Mean Hb-A2 in the iron deficient individuals was 4.1 ± 0.47% as compared to 5.1 ± 0.58% in the remaining 97 individuals without iron deficiency (p < 0.001). In the 120 individuals with ß-thalassaemia trait, mean Hb-A2 was 5.8% with range 3 - 6.8% and confidence interval was 95%. In 2 individuals with ß-thalassaemia trait, Iron deficiency was observed and showed Hb-A2 less than 3.5%. These could have been missed while screening by Hb-A2 estimation alone. Co-existence of Iron deficiency and ß-thalassaemia trait may mask the diagnosis of beta thalassaemia trait and such individuals can be missed during screening by Hb-A2 estimation alone.

Frequency of Gγ-globin promoter -158 (C>T) XmnI polymorphism in patients with homozygous/compound heterozygous beta thalassaemia.

BACKGROUND: Response to hydroxyurea therapy in homozygous or compound heterozygous beta thalassaemia (BT) has been reported as more favourable in the presence of XmnI polymorphism. The prevalence of XmnI polymorphism may vary with BT phenotypes and genotypes, and differs geographically in distribution. Prevalence of XmnI polymorphism is not known in northern Pakistan. OBJECTIVE: To determine the frequency of Gγ-globin promoter -158 (C>T) XmnI polymorphism (XmnI polymorphism) in patients with homozygous or compound heterozygous beta thalassaemia. MATERIALS: Polymerase chain reaction (PCR) for common beta thalassaemia mutations and Gγ-globin promoter -158 (C>T) XmnI polymorphism was performed on 107 blood samples of transfusion dependent beta thalassaemia (BT) patients in Pakistan. One hundred samples of unrelated BT traits and 94 samples of healthy subjects as controls were also analysed for BT mutations and XmnI polymorphism. RESULTS: Out of 301 DNA samples, XmnI polymorphism was detected in 71(24%); in normal controls, XmnI polymorphism was detected in 34/94 (36%) subjects; while in homozygous/compound heterozygous BT, it was detected in 14/107(13%) patients (Fisher's exact test, p=.0002). In heterozygous BT group, XmnI polymorphism was detected in 23/100 subjects (Fisher's exact test, p=.03 with normal controls, and p=.049 with homozygous/compound heterozygous BT). The most common BT genotype was Frame Shift (Fr) 8-9/Fr 8-9, and none of the patients with this genotype had XmnI polymorphism. The second most common genotype was IVSI-5/IVSI-5; 4/26 (15%). Cases with this genotype had XmnI polymorphism. CONCLUSION: XmnI polymorphism in homozygous/compound heterozygous BT group is 13%. The most common genotype associated with XmnI polymorphism was IVSI-5/IVSI-5.

Real time polymerase chain reaction in diagnosis of chronic myeloid leukemia.

OBJECTIVE: To compare the sensitivity and specificity of Real Time Polymerase Chain Reaction (RT-PCR) with conventional cytogenetics in diagnosis of chronic myeloid leukemia. STUDY DESIGN: A cross-sectional, analytical study. PLACE AND DURATION OF STUDY: The Armed Forces Institute of Pathology (AFIP), Rawalpindi, from December 2010 to January 2012. METHODOLOGY: A total number of 40 patients were studied, in which all were diagnosed as CML on peripheral blood and bone marrow aspiration. The subjects were tested for the presence of Philadelphia (Ph) chromosome by cytogenetics and BCR-ABL fusion gene by RT-PCR. 2-3 ml of venous blood was collected, half in sodium heparin (anti-coagulant) for cytogenetics and half in EDTA for PCR. For cytogenetics, cells were cultured for 72 hours in RPMI 1640 medium and examined by arresting in metaphase using Colchicine to identify Philadelphia chromosome. For PCR, RNA extraction was done by Tri Reagent LS (MRC, USA) and cDNA was synthesized using reverse transcriptase and gene specific primer. RT- PCR was done on ABI-7500. The positive samples were identified when fluorescence exceeded threshold limit. Results of cytogenetics and RT PCR were compared. RESULTS: Out of the 40 patients, PCR showed 37 (92.5%) were positive and 3 (7.5%) were negative for BCR-ABL fusion gene, whereas in cytogenetics 28 (70%) were positive for Ph chromosome and 12 (30%) were negative for Ph chromosome. Sensitivity and specificity of cytogenetics was 75.6% and 100% respectively. CONCLUSION: Real time PCR as compared to cytogenetics is less tedious, gives quick results, does not require multiple sampling due to culture failure and can be done on peripheral blood.

Immunoglobulin gene rearrangement in detection of minimal residual disease in acute lymphoblastic leukaemia.

BACKGROUND: Acute lymphoblastic leukaemia (ALL) is mainly a childhood malignancy but affects both children and adults. OBJECTIVE: The study was conducted to evaluate a qualitative PCR based method for detection of clonal immunoglobulin gene rearrangement as a marker of minimal residual disease in patients of acute lymphoblastic leukaemia at the end of induction. METHOD: It was a descriptive study conducted at Armed Forces Institute of Pathology, Rawalpindi from Aug 2009 to Feb 2010. For prospective analysis, genomic DNA was extracted from peripheral blood/bone marrow aspirates and unstained bone marrow smears. A total of 50 patients of acute lymphoblastic leukaemia who showed positive immunoglobulin gene rearrangement by qualitative PCR at the time of diagnosis were included. These patients were then investigated for minimal residual disease at the end of induction. PCR amplification of the IgH gene was done by a VH primer homologous with a highly conserved sequence near the 3' end of the FR3 region and a consensus sequence JH primer. Test for minimal residual disease was conducted by PCR amplification of DNA from remission marrow cells (at day 29 of chemotherapy) with the help of the primer sets used at the time of diagnosis. The amplified DNA was seen by electrophoresis on 6% polyacrylamide gel. RESULTS: A sharp clonal hand ranging from 90-200 bp indicated a positive reaction. Of 50 patients, 28 (56%) were positive for Ig gene rearrangement on PCR at the end of induction, 17 (34%) patients were found to be negative for minimal residual disease, 2 (4%) patients died during induction therapy, and 3 (6%) patients did not come for follow-up. CONCLUSION: Molecular approaches have allowed us to detect low level of residual disease which is not detected by cytomorphological methods. Minimal residual disease (MRD) by PCR used in this study would definitely help in monitoring of MRD in all patients with leukaemia.

Mixed donor chimerism in non-malignant haematological diseases after allogeneic bone marrow transplantation.

OBJECTIVE: To determine the frequency of mixed donor chimerism in patients of non-malignant haematological diseases after allogeneic bone marrow transplant. STUDY DESIGN: A cross-sectional, observational study. PLACE AND DURATION OF STUDY: Department of Haematology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from July 2010 to June 2011. METHODOLOGY: Donor chimerism was assessed in patients of aplastic anaemia and beta-thalassaemia major who underwent allogeneic bone marrow transplantation (BMT). Peripheral blood samples were used to assess chimerism status by analysis of short tandem repeats (STR). In patients where pre-transplant blood sample was not available, swab of buccal mucosa was used for pre-transplant STR profile. A standard set of primers for STR markers were used and the amplified DNA was resolved by gel electrophoresis and stained with silver nitrate. The percentage of donor origin DNA was estimated by densitometer. RESULTS: Out of 84 patients, 52 (62%) were males, while 32 (38%) were females. In patients of beta-thalassaemia major, 31 (62%) developed mixed donor chimerism (MC), 13 (26%) developed complete donor chimerism (CC) and 6 (12%) had graft failure. In aplastic anaemia, 17 patients (50%) achieved MC, 13 (38.2%) had CC and 4 (11.8%) developed graft failure. The combined frequency of mixed donor chimerism for both the diseases was 58.3%. D3S1358 was the most informative STR marker in these patients. CONCLUSION: Majority of the studied patients developed mixed donor chimerism following bone marrow transplantation, whereas only a minor percentage of the patients had graft failure. Analysis of D3S1358 was the most informative in assessing donor chimerism in patients who underwent BMT.

Frequency of Janus associated kinase 2 (V617F) mutation in patients of polycythemia vera.

OBJECTIVE: To determine the frequency of Janus associated kinase 2 ( JAK2) mutation in patients of polycythemia vera (PV). STUDY DESIGN: Descriptive cross-sectional. PLACE AND DURATION OF STUDY: Haematology Department, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from January 2008 to December 2009. METHODOLOGY: Forty-six consecutive patients of PV diagnosed by the conventional haematological criteria were included in the study. Blood samples of all patients were screened for G-T point mutation (V617F) in the JAK2 gene on chromosome 9 by an allele specific polymerase chain reaction (PCR). RESULTS: JAK2 V617F mutation was found in 43 out of 46 patients (93.5%) with PV. Among them, 30 were males (65.2%) and 16 were females (34.8%). Mean TLC in patients with PV was 16.5 ± 9.1 x 109/L, mean haemoglobin (Hb) was 17.8 ± 2.0 g/dl, mean platelet count was 531 ± 261 x 109/L, mean PCV was 57.9 ± 6.3 l/l, mean MCV was 78.8 ± 11.0 fl and mean MCH was 24.4 ± 4.8 pg. CONCLUSION: Peripheral blood mutation screening for JAK2 V617F can be incorporated into the initial work up of patients suspected to have polycythemia as this mutation is present in majority of such patients.

Feasibility and safety of transabdominal chorionic villus sampling.

BACKGROUND: Chorionic Villus Sampling (CVS) is the technique of choice for prenatal diagnosis prior to 12 weeks gestation. The objective of this study was to determine the feasibility, and pattern of complications following first trimester Trans-abdominal Chorionic Villus Sampling (TA-CVS). METHODS: This was a descriptive study conducted in the Obstetrics and Gynaecology Department Military Hospital (MH) Rawalpindi from Jan 2007 to July 2008. Couples at risk of giving birth to a child with genetic disorder were identified and counselled. Trans-abdominal Chorionic Villus Sampling was done using double needle technique under ultrasound guidance. Immediate and late complications were followed up. Data was analysed using SPPS-10. RESULTS: On 200 cases chorionic villus sampling was done as an outdoor procedure. Most common indication was thalassaemia trait 75 (37.5%). Most procedures were done between 12-13 weeks. All placental positions including 104 (52%) posterior and 71 (35.5%) anterior were approachable. Most aspirations were easy, however, in 30 (15%) the aspiration was difficult. Overall success rate was 100%. In 158 (79%) of the cases sample yield was good. One (0.5%) patient had vaginal bleeding and three (1.5%) had placental haematoma formation. Most patients (84%) experienced mild pain during the procedure. The procedure related miscarriage occurred in 2 (1%) patients while another patient developed this complication after 6 weeks. CONCLUSION: First trimester TA-CVS is an accurate and safe invasive prenatal diagnostic procedure. Placentas in almost any position can be approached without any significant risk to mother and the foetus.

Relapsed diffuse large B-cell lymphoma treated by reduced-intensity allogeneic stem cell transplantation with donor lymphocyte infusion.

A 42 years old male with relapsed diffuse large B-cell lymphoma was given second-line chemotherapy followed by reduced intensity allogeneic stem cell transplantation from HLA matched brother. Twelve weeks posttransplant, his disease relapsed evidenced by the appearance of lymphoma cells in the peripheral blood and declining donor chimerism. Donor lymphocyte infusion was given that induced complete lymphoma remission. The patient is well 3 years posttransplant with his disease in complete remission.

Response rate of Pakistani children with acute lymphoblastic leukaemia to Medical Research Council acute lymphoblastic leukaemia 97 chemotherapy protocol.

BACKGROUND: Acute lymphoblastic leukaemia (ALL), a malignancy of lymphoid lineage cells, has excellent prognosis in children. In Pakistan, a few studies highlighted the response of ALL to chemotherapy. The Present study was planned to see the response rate of Pakistani children with ALL to Medical Research Council ALL 97 (MRCALL97) chemotherapy protocol. This descriptive case series was conducted at the Department of Haematology, Armed Forces Institute of Pathology and the Department of Paediatric Oncology, Combined Military Hospital, Rawalpindi from 16th of February 2007 to 16th of August 2007. METHODS: Diagnosed children with ALL fulfilling the inclusion criteria were interviewed regarding history of the present, past illnesses, and family history. Physical examination was performed. Presenting clinical features, blood counts and blood and bone marrow blasts percentage were used to see the response on day 29 post chemotherapy. The data was recorded on a structured proforma for statistical analysis. RESULTS: A total of 33 patients were studied including 26 males and 7 females. Twenty-five patients belonged to age group 2-9 years, and 8 to < 2 or > 9 years, median age being 4.5 years. Presenting WBC count was < 50 x 10(9)/L in 30 patients and > 50 x 10(9)/L in 3 patients. At the end of induction, complete remission was achieved in 31 out of 33 (94%) patients while two patients did not achieve remission. CONCLUSION: Response rate of Pakistani children with ALL to chemotherapy was superior to the previously reported figures from Pakistan.

Association of Helicobacter pylori infection with idiopathic thrombocytopenic purpura.

OBJECTIVE: To determine the association of Helicobacter pylori infection in patients presenting with idiopathic thrombocytopenic purpura (ITP). METHODS: From March 2007 to March 2008, thirty adult patients with ITP and 30 age and sex matched healthy controls were investigated for the presence of H. pylori infection by Helicobacter pylori stool antigen (HpSA) an enzyme immunoassay (EIA) based method. The criteria for presence of H. pylori infection was a positive stool antigen test. RESULTS: H. pylori infection was found in 19 out of 30 patients with ITP (63.3%) which is well above the frequency of 13 out of 30 (43.3%) in controls. Calculated odds ratio was 2.25 which shows significant association of H. pylori infection with ITP. CONCLUSION: The study confirms the existence of an association between H. pylori infection and ITP. Therefore the screening for H. pylori infection and an attempt to eradicate bacterium in positive cases seems appropriate in patients with ITP at diagnosis.

Frequency of bcl-2 gene rearrangement in B-cell Non-Hodgkin's lymphoma.

OBJECTIVE: The objective of the study was to determine the frequency of bcl-2 gene rearrangement in B-cell Non-Hodgkin's lymphoma (NHL) and identify different breakpoints of bcl-2 gene. METHODS: Thirty cases of B-cell lymphoma (including 8 cases of follicular lymphoma, 19 cases of diffuse large B-cell lymphoma and 3 cases of T-cell rich B-cell lymphoma) were included in the study. Good quality of DNA was extracted in 4 cases from formalin fixed paraffin embedded tissue and in 26 cases from fine needle aspirate. The polymerase chain reaction was done for major break point region (mbr), minor cluster region (mcr) and intermediate cluster region (icr) of the bcl-2 gene. RESULTS: The bcl-2 gene rearrangement was identified in 23.3% of B-cell lymphoma, 50% of follicular lymphoma, 15% of diffuse large B-cell lymphoma and no bcl-2 rearrangement was identified in any of the T-cell rich B-cell lymphomas. Further analysis showed the icr breakpoint in 16.7% of B-cell lymphoma, 37.5% of follicular lymphoma and 10.5% of diffuse large B-cell lymphoma. Involvement of the mbr breakpoint was found in 6.7% of B-cell lymphoma, 12.5% of follicular lymphoma, and 5.3% of diffuse large B-cell lymphoma. Involvement of the mcr breakpoint was not seen in any of the cases. CONCLUSION: The bcl-2 gene rearrangement is quite frequent in follicular lymphoma, followed by diffuse large B-cell lymphoma. The commonest breakpoint in present series is icr followed by mbr. This indicates that primers for bcl-2 gene must include icr primer, whenever the bcl-2 gene is being evaluated for B-cell NHL in this part of the world and this might reduce the variability of frequency of bcl-2 gene rearrangement within and between different regions.

CD5-positive acute lymphoblastic leukemia.

CD5-positive B-ALL is a rare variant of Acute Lymphoblastic Leukemia (ALL). In literature, only three cases have been reported so far. This fourth case report describes a young lady who was diagnosed as ALL (L-2) on bone marrow examination and was found to be CD5 positive B-cell acute lymphoblastic leukemia on immunophenotyping. Cytogenetic analysis revealed translocation t(9:22).