Frequency of pyruvate kinase [PK] deficiency in neonates with haemolytic anaemia

Objective: To determine the frequency of pyruvate kinase deficiency in neonates presenting with haemolytic anaemia

Study Design: Cross sectional descriptive study

Place and Duration of Study: Haematology department, Armed Forces Institute of Pathology, Rawalpindi [AFIP] from Jan 2011 to Jan 2012

Material and Methods: Study was done in collaboration with neonatology department of Military Hospital. Informed consent from parents of neonates was obtained. Two hundred and twenty five neonates with haemolytic anaemia based on low haemoglobin [<14g/dl], raised reticulocyte counts [>5%] and indirect hyper bilirubinaemia [as per CDC nomogram for evaluation of hyper bilirubinaemia in term neonates] were selected. Qualitative pyruvate kinase enzyme assay was done using Bio vision PK assay kit. Estimation of enzyme was based on generation of pyruvate by addition of substrate with change in optical density [OD] of sample in presence of enzyme. Dilution of standard as recommended by manufacturer was made and standard graph was plotted. The OD was measured using wave length of 570 nm at two points [start and at 25 min]. Cut off limit of less than 25% activity was considered positive for pyruvate kinase deficiency. Confirmation was done by running in parallel negative and positive controls [provided]

Results: Seven [3.1%Confidence Interval: +/- 3.33] out of 225 patients were found deficient. Among these 4 were male and 3 were female neonates. The age range was between 1 to 3 weeks. The mean age of presentation was 1.89 +/- [0.832] weeks

Conclusion: We conclude that pyruvate kinase deficiency is not uncommon in our setup and all patients with congenital non-spherocytic haemolytic anaemia where cause cannot be established should be screened for pyruvate kinase deficiency

Seroprevalence of human T-Cell Lymphotropic Virus-1/2 in blood donors in Northern Pakistan: implications for blood donor screening

Objective: To determine the seroprevalence of Human T-cell Lymphotropic Virus-1/2 [HTLV-1/2] in blood donors in Northern Pakistan

Study Design: Descriptive study

Place and Duration of Study: Armed Forces Institute of Transfusion, Rawalpindi, from July to August 2013

Methodology: A total of 2100 blood donors were screened for anti-HTLV-1/2 antibodies during the study period, in a pool of six, on a highly sensitive, Chemiluminiscent Microparticle Immunoassay [CMIA] based system. The screening testreactive donors were recalled, counseled and interviewed, and a fresh sample was obtained for confirmatory testing. Confirmation was performed using additional immunoassays including Line Immunoassay [LIA]; with additional testing for HTLV-1 pvDNA PCR. Frequency and percentages were determined

Results: Four donors [0.19%] were repeatedly screening test-reactive and were subsequently confirmed to be HTLV-1 infected by line immunoassay and HTLV-1 pvDNA PCR. All four donors were male with mean age of 27 +/- 6.27 years. Two [50%] of the positive donors gave history of Multiple Sexual Partners [MSP]

Conclusion: HTLV-1 seroprevalence in Northern Pakistan blood donors was determined to be 0.19%. Large scale studies, including the cost effectiveness of screening blood donations for anti-HTLV-1/2 in Pakistan, are recommended

Real time polymerase chain reaction in diagnosis of chronic myeloid leukemia

To compare the sensitivity and specificity of Real Time Polymerase Chain Reaction [RT-PCR] with conventional cytogenetics in diagnosis of chronic myeloid leukemia. A cross-sectional, analytical study. The Armed Forces Institute of Pathology [AFIP], Rawalpindi, from December 2010 to January 2012. 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. 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. 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

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

To determine the frequency of mixed donor chimerism in patients of non-malignant haematological diseases after allogeneic bone marrow transplant. A cross-sectional, observational 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. 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. 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

BRAFV600E mutation in a case of hairy cell leukaemia

Hairy cell leukaemia is a clonal B cell lymphoproliferative disorder. Patient usually presents to clinician with cytopenias and splenomegaly. Common symptoms include abdominal discomfort, fatigue, generalized weakness, recurrent infections, bruising and bleeding complaints. We present a case of Hairy cell leukaemia with only 25% neoplastic cells in which BRAFV600E mutation was detected. Cytochemical stain, trephine biopsy and immunophenotyping were also consistent with Hairy cell leukaemia

One tube osmotic fragility test: a screening test for microcytic red cells

To evaluate the usefulness of One Tube Osmotic Fragility Test [OTOFT] for detection of microcytosis. Validation study. The study was carried out from July 2004 to November 2004 at Pathology Department PNS Shifa, Karachi. Five hundred and fifteen individuals were studied who reported to the reception of Pathology Dept. for blood complete picture. One drop of finger prick blood was added to a test tube containing 5 ml of 0.36% saline. The results were read at 10 minutes interval by visualizing a written material through the contents of the test tube. Three ml of blood was collected in the EDTA tube by venepuncture for determination of Haemoglobin [Hb], Mean Cell Volume [MCV] and Mean Cell Haemoglobin [MCH] by electronic haematology counter "Sysmex 4500". Out of total 515 subjects studied, OTOFT was positive in 130 [25.2%] and negative in 385 [74.8%] cases. OTOFT positive and negative groups revealed statistically significant difference [P-value of < 0.05] in MCV, MCH and Hb. OTOFT proved to be 92.5% sensitive and 95.2% specific for screening of microcytosis. It had positive predictive value of 85.38% and negative predictive value was 97.66%. The diagnostic accuracy was 94.6%. One Tube Osmotic Fragility Test [OTOFT] is a simple and cost effective test, which is highly sensitive as well as specific for screening of microcytosis. It may prove a useful tool in targeted and/or population screening for thalassaemia

Indigenous development of antibody screening cell panels at armed forces institute of transfusion [AFIT]

To prepare good quality screening cells reagent according to the standards, at Armed Forces Institute of Transfusion [AFIT]. Random group O donors, seronegative for HBsAg, HCV and HIV were selected if they resided in Rawalpindi or Islamabad and could be contacted. Micro column Gel technique was used to find out R[1]R[1], Ri[w]r, R[2]R[2] and rr phenotypes with or without K antigen. Repeat sample of these donors were phenotyped for minimum antigens required for reagent cells. Teams of three donors each were made on the basis of Rh, K antigens and homozygosity for E, Fy[a],Fy[b], Jk[a], Jk[b], S, and s antigens. The selected cells were added to preservative suspension containing neomycin and chloramphenicol and dispensed as 8% solution and labeled. Cells were submitted to quality control testing for 35 days shelf life and efficacy was compared with commercial cells. The cells of required phenotype were prepared according to UK guidelines and AABB standards with minor exceptions. Reagent cells had excellent quality confirmed by many quality control procedures and were comparable to commercial cells in efficacy. The cost saving was significant. AFTT can introduce type and screen policy and Maximum Surgical Blood Ordering Schedule using indigenously prepared cells, of good quality and at an affordable price. This will enhance serological safety of recipients and brings AHT near to adopting standard practice of pretransfusion testing

Frequency of glucose-6-phosphate dehydrogenase deficiency in some ethnic groups of Pakistan

To determine the frequency of G6PD deficiency in young healthy adult males of some ethnic groups in Pakistan. Design: Descriptive study. Place and Duration of Study: Study performed in Combined Military Hospital, Attock in collaboration with Armed Forces Institute of Pathology, Rawalpindi from October 2003 to January 2004. Patients and Asymptomatic and healthy adult males were included in the study. A brief clinical record including age, ethnic group, place of residence, and history of past illnesses including fever, episodes of recurrent jaundice were recorded. Met-hemoglobin reduction test for G6PD screening was performed. Hemoglobin, red cell indices and total leukocyte count of G6PD deficient cases were measured on Sysmex KX 32 hematology analyzer. Three thousand adult males with age between 17 years to 23 years were screened. G6PD deficiency was detected in 1.8%. Deficiency state was 1.07% in Kashmiris, 1.47% in Punjabis, 2.77% in Sindhis, and 3.17% in Pathans. Past history of recurrent jaundice was present in 5.7%. Mild anemia was present in 3.8%. Frequency of G6PD deficiency was 1.8% in young healthy adults with insignificant difference among various ethnic groups except in Pathans

Indigenous development of antibody screening cell panels at armed forces institute of transfusion [AFIT]

To prepare good quality screening cells reagent according to the standards, at Armed Forces Institute of Transfusion [AFIT]. Random group O donors, seronegative for HBsAg, HCV and HIV were selected if they resided in Rawalpindi or Islamabad and could be contacted. Micro column Gel technique was used to find out R1R1, R1wr, R2R2 and rr phenotypes with or without K antigen. Repeat sample of these donors were phenotyped for minimum antigens required for reagent cells. Teams of three donors each were made on the basis of Rh, K antigens and homozygosity for E, Fya,Fyb, Jka, Jkb, S, and s antigens. The selected cells were added to preservative suspension containing neomycin and chloramphenicol and dispensed as 8% solution and labeled. Cells were submitted to quality control testing for 35 days shelf life and efficacy was compared with commercial cells. The cells of required phenotype were prepared according to UK guidelines and AABB standards with minor exceptions. Reagent cells had excellent quality confirmed by many quality control procedures and were comparable to commercial cells in efficacy. The cost saving was significant. AFIT can introduce type and screen policy and Maximum Surgical Blood Ordering Schedule using indigenously prepared cells, of good quality and at an affordable price. This will enhance serological safety of recipients and brings AFIT near to adopting standard practice of pretransfusion testing

Red cell immunization in beta thalassaemia major

To find out the frequency, pattern and factors influencing red cell immunization secondary to multiple blood transfusions in patients of beta thalassaemia major. A cross-sectional study. Armed Forces Institute of Transfusion, Rawalpindi, in November 2002. One hundred and sixty-one patients suffering from beta-thalassaemia major and on regular blood transfusions were included in the study. Their blood samples were tested for blood grouping, direct antiglobulin test and antibody screening/identification using reagents of DiaMed-ID Gel microtyping system. The total rate of red cell immunization was found to be 6.84%. Red cell alloantibodies were detected in 4.97% patients, and belonged mainly to Rh system, with one example each of anti-K, anti-Js b and anti-Jk a. Direct antiglobulin test was positive in 3 patients [1.87%] with increased hemolysis. Two had warm panreactive IgG antibodies suggesting red cell autoimmunization. Red cells of the 3 rd patient showed sensitization with c-3d, with presence of an autoreactive cold agglutinin in the serum having a titre of 1:4. The red cell alloantibody formation was not influenced by age at first transfusion, number of blood transfusions and ethnicity. The rate of red cell alloimmunization in beta-thalassaemia major is relatively low in our setup and may be related to red cell homogeneity between the donor and recipient population. Routine pre-transfusion matching of blood, other than ABO and Rh "D" antigens is not recommended because of low rate of red cell alloimmunization, and high costs associated with such testing. Hyperhaemolysis, due to acquired red cell autoantibodies was found to be an important complication. Patients who develop this complication should be tested for presence of underlying alloantibodies and considered for immunosuppressive treatment

Transfusion associated graft versus host disease

Transfusion associated graft versus host disease [TA-GVHD] results from engraftment of viable donor T-lymphocytes in recipient that can not recognize or destroy them. It is seen in immunocompromised patients and pre-mature neonates. It can also occur in immunocompetent individuals receiving blood from first-degree relatives. It has emerged as single most common cause of death resulting from transfusion. Patients with B-cell malignancies appear to be especially at risk. TA-GVHD is associated with 80-90% mortality. Death most commonly occurs due to infection or haemorrhage secondary to pancytopenia. It is therefore important to prevent its occurrence. Prevention can be achieved either by complete removal of T-lymphocytes from donors blood or by abolishing their proliferating potentials. Available methods of leuko-depletion are not effective in preventing TA-GVHD. Only effective way is to inactivate T-lymhocytes. This can be achieved by irradiating blood product with gamma or X-ray irradiation. The concerns about malignant transformation of cells or reactivation of intracellular viruses have not been proved so far. Newer technologies for T-cell inactivation, which are not based on irradiation, are currently under trial. This is a review article