Cold Agglutinin Disease in a Patient of Pulmonary Tuberculosis.

We report a case of secondary cold agglutinin disease (CAD) due to pulmonary tuberculosis in a 68-year male patient who presented with acrocyanosis involving both upper limbs and greater portion of lower limbs. Direct Coombs' test was positive with mild anemia and the cold agglutinin titer was high. Sputum showed numerous acid fast bacilli per high power field. The patient was given standard anti-tuberculous treatment (ATT) and his symptoms gradually improved. After nine months of ATT, his haemoglobin improved, acrocyanosis resolved completely and cold agglutinin titer decreased in level.

Hepatitis B genotypes and surface antigen mutants present in Pakistani blood donors.

BACKGROUND: The prevalence of chronic Hepatitis B Virus (HBV) infection is 2-4% in the Pakistani population, defining Pakistan as an intermediate prevalence country. In this study, hepatitis B surface antigen (HBsAg) reactive blood donations were screened using a combination of serological and molecular methods to identify immune escape HBV mutant strains and to determine the HBV genotypes and subtypes present in Pakistan. METHODS: Blood donations were collected at the Armed Forces Institute of Transfusion (AFIT) located in northern Pakistan and the Hussaini Blood Bank (HBB) located in the south. From 2009 to 2013 a total of 706,575 donations were screened with 2.04% (14,409) HBsAg reactive. A total of 2055 HBsAg reactive specimens, were collected and screened using a monoclonal antibody based research assay to identify immune escape mutants followed by PCR amplification and DNA sequencing to identify the mutation present. DNA sequences obtained from 192 specimens, including mutant candidates and wild type strains, were analyzed for escape mutations, genotype, and HBsAg subtype. RESULTS: Mutations were identified in approximately 14% of HBsAg reactive donations. Mutations at HBsAg amino acid positions 143-145 are the most common (46%) with the mutation serine 143 to leucine the most frequently occurring change (28%). While regional differences were observed, the most prevalent HBV strains are subgenotypes of D with subgenotype D1/subtype ayw2 accounting for the majority of infections; 90.2% at AFIT and 52.5% at HBB. CONCLUSIONS: The high frequency of immune escape HBV mutants in HBV infected Pakistani blood donors highlights the need for more studies into the prevalence of escape mutants. Differences between vaccinated and unvaccinated populations, the correlation of escape mutant frequency with genotype, and impact of escape mutations in different genotype backgrounds on the performance of commercially available HBsAg assays represent avenues for further investigation.

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 test-reactive 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 pvDNAPCR. 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 pvDNAPCR. 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.

Impact of nucleic acid amplification test on screening of blood donors in Northern Pakistan.

BACKGROUND: The Armed Forces Institute of Transfusion located in Rawalpindi, Northern Pakistan, acts as a regional blood center with more than 50,000 donations collected annually. Nucleic acid amplification testing (NAT) was introduced in our institution in September 2012 for screening all seronegative blood donors. STUDY DESIGN AND METHODS: The study was conducted from September 21, 2012, to September 20, 2013. Samples from the seronegative donors were run on cobas s 201 platform (Roche) in pools of six. Reactive donors were followed up for further confirmatory testing to rule out false-positive results. Viral load estimation was done for all NAT-reactive donors. RESULTS: After serologic screening of 56,772 blood donors, 2334 were found to be reactive; 719 (1.27%) were reactive for hepatitis B surface antigen, 1046 (1.84%) for antibody to hepatitis C virus (anti-HCV), 12 (0.02%) for antibody to human immunodeficiency virus, and 557 (0.98%) for syphilis antibodies. A total of 27 NAT-reactive donors were confirmed after testing 54,438 seronegative donors, with an overall NAT yield of one in 2016 donors: 23 for hepatitis B virus (HBV) DNA (HBV NAT yield, 1:2367) and four for HCV RNA (HCV NAT yield, 1:13,609). The residual risk after NAT implementation, calculated for the first-time blood donors, was 62.5 and 4.4 per million donors for HBV and HCV, respectively. CONCLUSIONS: NAT has improved the safety of blood products at our transfusion institution. Confirmation of NAT results must always be done either on follow-up samples or on samples from the retrieved frozen plasma bag.

Frequencies of Duffy blood group alleles in Northern Pakistani donors.

OBJECTIVE: Find the allele frequencies of Duffy blood group antigens in donor population from northern Pakistan. DESIGN: Cross sectional study. PLACE AND DURATION OF STUDY: Armed Forces Institute of Transfusion (AFIT), Rawalpindi in year 2012. PATIENTS AND METHODS: A total of 1000 healthy, adult blood donors were included in the study. Blood samples were collected in ethylenediamine tetra aceticacid (EDTA) tube and then tested with anti sera Fy(a) and Fy(b) by the tube method. RESULTS: The allele frequencies of Duffy blood group antigens were calculated. The most common phenotype was Fy(a+b+) which was present in 552 (55.2%) donors followed by the Fy(a+b-) phenotype in 228 (22.8%) donors, while 178 (17.8%) were Fy(a-b+) and the least prevalent phenotype was Fy(a-b-) which was present in 42 (4.2%) of donors. CONCLUSION: The majority of our population is heterozygous for Duffy antigens a and b.

Effectiveness of blood donor questionnaire directed at risk factor for transfusion transmitted infections in Pakistani population.

BACKGROUND: Deferring blood donors who admit to high-risk behavior on questioning are likely to eliminate those in window period for transfusion transmitted infections (TTI). However, many questions have been implemented in some countries as part of donor history questionnaire, based on precautionary principle and not on evidence, and can result in increased donor losses. This study aims to identify effective risk-directed questions having high predictive value, in local context which can form part of blood donor deferral policies. For this, a case control study in a hospital blood bank having donation services was carried out prospectively over a period of three years. MATERIALS AND METHODS: Two hundred and twenty donors, who were repeatedly reactive for HBsAg, anti-HCV, anti-HIV with EIA, and syphilis with TPHA, were the cases. Eight hundred and eighty four controls were the donors who tested negative for all TTI test. All donors answered seven hepatitis risk directed questions and their responses and reactivity status for TTI were used for statistical analysis with SPSS ver. 15. RESULTS: Positive predictive value for history of jaundice at any age for HBsAg was 20%, while PPV for history of surgery in previous six months for both HBsAg and anti-HCVHCV was also around 20%, based on pretest probability of 7%. The post-test probability for these questions was around 30%. Odds ratios with 95% CI did not reveal any significant association of hepatitis with any of seven questions. Donor losses after deferring on basis of two questions were 5.3% per year, while deferral rate after all seven questions was 20%. CONCLUSIONS: Donors should be permanently deferred if there is history of jaundice at any age, while deferral period after surgery should be one year. Other risk-directed questions should not be used to defer donors. Donor deferral policies should be evidence based and questions with proven efficacy should be made part of donor history questionnaire to minimize donor losses.

Anti-hepatits B core antigen testing, viral markers, and occult hepatitis B virus infection in Pakistani blood donors: implications for transfusion practice.

BACKGROUND: The purpose of this study was to determine the seroprevalence of anti-hepatitis B core antigen (HBc) and the impact of its testing along with other markers of hepatitis B, hepatitis B virus (HBV) DNA, hepatitis C virus antibody (anti-HCV), and syphilis in Pakistani blood donors. STUDY DESIGN AND METHODS: The study design was cross-sectional. A total of 966 donors were selected randomly for testing of anti-HBc and HBV markers, including HBV DNA, of 94,177 blood donors who were routinely screened for hepatitis B surface antigen (HBsAg), anti-HCV, human immunodeficiency virus antibody (anti-HIV), Treponema pallidum hemagglutination assay (TPHA), and malarial parasites from 2003 to October 2005. RESULTS: The seroprevalence of various infectious markers was as follows: HBsAg, 2.16 percent; anti-HCV, 4.16 percent; anti-HIV, 0.004 percent; TPHA, 0.75 percent; and malaria, 0.002 percent. Anti-HBc prevalence in HBsAg-negative, HBV DNA-negative blood donors was 167 of 966 (17.28%), with 76 percent demonstrating anti-HBs positivity. Younger donors with mean age of 25 years were exposed to HBV to a lesser extent compared to those with a mean age of 29 years. Anti-HBc positivity was significantly higher in anti-HCV-reactive individuals. HBV DNA was detectable in 5 blood donors who were HBsAg-, anti-HBc-positive and were categorized as having occult HBV infection. CONCLUSIONS: The study shows that more than 17 percent of healthy, young blood donors in Pakistan are already exposed to HBV, with two-thirds showing anti-HBs levels of greater than 100 mIU per mL. One in 200 blood donors who are HBsAg-, anti-HBc-positive, however, have occult HBV infection, with likelihood of transmission of hepatitis B in recipients of blood components derived from them. HBsAg-negative individuals who are anti-HBc-negative and those who are anti-HBc-positive, anti-HBs-positive, and HBV DNA-negative should be selected as regular blood donors to minimize transmission due to occult hepatitis B infection.

Blood volumes of Pakistani male donors: implications for blood donation.

BACKGROUND: Safety of blood donors rest on withdrawing only appropriate quantities of blood. Adjusting the volumes drawn according to the average blood volumes of any population can ensure this. This requires knowledge of total blood volume of donors, which should ideally be measured by standard methods or derived by alternate suitable method. This observational, cross sectional study was undertaken to calculate blood volumes of Pakistani male donor using recommended equations and obtain safe donation volume limits for our population. METHODS: Height and weight of male Pakistani donors reporting to Combined Military Hospital blood bank was recorded by standardized method. Blood volumes were calculated by two different equations using body surface area. The data was entered in SPSS 10.0 version for Windows and statistical analysis done. RESULTS: Mean total blood volumes of 625 male donors calculated was 4819.2 ml with first equation and 4566.8 ml with second equation. 95% CI was between 4796.7 and 4841.6 with first equation and 4541.6 and 4591.9 with second equation. The maximum volumes of donation recommended for western population constitutes less than 12 % of calculated total blood volume of our population, with either equation. This is with in safe limits by any standard. CONCLUSION: 450 ml +/- 45 ml including samples in pilot tubes should be the recommended donation volume in Pakistani donors. The maximum volume being collected in other countries constitute safe limits for Pakistani donors as well. Equations showing better correlation with measured volumes should preferably be used to calculate blood volumes. Impact of collecting blood volumes recommended in this study, on blood donors, should be studied.

Primary splenic lymphoma.

A middle-aged lady presented with fever and splenomegaly and had been provisionally treated for malaria, typhoid and tuberculosis. Diagnostic splenectomy was performed which revealed diffuse large cell lymphoma, B type, localized to spleen. Patient had remission of disease after splenectomy.

Indigenous development of antibody screening cell panels at Armed Forces Institute of Transfusion (AFIT).

OBJECTIVE: To prepare good quality screening cells reagent according to the standards, at Armed Forces Institute of Transfusion (AFIT). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

OBJECTIVE: To find out the frequency, pattern and factors influencing red cell immunization secondary to multiple blood transfusions in patients of beta-thalassaemia major. DESIGN: A cross-sectional study. PLACE AND DURATION OF STUDY: Armed Forces Institute of Transfusion, Rawalpindi, in November 2002. PATIENTS AND METHODS: 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. RESULTS: 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-Jsb and anti-Jka. 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 3rd 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. CONCLUSION: 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.