Mutational Profiles of F8 and F9 in a Cohort of Haemophilia A and Haemophilia B Patients in the Multi-ethnic Malaysian Population.

BACKGROUND: Haemophilia A (HA) and Haemophilia B (HB) are X-linked blood disorders that are caused by various mutations in the factor VIII (F8) and factor IX (F9) genes respectively. Identification of mutations is essential as some of the mutations are associated with the development of inhibitors. This study is the first comprehensive study of the F8 mutational profile in Malaysia. MATERIALS AND METHODS: We analysed 100 unrelated HA and 15 unrelated HB patients for genetic alterations in the F8 and F9 genes by using the long-range PCR, DNA sequencing, and the multiplex-ligation-dependent probe amplification assays. The prediction software was used to confirm the effects of these mutations on factor VIII and IX proteins. RESULTS: 44 (53%) of the severe HA patients were positive for F8 intron 22 inversion, and three (3.6%) were positive for intron one inversion. There were 22 novel mutations in F8, including missense (8), frameshift (9), splice site (3), large deletion (1) and nonsense (1) mutations. In HB patients, four novel mutations were identified including the splice site (1), small deletion (1), large deletion (1) and missense (1) mutation. DISCUSSION: The mutational spectrum of F8 in Malaysian patients is heterogeneous, with a slightly higher frequency of intron 22 inversion in these severe HA patients when compared to other Asian populations. Identification of these mutational profiles in F8 and F9 genes among Malaysian patients will provide a useful reference for the early detection and diagnosis of HA and HB in the Malaysian population.

Factors influencing transfusion requirement in patients undergoing first-time, elective coronary artery bypass graft surgery.

CONTEXT: Coronary artery bypass graft (CABG) operation is associated with high frequency of allogeneic blood transfusion due to the acquired hemostatic challenges in patients undergoing CABG. However, allogeneic blood transfusion carries risks of infection, adverse reaction, and mortality as well as prolonged hospital stay and increased hospital cost. It is important to identify patients who require blood transfusion to mitigate their risk factors and reduce the chance of exposure to allogeneic blood. AIMS: This study was conducted to evaluate factors that influence the decision to transfuse red cell in first-time elective CABG patients. SETTINGS AND DESIGN: This was a cross-sectional study based on a retrospective record review. The study was done in the National Heart Institute. MATERIALS AND METHODS: All patients who underwent first-time elective CABG were included in this study. Variables analyzed include age, gender, body weight, preoperative hemoglobin (Hb) level, patients' comorbidities, and other clinical parameters. STATISTICAL ANALYSIS USED: Data were analyzed using SPSS software version 20. RESULTS: A total of 463 patients underwent first-time elective CABG during the period of the study. Three hundred and eighty-six (83.4%) patients received red cell transfusion. From multiple logistic regression analysis, only age (odds ratio [OR] = 1.040, 95% confidence interval [CI]: 1.003, 1.077, P = 0.032), body weight (OR = 0.951, 95% CI: 0.928, 0.974, P < 0.001), Hb level (OR = 0.500, 95% CI: 0.387, 0.644, P < 0.001), and cardiopulmonary bypass time (OR = 1.013, 95% CI: 1.004, 1.023, P < 0.001) were the significant independent predictors of red cell transfusion. CONCLUSIONS: By stratifying patients according to their risk factor for red cell transfusion, the high-risk patients could be recognized and should be enrolled into effective patient blood management program to minimize their risk of exposure to allogeneic blood transfusion.

A retrospective review of cryoprecipitate transfusion practice in Kuala Lumpur Hospital.

BACKGROUND: Cryoprecipitate is generally used to treat bleeding patients with hypofibrinogenemia, and the transfusion decision is guided based on published guidelines. AIM: This study aimed to evaluate the practice appropriateness in accordance to cryoprecipitate transfusion guidelines in Hospital Kuala Lumpur. METHODOLOGY: This cross-sectional study of 117 cryoprecipitates transfused adult patients was conducted in Kuala Lumpur Hospital from January to June 2012. The compliance of the indication of cryoprecipitate was considered as appropriate if indicated for patients who have hypofibrinogenemia (<1.0 g/L) with bleeding, or otherwise inappropriate if pretransfusion fibrinogen level was more than 1.0 g/L, pretransfusion fibrinogen level was not examined and posttransfusion fibrinogen level more than 1.5 g/L. RESULTS: Most of the cryoprecipitate prescriptions were found to be inappropriate, which read 81.2% (95% confidence interval = 0.740, 0.880). Patients who underwent neurovascular surgery were the major recipient of cryoprecipitate, but majority of the prescription was found not appropriate. The decision to transfuse cryoprecipitate was found mostly appropriate when was guided by fibrinogen (52.2%), but the percentage dropped to 10.6% when pretransfusion fibrinogen test was not performed. Regrettably, only 19.7% of total cryoprecipitate were given based on pretransfusion fibrinogen level.

Molecular basis of Rh blood group system in the Malaysian population.

BACKGROUND: Rh molecular studies have been previously mainly conducted in Caucasians and African population. There is a limited data on the molecular basis for Rh genotypes among Asians. AIMS: This study aims to characterize the Rh genes and frequency of the various RH genotypes among blood donors in National Blood Centre (NBC), Kuala Lumpur. MATERIALS AND METHODS: A total of 1014 blood samples were obtained from blood donors from four different ethnic groups (360 Malays, 434 Chinese, 164 Indians and 56 others). Serological and molecular analysis of all 1014 blood samples were performed. An automated deoxyribonucleic acid sequencing analysis was performed. RESULTS: Rh phenotypes and RH genotypes showed heterogeneity and significant association with ethnicities. Discrepancies in allele D, C/c and E/e between phenotypes and genotypes results were observed. Discrepancy results in allele D showed significant association with the ethnic groups of the blood donors in NBC. There were multiple novel mutations (23) and published mutations (5) found in this study. Significant associations between discrepancy results and mutations were found in allele D and C/c. CONCLUSION: Performing RH molecular analysis in Malaysian population provided the basic database for the distribution of Rh genotypes of donors from major ethnic groups in Malaysia.

WITHDRAWN: Rh genotypes among Malaysian blood donors.

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Factor IX mutations in haemophilia B patients in Malaysia: a preliminary study.

Haemophilia B is caused by coagulation defects in the factor IX gene located in Xq27.1 on the X chromosome. Identification of mutations contributing to defective factor IX may be advantageous for precise carrier and prenatal diagnosis. We studied 16 patients from 11 families, consisting of 8 patients of the Malay ethnic group, of which 6 were siblings. Factor IX mutations have not been previously reported in the Malay ethnic group. The functional region of the factor IX gene was sequenced and mutations were identified in either the exon or intronic regions in 15 of the patients. One novel mutation, 6660_6664delTTCTT was identified in siblings with moderate form of haemophilia B. Mutations identified in our patients when linked with disease severity were similar to findings in other populations. In summary, this preliminary data will be used to build a Malaysian mutation database which would facilitate genetic counseling.

Red cell phenotyping of blood from donors at the National blood center of Malaysia.

BACKGROUND: Human blood groups are polymorphic and inherited integral structures of the red cell membrane. More than 300 red cell antigens have been identified and further categorized into 30 major discrete systems. Their distribution varies in different communities and ethnic groups. AIMS: This work was set to determine the prevalence of red cell phenotypes in donors from the major ethnic groups in Malaysia, namely, Malays, Chinese, and Indians. MATERIALS AND METHODS: The work utilized the dextran acrylamide gel technique in which four types of gel cards were used to identify the blood groups of 594 subjects collected at the National Blood Transfusion Centre, Malaysia. RESULTS: Blood group O and CDe/CDe (R1R1) were the most common in all ethnic groups. The cde/cde (rr) was more prevalent amongst Indians. The rare phenotypes found were cDE/cDE(R2R2) and cDE/CDE(R2Rz). With the Lewis system, the distribution of Le(a-b+) was similar among the ethnic groups. The rarest phenotype Fy(a-b-) was discovered in two donors. Jk(a-b-) was found in seven Malays and in two Indians. In the MNSs system, MN was common in Malays and Chinese, while the MM was more common among Indians. The rare SS was found in 19 donors. Malay and Chinese subjects had high P1 Negative blood but Indians showed high P1 positive blood. Within the Kell System, the very rare KK type was found in six subjects. CONCLUSIONS: The results obtained serve as an established database for the distribution of red cell phenotypes based on the blood group systems of donors from the major ethnic groups in Malaysia.

Quality indicators for discarding blood in the National Blood Center, Kuala Lumpur.

BACKGROUND AND OBJECTIVE: The implementation of quality system and continuous evaluation of all activities of the Blood Transfusion Services (BTS) can help to achieve the maximum quantity and quality of safe blood. Optimizing blood collection and processing would reduce the rate of discard and improve the efficiency of the BTS. The objective of this study is to determine the rate of discard of blood and blood component and identify its reasons at the National Blood Centre (NBC), Kuala Lumpur, during the year of 2007 in order to introduce appropriate intervention. STUDY DESIGNS AND METHODS: Data on the number of discarded whole blood units and its components, reasons for discard, and the number of blood components processed as well as the number of collected blood units were obtained from the Blood Bank Information System - NBC database. These were analyzed. RESULTS: The total number of blood units collected in 2007 was 171169 from which 390636 units of components were prepared. The total number of discarded whole blood units and its components was 8968 (2.3%). Platelet concentrate recorded the highest of discard at 6% (3909) followed by whole blood at 3.7% (647), fresh frozen plasma (FFP) at 2.5% (2839), and cryoprecipitate at 2% (620). The rate of discarded packed red blood cells RBCs, plasma aphaeresis, and PLT aphaeresis was less than 1% at 0.6% (902), 0.6% (37), and 0.29% (14), respectively. RBC contamination of PLT and plasma were the major cause of discard at 40% (3558). Other causes include leakage (26% - 2306), lipemia (25% - 2208), and underweight (4% - 353). CONCLUSION: Good donor selection, training and evaluation of the staff, as well as implementation of automation will help to improve processes and output of BTS. This would reduce discard of blood components and wastage caused by non conformance.

Study of the D-- phenotype reveals erythrocyte membrane alterations in the absence of RHCE.

Red cells with the D-- phenotype do not express the RHCE protein because of mutations in both alleles of the RHCE gene. At present, little is known of the effect this has on the normal function of erythrocytes. In this study a group of five families belonging to a nomadic tribe in Malaysia were identified as carriers of the D-- haplotype. Analysis of homozygous individuals' genomic DNA showed two separate novel mutations. In four of the families, RHCE exons 1, 9 and 10 were present, while the 5th family possessed RHCE exons 1-3 and 10. Analysis of cDNA revealed hybrid transcripts, suggesting a gene conversion event with RHD, consistent with previously reported D-- mutations. Immunoblotting analysis of D-- erythrocyte membrane proteins found that Rh-associated glycoprotein (RHAG) migrates with altered electrophoretic mobility on sodium dodecyl sulphate polyacrylamide gel electrophoresis, consistent with increased glycosylation. Total amounts of Rh polypeptide in D-- membranes were comparable with controls, indicating that the exalted D antigen displayed by D-- red cells may be associated with altered surface epitope presentation. The adhesion molecules CD44 and CD47 are significantly reduced in D--. Together these results suggest that absence of RHCE polypeptide alters the structure and packing of the band 3/Rh macrocomplex.

Recombinant factor VIIa analog (vatreptacog alfa [activated]) for treatment of joint bleeds in hemophilia patients with inhibitors: a randomized controlled trial.

BACKGROUND: A recombinant factor VIIa analog (NN1731; vatreptacog alfa [activated]) was developed to provide safe, rapid and sustained resolution of bleeds in patients with hemophilia and inhibitors. PATIENTS/METHODS: This global, prospective, randomized, double-blinded, active-controlled, dose-escalation trial evaluated and compared one to three doses of vatreptacog alfa at 5, 10, 20, 40, and 80 lg kg(-1) with one to three doses of recombinant FVIIa (rFVIIa) at 90 lg kg(-1) in the treatment of acute joint bleeds in hemophilia patients with inhibitors. The primary endpoint comprised adverse events; secondary endpoints were evaluations of immunogenicity, pharmacokinetics, and efficacy. RESULTS AND CONCLUSIONS: Overall, 96 joint bleeds in 51 patients (> 12 years of age) were dosed. Vatreptacog alfa was well tolerated, with a low frequency of adverse events. No immunogenic or thrombotic events related to vatreptacog alfa were reported. A high efficacy rate of vatreptacog alfa in controlling acute joint bleeds was observed; 98% of bleeds were controlled within 9 h of the initial dose in a combined evaluation of 20­80 lg kg(-1) vatreptacog alfa. The efficacy rate observed for rFVIIa (90%) is consistent with data from published clinical trials. The trial was not powered to compare efficacy, and further trials are needed to investigate the efficacy of vatreptacog alfa as compared with that of rFVIIa. The trial was registered at ClinicalTrials.gov ( REGISTRATION NUMBER: NCT00486278).

Occult hepatitis B infection in blood donors from South East Asia: molecular characterisation and potential mechanisms of occurrence.

OBJECTIVE: To investigate the molecular basis of occult hepatitis B virus (HBV) infection (OBI) in Asian blood donors. DESIGN: OBI donors from Hong Kong, Malaysia, Singapore, Taiwan and Thailand were tested for HBV serological markers, and strains were molecularly characterised. RESULTS: Among 138 confirmed OBI carriers (median age 47 years), HBV genotypes B and C were dominant (60% and 34%, respectively) in agreement with the genotype distribution in chronically infected donors in the region. Viral load ranged between unquantifiable and 3670 IU/ml (median 11 IU/ml). Eleven per cent of OBIs showed an unusual anti-HBs-only serological profile without evidence of past vaccination for most of these individuals. Occult HBV strains showed a higher genetic diversity than strains from matched hepatitis B surface antigen (HBsAg)+ donors, irrespective of genotype. No unique genetic signature or evidence of reduced replication competence was found. Mutations in the vicinity of the pre-S2/S splice donor site were common in OBI(B) (44%) and OBI(C) (36%) strains. S regions from four OBI cases were transfected in HuH7 cells. Results showed limited HBsAg secretion and suggested that mutations disrupting the splice donor site structure may affect pre-S2/S mRNA splicing. CONCLUSIONS: There is indirect evidence that incomplete immune control is involved in the occurrence of OBI in Asian blood donors infected with genotypes B and C as observed in Europe with genotype A2 but to a lower extent than with genotype D. A post-transcriptional mechanism may play a role in HBsAg expression in some OBIs irrespective of HBV genotype.

Hemovigilance in developing countries.

Hemovigilance like quality systems and audits has become an integral part of the Blood Transfusion Service (BTS) in the developed world and has contributed greatly to the development of the blood service. However developing countries are still grappling with donor recruitment and efforts towards sufficiency and safety of the blood supply. In these countries the BTS is generally fragmented and a national hemovigilance program would be difficult to implement. However a few developing countries have an effective and sustainable blood program that can deliver equitable, safe and sufficient blood supply to the nation. Different models of hemovigilance program have been introduced with variable success. There are deficiencies but the data collected provided important information that can be presented to the health authorities for effective interventions. Hemovigilance program modeled from developed countries require expertise and resources that are not available in many developing countries. Whatever resources that are available should be utilized to correct deficiencies that are already apparent and obvious. Besides there are other tools that can be used to monitor the blood program in the developing countries depending on the need and the resources available. More importantly the data collected should be accurate and are used and taken into consideration in formulating guidelines, standards and policies and to affect appropriate interventions. Any surveillance program should be introduced in a stepwise manner as the blood transfusion service develops.

Heterosubtypic anti-avian H5N1 influenza antibodies in intravenous immunoglobulins from globally separate populations protect against H5N1 infection in cell culture.

With antigenically novel epidemic and pandemic influenza strains persistently on the horizon it is of fundamental importance that we understand whether heterosubtypic antibodies gained from exposures to circulating human influenzas exist and can protect against emerging novel strains. Our studies of IVIG obtained from an infection-naive population (Australian) enabled us to reveal heterosubtypic influenza antibodies that cross react with H5N1. We now expand those findings for an Australian donor population to include IVIG formulations from a variety of northern hemisphere populations. Examination of IVIGs from European and South East-Asian (Malaysian) blood donor populations further reveal heterosubtypic antibodies to H5N1 in humans from different global regions. Importantly these protect against highly pathogenic avian H5N1 infection in vitro, albeit at low titres of inhibition. Although there were qualitative and quantitative differences in binding and protection between globally different formulations, the heterosubtypic antibody activities for the respective IVIGs were in general quite similar. Of particular note because of the relative geographic proximity to the epicentre of H5N1 and the majority of human infections, was the similarity in the antibody binding responses between IVIGs from the Malayan peninsula, Europe and Australia. These findings highlight the value of employing IVIGs for the study of herd immunity, and particularly heterosubtypic antibody responses to viral antigens such as those conserved between circulating human influenzas and emerging influenza strains such as H5N1. They also open a window into a somewhat ill defined arena of antibody immunity, namely heterosubtypic immunity.

Hemophilia treatment in developing countries: products and protocols.

The most important aspect of management of hemophilia is to provide adequate replacement of safe clotting factor concentrates to prevent or treat bleeding episodes. There has been considerable progress in many countries in the developing world with regard to this aspect of care. However, very little data are available in the literature on the types of products being used for factor replacement and the doses being administered for control or treatment of bleeding in different countries. These data are important to document because only then can data from different centers be compared. This article provides data from seven countries: Korea, Malaysia, Thailand, Venezuela, Argentina, Iran, and India. It shows that there is wide variability not only in the types of products used (plasma to recombinant factor concentrates) but also in the doses administered (minimal to very high) for similar indications. Prospective documentation of data on musculoskeletal outcome at these centers and correlation with dose of factor replacement could help identify different models of care. Comparing such data and collating the experience in different countries could be useful for optimizing care and establishing cost-effective models. The combined experience in the developing world in providing hemophilia services should be used to define standards of care that are practical and to set achievable goals.