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Healthcare workers run the risk of contracting COVID-19 during the course of their work if required precautions and usage of appropriate personal protective equipment is not adhered to In the transfusion testing laboratory, indirect exposure to COVID-19 may result from environmental contamination that may occur through surface contact or be airborne Handling of potentially contaminated surfaces such as sample tubes and sample packaging, and subsequent self-inoculation through the mucous membranes of mouth, nose and eyes may occur Information on the risk of indirect contact transmission of COVID-19 from such surfaces is limited When risk assessments are conducted in the laboratory, assumptions often need to be based on studies derived from other coronaviruses and respiratory viruses such as HCoV-229E, SARS-CoV, MERS-CoV and Influenza The other mode of potential indirect transmission is through aerosols generated from certain laboratory processes The risk of inhaling aerosolized blood containing COVID-19 viral RNA is unresolved Laboratories need to perform risk assessment of their activities before appropriate decisions can be made with regards to COVID-19 exposure risk reduction or elimination

Background Antibodies to Mia , MUT, and Mur are among the most frequently identified alloantibodies in Southeast Asia. Understanding the characteristics of these antibodies in terms of induction and evanescence would aid in optimizing methods for their detection. Study design and methods Antibody testing results between the years 2013 and 2015 with relevant patient demographic data and red blood cell (RBC) transfusion history were retrieved. Cumulative alloimmunization incidence and evanescence to MUT and Mur were estimated by Kaplan-Meier analysis in relation to the number of RBC units transfused and time. Results Of 70,543 selected patients, 6186 nonalloimmunized subjects with available antibody testing results posttransfusion were identified. Cumulative alloimmunization incidence for MUT increased from 0.12% (95% confidence interval [CI], 0.03-0.21) to 0.63% (95% CI, 0.25-1.01), while for Mur it increased from 0.04% (95% CI, 0-0.09) to 0.42% (95% CI, 0.05-0.79) when a patient was transfused 2 RBC units as compared to 12. Both antibodies had high evanescence rates and at 1 year, anti-MUT and -Mur will be detected in only 45% (95% CI, 35%-57%) and 27% (95% CI, 17%-43%), respectively, of previously positive patients. MUT and Mur immunogenicity was estimated to be 1.7 and 1.2 times higher than E when their rate of evanescence was taken into account. Conclusion Antibodies to MUT and Mur develop following multiple RBC exposures. Immunogenicity of MUT/Mur and evanescence rates of the corresponding antibodies is higher compared to anti-E. Appropriate selection of antibody screening cells is needed in view of the high prevalence, immunogenicity, and evanescence of the antibodies.

The majority of allelic variants conferring red cell blood group antigens are attributable to single nucleotide polymorphisms, which are immensely amenable to routine genotyping methods such as PCR-RFLP, SSP, SSO and fluorescent-based real-time PCR assays. These techniques are feasible in a low- to medium-throughput setting. Multiplexing and use of solid surfaces or beads for probe capture have further improved the throughput and turnaround time allowing multiple alleles to be interrogated simultaneously. Novel approaches, as in the use of MALDI-TOF mass spectrometry, appear as an attractive option for accurate, cost-effective, high-throughput SNP genotyping within major blood centres. Massively parallel targeted sequencing is likely the next step in the evolution of RBC genotyping. In the hospital setting, genotyping is unlikely to supplant serological typing but would rather be complementary to it. Scenarios where it would play an important role would be in the transfusion management of transfusion dependant patients such as in sickle cell anaemia or in situations where RBC phenotyping cannot be reliably undertaken as in auto-immune haemolytic anaemia and haemolytic disease of foetus and newborn. Genotyping would also be useful where anti-sera are not readily available or are of weak potency. Molecular typing is likely to, however, show its greatest potential in donor genotyping to establish pools of fully typed donors as well as identify donors with rare blood groups. Such an exercise will facilitate prompt and efficient delivery of best-matched blood for patients.

Despite the large number of serologically identified red cell antigens to date, it is reassuring to note that in the common clinical scenario, the transfusion laboratory would only likely deal with a narrow range of clinically significant antibodies to specific antigens of blood group systems. Accurate ABO and RhD typing still remains the most crucial step in pretransfusion testing followed by the antibody screen. The correct identification of single antibodies or multiple antibodies and whether they are alloantibodies or autoantibodies is necessary so that corresponding antigen-negative red cells can be supplied. Clinically insignificant antibodies or what some would denote as ‘nuisance’ antibodies would need to be differentiated from clinically significant antibodies. When discrepancies occur between forward and reverse ABO typing, it is crucial that laboratories resolve the discrepancy before a specific ABO blood group is assigned. The inclusion of O cells in reverse grouping, anti-H and anti-A1 lectins in the forward, performing the typing on a range of temperatures using washed red cells and careful interpretation of agglutination patterns, would usually provide an answer. On occasions, a saliva test may be necessary and rarely molecular typing. With regard to RhD typing, test systems should identify RhD-negative and D-variant individuals so that D-negative units can be issued to them. Testing with antisera from different clones, the use of adsorption–elution techniques and RhD.CcEe typing aids in coming to a conclusion, before molecular testing becomes necessary. Extended red cell phenotyping is indicated if the patient is at risk for developing a red cell antibody.

As an East-Asian international study, we evaluated erythrocyte alloimmunity by gender and history of transfusion or pregnancy. In total, data from more than 1,826,000 patients were analyzed, from whom 26,170 irregular erythrocyte antibodies were detected in 22,653 cases. Antibody frequencies in these cases were as follows: anti-E, 26.8%; anti-Lea, 20.0%; anti-P1, 7.1%; anti-M, 6.4%; anti-Mia, 5.6%; anti-c + E, 5.6%; anti-Leb, 4.6%; anti-D, 2.8%; anti-Fyb, 2.6%; anti-Lea+Leb, 2.5%; anti-Dia, 2.0%; and others. For pregnant patients, anti-D (12.7%) was statistically more frequent. For transfused patients, anti-E (37.3%), anti-c + E (9.5%), anti-C + e (3.3%) and anti-Jka (3.1%) were significantly more frequent.

Autoimmune haemolytic anaemia (AIHA) occurs as a result of antibodies directed against self-red blood cell (RBC) antigens, leading to the premature destruction of RBC. RBC destruction may occur within the vasculature, often mediated by the membrane-lysing complex, which is generated upon complement activation or occur extravascularly, within the reticulo-endothelial system that expresses FcγR and C3 receptors that bind antibody and complement coated RBC. The laboratory hallmark of AIHA is a positive direct antiglobulin test (DAT) although it has to be recognized that occasional cases of DAT-negative AIHA may occur. Secondary causes for AIHA such as an underlying autoimmune disorder, infections or malignancies should be considered as part of the investigation process for AIHA. Immunohaematology investigations for AIHA should always include a DAT using monospecific anti-IgG and anti-C3d. On occasions, further testing with anti-IgA or -IgG subtypes may be necessary. While the DAT provides information on bound RBC antibodies, the indirect antiglobulin test (IAT) using screening and antibody identification cells will provide information on the specificity of circulating antibody. Often this will give a pan-reactive pattern although it is not unusual to identify coincident autoantibodies with defined red cell specificity. Negative IAT with screening and identification cells in the presence of a positive DAT should raise suspicion of drug-induced AIHA. In patients who may have been potentially alloimmunized, further procedures should be performed to exclude the coincident presence of alloantibodies as failure to recognize alloantibodies may result in an immediate or delayed haemolytic transfusion reaction if red cell transfusions are initiated. Elution and auto- or allo-adsorbtion techniques are useful to separate allo- and autoantibodies. Complete red cell phenotyping should be concurrently performed to aid in identifying antibody specificities. Heavily antibody-coated red cells may be difficult to phenotype with some anti-sera, in which case, molecular based red cell genotyping should be initiated. Molecular based red cell typing is also particularly useful where the patient has recently been transfused and the initial red cell phenotype of the patient is not known. The primary management of the patient with AIHA is amelioration of the underlying condition and suppression of immune mediated haemolysis. This is usually achieved with administration of steroids or immunosuppressive agents. Splenectomy may be an effective second line treatment. Rituximab is increasingly becoming an effective treatment option in patients who are steroid-refractory and not suitable for splenectomy. Red cell transfusions in patients with AIHA should be undertaken carefully although they should never be denied blood transfusions because of inability to find compatible units. As far as possible, phenotype matched red cells, cross-match compatible with the patient's autoadsorbed serum should be transfused. If coincident alloantibodies are identified, antigen-negative red cells will need to be selected.

S. Panzer, S. Engelbrecht, M. F. Cole-Sinclair, E. M. Wood, S. Wendel, S. Biagini, Z. Zhu, J.-J. Lefrere, G. Andreu, T. Zunino, J.-J. Cabaud, P. Rouger, O. Garraud, K. Janetzko, M. Muller-Steinhardt, P. van der Burg, A. Brand, P. Agarwal, T. Triyono, A. Gharehbaghian, N. Manny, O. Zelig, A. Takeshita, Y. Yonemura, H. Fujihara, K. E. Nollet, H. Ohto, K.-S. Han, V. S. Nadarajan, G. Berlin, S. G. Sandler, R. G. Strauss & H. W. Reesink

Acute promyelocytic leukemia with concurrent myeloid sarcoma is a rare clinical event. Herein we describe a patient that presented with back pain and bilateral leg weakness caused by spinal cord compression due to extramedullary deposition of leukemic cells. Acute promyelocytic leukemia was suspected based on immunophenotypic findings of malignant cells in bone marrow aspirate. The diagnosis was confirmed by the presence of PML-RARα fusion copies. MRI showed multiple hyperintense changes on the vertebral bodies, together with intraspinal masses causing spinal cord compression. The patient immediately underwent radiotherapy, and was treated with all-trans retinoic acid and idarubicin. Reassessment MRI showed complete resolution of all intraspinal masses and the disappearance of most of the bony lesions. Post-treatment bone marrow aspirate showed complete hematological and molecular remission. The motor power of his legs fully recovered from 0/5 to 5/5; however, sensory loss below the T4 level persisted.Eş zamanlı miyeloid sarkomlu akut promiyelositik lösemi nadir bir klinik olaydır. Burada ekstramedüller lösemik hücre birikimi nedeniyle spinal kord kompresyonu sonucunda bel ağrısı ve bilateral bacak zayıflığı ile gelen bir hastayı tanımlıyoruz Kemik iliği aspiratında malign hücreler immünofenotipik bulguları temelinde akut promiyelositik lösemiden şüphelenildi. Tanı PML-RARα füzyon kopyalarının varlığıyla doğrulandı. MRG, vertebral cisimlerde çok sayıda hiperintens değişiklik ve beraberinde spinal kord kompresyonuna neden olan intraspinal kitleler gösterdi. Hastaya hemen radyoterapi başlandı ve all-trans retinoik asit ve idarubisin ile tedavi edildi. Tekrar değerlendirmede MRG tüm intraspinal kitlelerin tam olarak geçtiğini ve kemik lezyonlarının çoğunun kaybolduğunu gösterdi. Tedavi sonrası kemik iliği aspiratı tam hematolojik ve moleküler remisyon gösterdi. Bacaklarda motor güç 0/5’ten 5/5’e tam olarak iyileşti ancak T4 seviyesi altındaki duyu kaybı devam etti.

We investigated the role of lipocalin-2 (LCN-2) and its receptor (SLC22A17) in mediating clonal dominance in a patient with both BCR-ABL and JAK2-V617F mutations. LCN-2 mRNA showed a near 50-fold increase in expression, accompanied by down-regulation of SLC22A17, coinciding with increase in BCR-ABL transcripts, loss of JAK2-V617F and change of clinical phenotype from polycythaemia vera to chronic myeloid leukaemia. These changes were reversed after commencing imatinib mesylate. Consistent with experimental studies, BCR-ABL+ cells express LCN-2 leading to suppression of BCR-ABL- cells and explain their eventual dominance when occurring together with JAK2-V617F.

Background and Objectives Appropriate screening for irregular red-cell antibodies is essential for ensuring transfusion compatibility and for antenatal management of mothers at risk of haemolytic disease of the foetus and newborn. Screening for all relevant antibodies is, however, limited by screening cells that do not express antigens present in the patient and donor population. Technology to artificially incorporate antigens into red cells is currently available and may be an option for customizing screening cells. Materials and Methods We sought to identify retrospectively the changing patterns of alloantibody prevalence in our multiethnic population on change of screening cells. Antibody screening records of 143 501 patients tested from 2004 to 2010 were retrieved and divided into two groups: period-1 (2004–2008) and period-2 (2009–2010). During period-1, standard screening cells were used while in period-2, MUT+Mur+ KODE™ transformed red cells (kodecytes) were used. Results Four per cent of samples tested during period-2 were positive on antibody screening compared to 3·2% in period-1. Specific antibodies, excluding anti-D, were identified in 1·66% and 1·52% of patients in period-2 and -1, respectively. When confined to antibodies of clinical significance only, period-2 showed higher alloantibody prevalence of 1·16% as compared to 0·66% in period-1. Antibodies to glycophorin variants of MNS (vMNS) were more commonly detected while antibodies to Lewis antigens declined during period-2. Conclusion Antibodies to vMNS antigens are common in South and East Asian populations and are often missed when using standard screening cells. Use of specifically engineered screening cells to express red-cell antigens artificially is beneficial in detecting the diverse alloantibodies present in our population.

Individuals with alpha-thalassaemia (ATT), beta-thalassaemia (BTT) and HbE trait (HET) are often initially identified based on haematological parameters. However, the values of these parameters usually overlap with iron deficiency anaemia (IDA) and anaemia of chronic disease (ACD). We evaluated the use of RBC-Y in 156 normal individuals and 332 patients; ATT (n = 37), BTT (n = 61), HET (n = 25), HbH disease (n = 5), ACD (n = 67), IDA (n = 83) and ACD with IDA (n = 54). Diagnostic efficiency was analysed by receiver operating characteristics (ROC). MCH was better compared with RBC-Y in discriminating normal from abnormal with sensitivity and specificity of 94% at a cut-off of 26 pg. The Green and King (G&K) index performed the best in discriminating carriers from IDA and ACD with area under the ROC curve (AUC(ROC)) of 0.81. However, if ACD was excluded, RBC-Y/MCV was a good discriminator for carriers from IDA with AUC(ROC) = 0.845. In general screening of populations with ATT, BTT and HET, we propose that hypochromic individuals be first identified by MCH

The co-occurrence of JAK2 V617F mutation with BCR-ABL reciprocal translocation is uncommon. We report a 60-year-old man who initially presented with phenotype of polycythemia vera (PV), which evolved into chronic myeloid leukemia and back to PV once treatment with imatinib was commenced. JAK2 V617F mutation and BCR-ABL fusion transcripts were detected in the initial sample. However, JAK2 V617F alleles diminished when BCR-ABL mRNA burden increased and reappeared once the patient was commenced on imatinib. The dynamic interaction between JAK2 V617F and BCR-ABL implies that two independent clones exist with the JAK2 V617F clone only achieving clonal dominance when BCR-ABL positive clones are suppressed by imatinib.

Non-HLA gene polymorphisms have been shown to be associated with the risk of graft-versus-host disease (GVHD) and outcome of allogeneic haematopoietic stem cell transplantation (AHSCT). This study aims to investigate the role of IL6, TNFα, IL10, IL2 and IL12 gene polymorphisms in the outcome of AHSCT in a South East Asian population. A total of 67 patients and 59 donors who underwent HLA-identical matched sibling AHSCT were available for analysis. There was no significant association between the different cytokine genotypes of patients with the incidence and severity of acute GVHD. Patients with IL2 166∗T allele and patients who received donor stem cells who had IL2 166∗G allele appeared to have reduced incidence of cGVHD. Patients who received donor stem cells with IL12 1188∗C allele are found to be associated with better disease free survival. These results suggest a possible role of IL2 and IL12 gene polymorphisms in the outcome of AHSCT in a South East Asian population.

A letter to the editor is presented which discusses the introduction of imatinib mesylate (IM) in the medical case of an 18-year old man with in-frame mutations and blast transformation of chronic myeloid leukemia (CML).

Tyrosine kinase inhibitors (TKIs) elicit high response rates among individuals with kinase-driven malignancies, including chronic myeloid leukemia (CML) and epidermal growth factor receptor-mutated non-small-cell lung cancer (EGFR NSCLC). However, the extent and duration of these responses are heterogeneous, suggesting the existence of genetic modifiers affecting an individual's response to TKIs. Using paired-end DNA sequencing, we discovered a common intronic deletion polymorphism in the gene encoding BCL2-like 11 (BIM). BIM is a pro-apoptotic member of the B-cell CLL/lymphoma 2 (BCL2) family of proteins, and its upregulation is required for TKIs to induce apoptosis in kinase-driven cancers. The polymorphism switched BIM splicing from exon 4 to exon 3, which resulted in expression of BIM isoforms lacking the pro-apoptotic BCL2-homology domain 3 (BH3). The polymorphism was sufficient to confer intrinsic TKI resistance in CML and EGFR NSCLC cell lines, but this resistance could be overcome with BH3-mimetic drugs. Notably, individuals with CML and EGFR NSCLC harboring the polymorphism experienced significantly inferior responses to TKIs than did individuals without the polymorphism (P = 0.02 for CML and P = 0.027 for EGFR NSCLC). Our results offer an explanation for the heterogeneity of TKI responses across individuals and suggest the possibility of personalizing therapy with BH3 mimetics to overcome BIM-polymorphism-associated TKI resistance.

Appropriate screening for irregular red-cell antibodies is essential for ensuring transfusion compatibility and for antenatal management of mothers at risk of haemolytic disease of the foetus and newborn. Screening for all relevant antibodies is, however, limited by screening cells that do not express antigens present in the patient and donor population. Technology to artificially incorporate antigens into red cells is currently available and may be an option for customizing screening cells.We sought to identify retrospectively the changing patterns of alloantibody prevalence in our multiethnic population on change of screening cells. Antibody screening records of 143 501 patients tested from 2004 to 2010 were retrieved and divided into two groups: period-1 (2004-2008) and period-2 (2009-2010). During period-1, standard screening cells were used while in period-2, MUT+Mur+ KODE(™) transformed red cells (kodecytes) were used.Four per cent of samples tested during period-2 were positive on antibody screening compared to 3·2% in period-1. Specific antibodies, excluding anti-D, were identified in 1·66% and 1·52% of patients in period-2 and -1, respectively. When confined to antibodies of clinical significance only, period-2 showed higher alloantibody prevalence of 1·16% as compared to 0·66% in period-1. Antibodies to glycophorin variants of MNS (vMNS) were more commonly detected while antibodies to Lewis antigens declined during period-2. Antibodies to vMNS antigens are common in South and East Asian populations and are often missed when using standard screening cells. Use of specifically engineered screening cells to express red-cell antigens artificially is beneficial in detecting the diverse alloantibodies present in our population.

Summary Introduction: The glucose-6-phosphate dehydrogenase (G6PD) fluorescent spot test (FST) is a useful screening test for G6PD deficiency, but is unable to detect heterozygote G6PD-deficient females. We sought to identify whether reporting intermediate fluorescence in addition to absent and bright fluorescence on FST would improve identification of mildly deficient female heterozygotes. Methods: A total of 1266 cord blood samples (705 male, 561 female) were screened for G6PD deficiency using FST (in-house method) and a quantitative enzyme assay. Fluorescence intensity of the FST was graded as either absent, intermediate or normal. Samples identified as showing absent or intermediate fluorescence on FST were analysed for the presence of G6PD mutations using TaqMan@SNP genotyping assays and direct nucleotide sequencing. Results: Of the 1266 samples, 87 samples were found to be intermediate or deficient by FST (49 deficient, 38 intermediate). Of the 49 deficient samples, 48 had G6PD enzyme activity of ≤ 9.5 U/g Hb and one sample had normal enzyme activity. All 38 intermediate samples were from females. Of these, 21 had G6PD activity of between 20% and 60%, and 17 samples showed normal G6PD activity. Twenty-seven of the 38 samples were available for mutation analysis of which 13 had normal G6PD activity. Eleven of the 13 samples with normal G6PD activity had identifiable G6PD mutations. Conclusion: Glucose-6-phosphate dehydrogenase heterozygote females cannot be identified by FST if fluorescence is reported as absent or present. Distinguishing samples with intermediate fluorescence from absent and bright fluorescence improves detection of heterozygote females with mild G6PD deficiency. Mutational studies confirmed that 85% of intermediate samples with normal enzyme activity had identifiable G6PD mutations.

The outcome of treating chronic myeloid leukemia (CML) with imatinib mesylate (IM) is inferior when therapy is commenced in late chronic or accelerated phase as compared to early chronic phase. This may be attributed to additional genomic alterations that accumulate during disease progression. We sought to identify such lesions in patients showing suboptimal response to IM by performing array-CGH analysis on 39 sequential samples from 15 CML patients. Seventy-four cumulative copy number alterations (CNAs) consisting of 35 losses and 39 gains were identified. Alterations flanking the ABL1 and BCR genes on chromosomes 9 and 22, respectively, were the most common identified lesions with 5 patients losing variable portions of 9q34.11 proximal to ABL1. Losses involving 1p36, 5q31, 17q25, Y and gains of 3q21, 8q24, 22q11, Xp11 were among other recurrent lesions identified. Aberrations were also observed in individual patients, involving regions containing known leukemia-associated genes; CDKN2A/2B, IKZF1, RB1, TLX1, AFF4. CML patients in late stages of their disease, harbor pre-existing and evolving sub-microscopic CNAs that may influence disease progression and IM response.

Altitude training is sometimes employed by elite endurance athletes to improve their sea level performance. This improvement results from the increased red cell mass consequent upon the boost in erythropoietin (EPO) level that occurs as a response to the relatively hypoxic environment at high altitudes. We measured serum EPO levels together with various red cell and reticulocyte parameters including immature reticulocyte fraction (IRF) in eight national track-endurance cyclists, resident at sea-level, prior to and upon return from an altitude of approximately 1905 m. Reticulocytes and soluble transferrin receptor (sTfR) were significantly increased with reduction in ferritin levels immediately on return from high altitude indicating increased erythropoietic activity. IRF in particular showed a significant peak immediately on return but decline to sub-baseline levels by day 9, and recovery to baseline by day 16. Our results indicate that IRF is a sensitive marker of erythropoietic status in athletes undergoing altitude training and subsequent loss of EPO stimuli on return to sea level.

The objective of this study was to identify haematological parameters useful in screening for iron deficiency among blood donors. Iron deficiency is a common complication of blood donation and often goes unrecognized until anaemia develops. Biochemical markers such as soluble transferrin receptor (TfR), ferritin and log(TfR/F) have been proposed as more valid indicators of body iron status. Red blood cell (RBC) parameters are, however, more easily measured and have also been proposed as indicators of iron depletion. We measured ferritin and TfR in 192 blood donors together with RBC analysis, performed on two haematology analysers. Thirteen donors had parameters suggestive of haemoglobinopathy and were excluded from further analysis. Overall, 10% (18/179) of the remaining donors had iron deficiency, as defined by log(TfR/F) exceeding the 95th percentile of the value in the population of first-time donors. Using receiver operating characteristic analysis, the sensitivity of ferritin was 100%, with a specificity of 90% at a cut-off of 15 mug L(-1). The sensitivity and specificity of RBC-Y at a cut-off of 152 for detecting iron deficiency were 81 and 89%, respectively. Haemoglobin content of reticulocytes, meanwhile, showed sensitivity of 69% and specificity of 93% when a cut-off of 28 pg was used. Both measures compare favourably with haemoglobin which only showed a sensitivity of 50%, although specificity was 91% at a cut-off value of 125 g L(-1). The parameter RBC-Y can be useful as a screening measure for iron deficiency in blood donors.

Chronic neutrophilic leukaemia is a rare myeloproliferative disease characterised by splenomegaly, sustained neutrophilia, raised vitamin B12 level and absence of the Philadelphia chromosome. We report a 74-year-old man who presented first with Sweet's syndrome and subsequently leukocytosis. He had splenomegaly, a raised vitamin B12 level, serum uric acid and neutrophil alkaline phosphatase score. Cytogenetic study of the marrow was normal and peripheral blood for BCR-ABL gene transcript was not detectable. He subsequently passed away with bronchopneumonia.

The use of tyrosine kinase inhibitors (TKI) to target oncogenic kinases has led to remarkable responses in patients with chronic myeloid leukemia (CML) and EGFR-mutated non-small cell lung cancer (EGFR NSCLC). However, a significant subset of patients have a minimal or very brief response. It has been suggested that germline polymorphisms may account for this upfront TKI resistance, and that identifying such polymorphisms will allow personalization of targeted therapy to achieve optimal responses in patients. Using paired-end DNA sequencing, we discovered a common (12.3% carrier rate) deletion polymorphism in intron 2 of the BIM gene. BIM is a pro-apoptotic member of the BCL2 family of proteins, and is required for TKIs to induce apoptosis in many cancers. We investigated the effects of the polymorphism on BIM function and TKI resistance in CML and EGFR NSCLC. Inspection of BIM gene structure suggested the polymorphism would result in mutually exclusive splicing of exon 3 (E3) and 4 (E4). Importantly, such an event is predicted to affect TKI sensitivity, since the pro-apoptotic BH3 domain is found only in E4. Using minigenes, we confirmed the deletion favored splicing of E3 over E4 by 5-fold (p=0.008), and that the deletion contained a cis-acting splicing suppressor. Next, using Zn finger nuclease-editing, we recreated the polymorphism in TKI sensitive CML (K562) and EGFR NSCLC (PC9) cell lines. Polymorphism-containing subclones had increased E3/E4 transcript ratios, decreased expression of BH3-containing BIM protein and defective apoptotic signaling, and were intrinsically TKI resistant. Importantly, while manipulation of E3-containing transcript levels did not alter the resistance phenotype, pharmacologic restoration of BH3 function (using a BH3 mimetic ABT-737) restored apoptotic signaling as well as TKI-sensitivity. Finally, we determined if the polymorphism predicted for inferior clinical responses in TKI-treated CML and EGFR NSCLC patients. In 203 CML patients, the polymorphism predicted inferior imatinib responses (defined by EuropeanLeukemiaNet criteria) among those with the polymorphism vs those without (odds ratio=2.94, p=0.02, 95% CI 1.17-7.43). In 141 EGFR NSCLC patients, the polymorphism predicted a shorter PFS of 6.6 vs 11.9 months (p=0.0027), and was independently prognostic for poorer PFS (hazard ratio=2.14, p=0.0026, 95% CI 1.30-3.50). In summary, by altering BIM splicing, the BIM polymorphism is sufficient to cause intrinsic TKI resistance in vitro, and predicts inferior TKI responses in patients. Upfront testing of CML and EGFR NSCLC patients for the BIM polymorphism may identify individuals at risk for developing clinical TKI resistance. Our results also offer an explanation for the heterogeneity of TKI responses among CML and EGFR NSCLC patients, and suggest the possibility of personalizing therapy with BH3 mimetics to improve TKI responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1911. doi:1538-7445.AM2012-1911

Abstract 3144 Poster Board III-81 Purpose Allo-immunization to erythrocytes is an important issue with regard to blood transfusions, albeit perhaps less prominent than other possible adverse effects. Several studies including pregnant and transfused cases have been conducted in US and European counties. However, a sufficient international or interracial comparative study has not been performed yet. Asian populations contain a wide variety of genetics and circumstances. They may differ in immune responses to allo-antigens that are common among American and European populations. Because no confirmed nor detail information exists for many Asian populations, collaborative international study concerning these issues will improve blood transfusions and transplantations not only in Asia but also the world. (Method) Forty-eight institutes, including those in Japan (29), Korea (15), Hong Kong (1), Singapore (1), Malaysia (1) and Thailand (1), participated in this first cooperative study of Asian population examining allo-immunity to erythrocytes. The total number of independent cases included more than 866,000 patients. Ab screening methods adopted in these institutes included gel columns, beads columns, traditional tubes or some combination of these three. If a case was tested multiple times, we counted it as one case. Multiple antibodies detected during the study in one patient were separately analyzed. Patients with unconfirmed histories were excluded from the study. We analyzed and compared the frequencies of irregular antibodies (Abs) to erythrocyte antigens between patients of different gender, patients pregnant or not, and patients with and without previous blood transfusion. Results Abs were determined in 8,880 patients (male /female: 3,528 /5,482), including 1801 /2418 from Japan, 946 /1477 from Korea, 287 /688 from Malaysia, 214 /408 from Thailand, 237 /375 from Hong Kong, and 11 /15 from Singapore. Of note, anti-D Ab was more frequently detected in females (p Conclusion Although there have been several previous reports on allo-immunity in pregnant and transfused cases, this is the first international collaborative study in allo-immunity to erythrocyte antigens in relation to gender, pregnancy and blood transfusion status in Asia. Anti-D Ab was frequently detected in pregnant female patients. Anti-E, -Jka and -C Abs were frequently detected in patients that had received blood transfusion. However, the frequency varies among Asian countries. These data will contribute to the international body of knowledge concerning allo-immunity, allowing for advancements in successful blood transfusion in Asia and the world. The number of cases might be small in the light of total Asian populations, therefore, we should continue and spread the study. Disclosures No relevant conflicts of interest to declare.

Abstract 2112 Poster Board II-89 (Purpose) Asian populations have a wide variety of genetics and circumstances. Therefore, immune responses to allo-antigens may differ from those that are common among American and European populations. However, we have little information from Asian populations, and collaborative international study is required to improve the success of blood transfusions and transplantations. Therefore, we tried to analyze allo-antibodies (Abs) to erythrocyte antigens in patients from Asian countries. (Method) Forty-eight institutes, including those in Japan (29), Korea (15), Hong Kong (1), Singapore (1), Malaysia (1) and Thailand (1), participated to this cooperative study of allo-antibodies to erythrocyte antigens in Asian populations. We investigated the number of blood grouping tests, cross matching tests, and erythrocyte irregular Ab analyses as well as information of institutes including total number of bed, operations and transfusions performed. We also investigated methods adopted for screening and detecting irregular Abs to erythrocyte antigens. Furthermore, we studied the frequencies of irregular Abs to erythrocytes (D, C ,c, E, e, f, Ce, P1, M, N, S, s, Mia, Lea, Leb, Jka, Jkb, Jk3, Fya, Fyb,K, k, Kpa, Kpb, Jsa, Jsb, Dia, Dib, Lua, Lub, Xga and H), and compared them among Asian countries. The total number of independent cases included more than 866,000 patients. If a case was analyzed multiple times, we counted it as one case. Multiple antibodies detected in a patient were separately summarized. (Results) Ab screening methods adopted in these institutes included gel columns, beads columns, traditional tubes or some combination of these three. There was no difference in the methods including the erythrocyte panel adopted. Among the serum of 8,880 patients (male /female: 1 /1.55) 10,069 antibodies were detected. The numbers of these patients registered in each country are as follows; 4,222 in Japan, 2,423 in Korea, 975 in Malaysia, 622 in Thailand, 612 in Hong Kong and 26 in Singapore. On average, anti-E Ab was detected in 25.7% (min-max country, 14.3-32.2%) of patients, anti-Lea 20.9 % (3.2-30.4), anti-Mia 7.3% (0-49.4), anti-P1 6.6 % (0-10.6), anti-M 5.6 % (0-7.0), ant-c+E 4.9 % (1.6-8.5), anti-Leb 4.3 % (0-7.6), anti-D 4.0 % (0-16.2), anti-Fyb 2.2 % (0-3.7), anti-Dia 1.9 % (0-3.3), anti-Jka 1.7 % (0.8-7.7), anti-C+e 1.5 % (0-3.3), anti-Lea+Leb 1.4 % (0-5.8), anti-S 1.1 % (0-1.9), anti-c 1.1 % (0-2.3) and other Abs 9.9 %. Anti-D Ab was frequently observed in patients from Korea and Malaysia compared to Thailand, Hong Kong and Japan. Anti-Lea Ab was frequently observed in patients from Malaysia, Japan, Singapore and Korea. Anti-Dia Ab was observed in patients from Japan and Korea. Anti-Mia Ab was frequently observed in patients from Thailand and Hong Kong, followed by Malaysia and Singapore, and rarely from Japan and Korea. (Conclusion) This is the first international collaborative study on allo-immunity to erythrocyte antigens in Asian populations. There are some important differences among Asian countries with regard to the presence of anti-D, -Lea, -Dia and -Mia Abs. Further, the proportions of detected Abs, (such as anti-K, -D and -MiaAbs) differ from those reported in previous studies of patients from US and European counties (Winters et al, Hoeltge et al, Walker et al, etc). These new data will contribute to the advancement in successful blood transfusion in Asia and the world. Furthermore, the study will strengthen the network of international collaboration in future. Disclosures: No relevant conflicts of interest to declare.

Introduction Serum measurement of free light chain (FLC) has been used for diagnosis and monitoring of patients with plasma cell dyscrasias. The aim of the study was to determine the prognostic value of FLC in multiple myeloma (MM) and to identify other independent prognostic factors of MM. Methods Biochemical and clinical data of 59 MM patients (age, Ig isotype, M protein, haemoglobin, platelet, serum calcium, creatinine, albumin, plasma cell infiltration and bone lesions) were retrieved. FLC and β 2 microglobulin (β 2 M) were measured in the sera collected at the time of diagnosis. Survival time was calculated by the Kaplan–Meier method and Cox regression analysis was done to analyse the prognostic indicators. Results The majority of MM patients were of IgG isotype (74.6%). Fifty-seven of 59 (97%) MM patients had abnormal FLC ratio. Median survival time was 46 months. Cox univariate analysis revealed that albumin, calcium, creatinine, FLC ratio, international staging system, and β 2 M were significantly associated with survival ( p = 0.02, 0.002, 0.045, 0.04, 0.045, 0.013, respectively). Cox multivariate analysis revealed that calcium, β 2 M and FLC ratio were independent prognostic predictive factors ( p = 0.022. 0.006 and 0.011, respectively). Conclusions Serum FLC ratio is an independent prognostic indicator for MM in addition to calcium and β 2 M.

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is a X-linked disorder causing neonatal jaundice and haemolysis on oxidative stress. We compared the currently used fluorescent spot test (FST) against a quantitative assay (OSMMR2000-D) and molecular testing methods for screening and identification of G6PD deficient neonates. A total of 1371 cord blood samples were tested using the semiquantitative FST and quantitative G6PD assay. Positive FST samples were subjected to allelic discrimination assays for G6PD Viangchan, Mediterranean, Canton and Kaiping, which encompass over 75% of the G6PD mutations detected in our population. Positive FST results were seen in 86 (6.3%) of the samples tested. Mean G6PD level using the quantitative assay was 15.3 U/g haemoglobin (Hgb) with 60% and 20% enzyme activity 9.2 and 3.1 U/g Hgb, respectively. Twenty-one of the 86 samples detected as positive by FST had activity above 9.2 U/g Hgb, 10 of which were identified as harbouring one of the four mutations tested. The quantitative assay used in this study, using cord blood samples may cause false negative results if limits are established as proposed. We propose that the FST remains an effective screening tool for G6PD deficiency and can be supplemented by rapid real time molecular identification methods for confirmation.

Abstract 1666 The use of the tyrosine kinase inhibitor (TKI), imatinib (IM), to target the oncogenic BCR-ABL kinase has resulted in profound responses in patients with chronic phase (CP) chronic myelogenous leukemia (CML). However, a subset of patients do not respond to TKIs, and are deemed to have primary resistance. Importantly, patients with European LeukemiaNet (ELN)-defined ‘failure’ or ‘suboptimal response’ are at increased risk of poorer long-term outcomes. Little is known about mechanisms underlying primary resistance, where only a minority of patients have BCR-ABL kinase domain (KD) mutations. Interestingly, East-Asian CML patients are reported to have lower complete cytogenetic response rates compared to the West (∼50 vs 80% respectively, Au et al. 2009). We used massively parallel DNA sequencing of paired-end ditags to identify genetic factors associated with resistance in CML patient samples. We discovered a novel deletion polymorphism in the BIM gene that correlated with resistance, and which represented a common polymorphism in normal East-Asian (12.3% carriers), but not African or Caucasian (0%), populations (n=2465). BIM is a pro-apoptotic BCL2 family member, and plays a central role in CML pathophysiology. Here, BCR-ABL suppresses FoxO3a-mediated BIM transcription to maintain a survival advantage, while preventing BIM expression following BCR-ABL inhibition results in TKI resistance. Inspection of BIM gene structure suggested the polymorphism would result in mutually exclusive splicing of exon 3 (E3) vs 4 (E4), leading to decreased expression of BIM transcripts encoding the pro-apoptotic BH3 domain (found only in E4). To test this hypothesis, we constructed a minigene to measure E3 vs E4 splicing, and found the polymorphism decreased splicing to E4 over E3 by >5-fold. Importantly, primary CML cells exhibited the same phenomenon, since polymorphism-containing samples expressed lower levels of E4- vs E3-containing transcripts (p=0.008), while general BIM transcription was unaffected. Our observations suggested a novel mechanism for intrinsic TKI resistance. Here, upon IM exposure, polymorphism-containing CML cells would favor induction of E3- vs E4-containing BIM transcripts, decreased expression of BH3-containing BIM isoforms, and impaired apoptosis. To facilitate these studies, we identified a Japanese CML cell line, KCL22, which contained the polymorphism, and confirmed it had a decreased E4/E3 transcript ratio compared to cells without the polymorphism. KCL22 cells also had decreased induction of E4-containing transcripts following IM, as well as lower levels of BIMEL protein, a major BH3-containing BIM isoform. Consistent with prior reports, KCL22 cells were resistant to IM, despite effective BCR-ABL inhibition, and had impaired apoptotic signalling upon IM exposure. Importantly, and as predicted by our model, pharmacologic restoration of BH3 activity (using the BH3-mimetic drug, ABT-737) sensitized cells to IM-induced death. Next, we used zinc finger nuclease-facilitated gene targeting to precisely create the polymorphism in the BIM gene of IM-sensitive K562 CML cells. We generated subclones that were heterozygous or homozygous for the polymorphism, and confirmed a decreased E4/E3 ratio in these cells in a polymorphism-dosage-dependent manner. Polymorphism-containing cells exhibited decreased induction of E4-containing transcripts following IM exposure, as well as impaired upregulation of BIMEL protein, and diminished apoptotic cell death. As in KCL22 cells, ABT-737 enhanced the ability of IM to activate apoptosis in polymorphism-containing cells. Using an expanded East-Asian CML cohort (n=203), we found the polymorphism correlated with TKI resistance (defined as ‘failure’ or ‘suboptimal’ per ELN criteria) in CP patients treated with 400 mg IM daily (p=0.02). Further, patients with the polymorphism were more likely to have resistance in the absence of a KD mutation than those without (OR=2.24, 95% CI of 1.22–4.12). In sum, we have found an East-Asian polymorphism in BIM that is associated with intrinsic resistance to TKIs. Screening for this polymorphism may be useful in identifying patients at risk of TKI resistance; a resistance we show can be overcome by BH3 mimetics. Our findings may also apply to other cancers and proliferative disorders in which drug-sensitivity is BIM-dependent. Disclosures: No relevant conflicts of interest to declare.