Comparing transfusion reactions between pre-storage and post-storage leukoreduced apheresis platelets: an analysis using propensity score matching.

Transfusion reactions induced by platelet transfusions may be reduced and alleviated by leukocyte reduction of platelets. Although leukoreduction of apheresis platelets can be performed either pre-storage or post-storage, seldom studies directly compare the incidence of transfusion reaction in these two different blood products. We conducted a retrospective study to compare the transfusion reactions between pre-storage and post-storage leukoreduced apheresis platelets. We reviewed the general characteristics and the transfusion reactions, symptoms, and categories for inpatients who received pre-storage or post-storage leukoreduced apheresis platelets. Propensity-score matching was performed to adjust for baseline differences between groups. A total of 40,837 leukoreduction apheresis platelet orders were reviewed. 116 (0.53%) transfusion reactions were reported in 21,884 transfusions with pre-storage leukoreduction, and 174 (0.91%) reactions were reported in 18,953 transfusions with post-storage leukoreduction. Before propensity-score matching, the odds ratio for transfusion reactions in the pre-storage group relative to the post-storage group was 0.57 (95% confidence interval [CI] 0.45-0.72, P < 0.01); the odds ratio after matching was 0.63 (95% CI 0.49-0.80, P < 0.01). A two-proportion z-test revealed pre-storage leukoreduction significantly decreases the symptoms of chills, fever, itching, urticaria, dyspnea, and hypertension as compared with those in post-storage leukoreduction. Pre-storage leukoreduced apheresis platelet significantly decreased febrile non-hemolytic transfusion reaction as compared with post-storage groups. This study suggests pre-storage leukoreduction apheresis platelet significantly decreases the transfusion reaction as compared with those in post-storage leukoreduction.

The TVGH-NYCU Thal-Classifier: Development of a Machine-Learning Classifier for Differentiating Thalassemia and Non-Thalassemia Patients.

Thalassemia and iron deficiency are the most common etiologies for microcytic anemia and there are indices discriminating both from common laboratory simple automatic counters. In this study a new classifier for discriminating thalassemia and non-thalassemia microcytic anemia was generated via combination of exciting indices with machine-learning techniques. A total of 350 Taiwanese adult patients whose anemia diagnosis, complete blood cell counts, and hemoglobin gene profiles were retrospectively reviewed. Thirteen prior established indices were applied to current cohort and the sensitivity, specificity, positive and negative predictive values were calculated. A support vector machine (SVM) with Monte-Carlo cross-validation procedure was adopted to generate the classifier. The performance of our classifier was compared with original indices by calculating the average classification error rate and area under the curve (AUC) for the sampled datasets. The performance of this SVM model showed average AUC of 0.76 and average error rate of 0.26, which surpassed all other indices. In conclusion, we developed a convenient tool for primary-care physicians when deferential diagnosis contains thalassemia for the Taiwanese adult population. This approach needs to be validated in other studies or bigger database.

The landscape of BCR-ABL mutations in patients with Philadelphia chromosome-positive leukaemias in the era of second-generation tyrosine kinase inhibitors.

BCR-ABL mutations are associated with resistance to tyrosine kinase inhibitors (TKIs) in Philadelphia chromosome-positive leukaemia. The emergence of these mutations in the era of second-generation TKIs, such as dasatinib and nilotinib, remains an evolving field. We conducted a retrospective study to quantitatively characterize the BCR-ABL transcript and mutation status during treatment with first-generation and second-generation TKI therapies. BCR-ABL mutations were detected by direct sequencing for patients with Philadelphia chromosome-positive leukaemia receiving TKI therapies. The efficacy of TKI therapy was quantitatively assessed by calculating the log reduction of BCR-ABL transcripts, which was measured using real-time quantitative polymerase chain reaction. Fisher's exact test was performed to analyse the associations of log reduction <3 and mutation status. We found 35 patients harbouring 55 mutations of 43 different types, of which 30% occurred in patients receiving imatinib, 27% in nilotinib, and 43% in dasatinib. We found a novel germline mutation, N336 N (AAC➔AAT), and two novel frameshift mutations, Asn358Thr fs*14 and Gly251Ala fs*16. T315I was the most common missense mutation, followed by V299L and F317L. Intron 8 35-bp insertion was the most frequent frameshift mutation. Both missense and multiple BCR-ABL mutations were significantly associated with worse molecular response compared with the molecular response of patients without mutation. Missense mutations, rather than frameshift, were associated with less log reduction, while the T315I, F317L, and T315A mutations were significantly correlated with poor log reduction. Collectively, amino acid substitutions at T315I, F317L, and T315A accounted for the majority of missense mutations and the loss of major molecular response. Mutation analysis is essential for patients receiving TKI therapy who exhibit an unfavourable response. The present study provided a landscape of BCR-ABL mutations in the era of second-generation TKIs.

Association of IL-6 C-572G Gene Polymorphism with Anti-E Production.

BACKGROUND: Interleukin 6 (IL-6) is involved in regulation of immunoglobulin production. The aim of this study was to investigate the association between IL-6 single nucleotide polymorphisms (SNPs) in the IL-6 promoter and anti-E in red blood cell (RBC) transfusion recipients. METHODS: 50 healthy subjects, 54 patients with RBC alloantibody anti-E (responders), and 45 patients without alloantibody (non-responders) were recruited. All patients were E antigen-negative. RESULTS: All healthy subjects and patients had GG at -174 position of IL-6 gene. In our healthy subjects, the frequency of the -572 CC genotype was 58%, that of the -572 CG genotype 38%, and that of the -572 GG genotype 4%. The frequency of G allele of -572 SNP in responders was significantly higher than that in non-responders, (31.5 vs. 16.7%; p = 0.020). The frequency of -572 G-positive genotypes (CG and GG) in responders was also significantly higher than that in non-responders, (55.6 vs. 31.1%; p = 0.016). The relative risk of RBC alloimmunization for patients with the -572 G-positive genotype was significantly higher than that of patients with the -572 CC genotype, (1.771 vs. 0.640; p = 0.016). CONCLUSION: IL-6 C-572G gene polymorphism is significantly associated with anti-E production, with the allele G as a risk allele.

The polymorphism of CAG repeats in the androgen receptor gene and breast cancer mortality.

BACKGROUND: The polymorphic CAG repeats of the androgen receptor (AR) gene have been suggested to affect the risk of breast cancer, but the results are controversial. In addition, the relationship between patients' CAG genotype and the prognosis has not been investigated. OBJECTIVE: The purpose of this study is to access the association between the polymorphic CAG repeats and the incidence and prognosis of breast cancer. METHODS: One hundred and fifty-six breast cancer cases and 108 healthy controls from Taipei Veteran General Hospital were enrolled. The length of CAG repeats was analyzed among by means of PCR amplification. The logistic regression model was used for cross-sectional analyses of prevalent breast cancer.Furthermore, we categorized the cases according to the average length of both CAG alleles (CAGn ≥ 23 versus < 23). Outcomes were disease-free survival and mortality. The Cox proportional hazards model and Kaplan-Meier estimate were used for survival analysis. RESULTS: The median age was 56 (51-64) and 46 (37-52) in breast cancer patients and healthy controls, respectively. The median of CAGn was 22.5 (21.5-24) in study group and 23 (21.5-24) in controls. Our study showed the length of CAG repeats did not contribute to breast cancer or benign breast tumors (HR 1.01; 95% CI, 0.90-1.13). In the median follow-up of 6.59 years, we found the CAGn ≥ 23 (n = 75) could be a poor prognosis (adjust HR, 3.08; 95% CI, 1.42-6.67, p = 0.004). CONCLUSION: The CAG polymorphism is not associated with development of breast cancer, but patients with more CAG repeats of the AR gene are prone to poor prognoses.

Clinical application of a flow cytometric direct antiglobulin test.

BACKGROUND: The clinical application of flow cytometric direct antiglobulin test (FC-DAT) has rarely been evaluated for patients with various diseases including immune and nonimmune hemolytic anemia. STUDY DESIGN AND METHODS: Blood samples from 380 patients with a variety of diseases were studied using the tube direct DAT and FC-DAT. The results of tube DAT and FC-DAT were compared. The predictive values of DAT for hemolysis were evaluated. RESULTS: Of 57 patients with autoimmune hemolytic anemia (AIHA), 6 of the 17 with a negative tube DAT (immunoglobulin G [IgG]) had a positive FC-DAT (IgG) and 23 of the 36 patients with a negative tube DAT (complement 3d [C3d]) had a positive FC-DAT (C3d). In 57 patients with AIHA, the incidence of positive results of FC-DAT (IgG) and tube DAT (IgG) were similar (42 positive vs. 40 positive); but in 323 patients without AIHA, the incidence of positive FC-DATs (IgG) was higher than that of tube DAT (IgG; 47 positive vs. 9 positive). The higher incidence of positive FC-DAT (C3d) than that of tube DAT (C3d) was seen in patients with AIHA (42 positive vs. 21 positive) as well as in patients without AIHA (61 positive vs. 5 positive). Both DAT (IgG) and DAT (C3d) positive has highest positive predictive value for hemolysis, followed by DAT (IgG) alone positive and DAT (C3d) alone positive. CONCLUSIONS: FC-DAT is a complementary test for diagnosing AIHA. There is a synergistic effect of the red blood cell-bound IgG and complement in predicting hemolysis.

Two novel Jk(null) alleles derived from 222C>A in Exon 5 and 896G>A in Exon 9 of the JK gene.

BACKGROUND: Polynesian Jk(null) is well known for its mutation as Intron 5 g>a at the 3' splice acceptor site. After sequencing analysis, however, it was noticed that only three of eight samples with the Jknull phenotype carried typical homozygous Polynesian Jk(null) mutation. Five others were noted to be unreported heterozygous Polynesian Jk(null) mutation. An investigation was then conducted to characterize the underlying mechanism leading to this particular Jk(null) genotype. STUDY DESIGN AND METHODS: Genomic DNA covering 5'-untranslated region exons and intervening introns of the JK gene was amplified by polymerase chain reaction, and the fragments were directly sequenced. The sequencing results were compared with those published in literature and related biologic Web sites. RESULTS: In all five samples with a heterozygous Polynesian Jk(null) mutation, additional mutations were identified. Two samples carried missense mutations: 222C>A (Asn74Lys) in Exon 5 and 499A>G (Met167Val) in Exon 7. Three others had missense mutation 896G>A (Gly299Glu) in Exon 9. These substituted amino acids were located either near or at transmembrane domains, respectively. In addition, two polymorphic nucleotides at positions -103 (a>g) and -119(c>a) from the 3' end of Intron 1 were also Polynesian mutation-related. CONCLUSIONS: In contrast to the typical homozygous Polynesian Jk(null) mutation, two novel heterozygous Jk(null) alleles were noted to be associated with the Jknull phenotype. One carried missense mutation 222C>A in Exon 5, and the other had 896G>A missense mutation in Exon 9. These findings may have implications in designing a molecular screening assay for people with the Jknull phenotype.

A Novel Ael allele derived from a unique 816insG in Exon 7 of the ABO gene.

The ABO blood group system is the most important blood group system in transfusion medicine. In addition to the major A, B and O alleles, many rare alleles with weak expression of the A or B antigens on RBCs have been defined. We report here the molecular analysis of a novel A(el) allele. Exons 6 and 7 of the ABO gene were PCR-amplified, cloned and sequenced for the propositus, Mr C, who is a 56-year-old Taiwanese male and was incidentally observed to have an A(el) phenotype. His direct family members including wife, son and daughter were subsequently enrolled for further study. Three hundred random blood donors of AB phenotype served as control. A novel A(el) allele was uncovered from the propositus and his daughter, of which a unique 816insG mutation occurred on the A102 background that results in a frame shift leading to a 37-amino acid longer polypeptide than the normal A1 transferase, a finding similar to that of Ael01 allele with 804insG. We found that the C family carried a novel A(el) allele that differs molecularly from seven A(el) alleles reported in the literature.

Y-chromosome short tandem repeats analysis to complement paternal lineage study: a single institutional experience in Taiwan.

BACKGROUND: Highly polymorphic autosomal short-tandem-repeat (STR) analysis can be useful in most kinship testing. Y-chromosome-specific STRs, in contrast, have been increasingly applied for the verification of equivocal paternal genetic transmissions. STUDY DESIGN AND METHODS: A total of 338 unrelated males were first typed for the 9-loci Y-STR European minimal haplotype (minHt). Samples with haplotypes that were found at least two times were subject to further study by a commercially available 17-Y-STR multiplex set (AmpFlSTR Yfiler). A separate clinical study for 113 various kinship identifications of male genetic transmission were then conducted by a panel consisting of 18 autosomal STRs and complemented by both Y-STR multiplex sets and their respective results compared. RESULTS: For the 338 individuals, a total of 270 haplotypes were identified after the minHt study, of which 234 were unique. Among the rest of the 104 samples, AmpFlSTR Yfiler identified 82 other unique haplotypes. Altogether, 324 different haplotypes were observed; 316 (97.5%) were unique whereas 8 were shared by two to seven times. The haplotype diversities for the minHt and the AmpFlSTR Yfiler were 99.75 and 99.96 percent, respectively, whereas the powers of discrimination (PDs) were 79.88 and 95.86 percent, respectively. Despite a lower PD for minHt, there was no discrepancy on the clinical setting for personal identification between the two Y-STR sets in an allegedly true male lineage transmission involving 66 cases with 24 father-son, 19 siblings, 9 uncle-nephew, 8 grandfather-grandson, 3 cousins, and 3 half-siblings. For 47 other cases with a false allegation of paternity, exclusion was made for all without ambiguity by either Y-STR panel. CONCLUSION: The 9-loci minHt Y-STR set is adequate to complement conventional autosomal STRs for kinship studies where Y-lineage transmission is concerned.

Unappreciated HLA antibodies in adult immune thrombocytopenic purpura.

BACKGROUND/PURPOSE: Immune thrombocytopenic purpura (ITP) is an autoimmune disease. Platelet refractoriness is frequently seen in patients with ITP. Platelets express platelet-specific antigens and human leukocyte antigens (HLA). Platelet antibodies to platelet-specific antigens and HLA may be present, but HLA antibodies in patients with ITP have rarely been reported. METHODS: Sera from 44 adult patients with ITP were screened for platelet antibodies by two flow cytometric assays. In method I, platelets from normal donor platelets were used as target cells to screen both platelet-specific antibodies and HLA class I antibodies. In method II, the FlowPRA Class I Screening Test kit was used to screen HLA class I antibodies. Fluorescein isothiocyanate (FITC)-conjugated sheep anti-human IgG Fc was used as the staining reagent in both methods. The negative serum control was from one of the normal males with AB blood group who had never received a transfusion. Sera from a pool of five highly sensitized patients were used as the positive control. RESULTS: Of the 44 sera from patients with ITP, 31 (70.5%) were method I positive, and 28 (63.6%) were method II positive. There was no significant difference between the results of method I and method II (p = 0.439). The distribution of the results of these two tests was: both tests positive in 22 sera, method I positive and method II negative in nine sera, method I negative and method II positive in six sera, and both tests negative in seven sera. The mean platelet counts of patients with positive (41.0 +/- 40.0 x 10(9)/L) and negative (40.4 +/- 26.8 x 10(9)/L) tests by method I did not differ significantly (p = 0.643). The mean platelet counts of patients with (36.7 +/- 31.5 x 10(9)/L) and without (48.1 +/- 43.6 x 10(9)/L) HLA class I antibodies did not differ significantly (p = 0.59). CONCLUSION: HLA class I antibodies are frequently found in ITP. The screening of platelet antibodies including platelet-specific antibodies and unappreciated HLA class I antibodies is warranted in patients with ITP.

Screening for platelet antibodies in adult idiopathic thrombocytopenic purpura: a comparative study using solid phase red cell adherence assay and flow cytometry.

BACKGROUND: Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder caused by antiplatelet autoantibodies. In this study, we compared 2 methods for screening serum platelet antibodies in patients with ITP. METHODS: A total of 44 adult patients were clinically classified with ITP. We used 2 indirect tests to detect human leukocyte antigen antibodies and/or platelet-specific antibodies in their sera. In method I, we used solid phase red cell adherence (SPRCA) assay. In method II, by flow cytometry, platelets from plateletpheresis components were used as target cells, and fluorescein isothiocyanate-conjugated sheep anti-human IgG Fc was used as the staining reagent. Positive results were defined as any test with the percentage of fluorescence exceeding the reference range by 3% or more in method II. Direct tests detecting platelet-associated IgG on platelets of patients with ITP were done by flow cytometry. RESULTS: Serum specimens from 44 adult patients with ITP (28 female, 16 male) were tested. SPRCA assay could only detect platelet antibodies in 22 patients (50%). By method II, 31 serum specimens (70.5%) yielded positive results. There was a difference between the results of the SPRCA test and method II, with a high degree of significance (p < 0.001) by the McNemar test. No significant difference in platelet counts was observed for patients with and without discernible platelet antibodies by SPRCA assay (p = 0.90). The direct test was positive in 12 patients (66.7%) out of 18 ITP patients tested. CONCLUSION: Flow cytometry is more sensitive than SPRCA assay for detecting platelet antibodies. Detection of platelet antibodies is useful in explaining the immune mechanism and platelet transfusion refractoriness in ITP.

A novel cis-AB allele derived from a unique 796C>A mutation in exon 7 of ABO gene.

BACKGROUND: The cis-AB phenotype is very rare, and only three genotypes that correspond to specific ABO allele changes have been reported. Cis-AB01 involves the A102 allele with a nonsynonymous substitution G803C in exon 7, whereas cis-AB02 and cis-AB03 involve different nonsynonymous substitutions A796C and C700T, respectively, on the B101 allele background. The nucleotide substitutions give rise to a change of the respective glycosyltransferase, resulting in varying bifunctional AB transferase activities. STUDY DESIGN AND METHODS: Two cis-AB phenotypes were identified in a Taiwanese C. family and two unrelated individuals, respectively. Serologic studies, molecular cloning, and sequencing of exon 6 and exon 7 were carried out to determine their respective phenotypic characteristics and cis-AB alleles. A cohort of 300 AB-phenotype, healthy random individuals served as controls. RESULTS: A novel cis-AB allele is uncovered out of the three family members, of which a 796C>A substitution occurs predicting an amino acid change at residue 266 of leucine to methionine on the background of A102 allele. It is serologically like cis-AB03, an A2B phenotype, but molecularly different. Both of the two unrelated individuals are of cis-AB01 allele, and all of the 300 AB blood group controls are excluded cis-AB phenotype. CONCLUSION: The C. family described carries a novel cis-AB allele that differs molecularly from all previously reported cis-AB alleles.