The clinical and prognostic relevance of driver mutations in 203 Taiwanese patients with primary myelofibrosis.

AIMS: We investigated the clinical and prognostic relevance of the mutational status of driver genes with allele burden and endogenous erythroid colony (EEC) growth in 203 Taiwanese patients with primary myelofibrosis (PMF). METHODS: Pyrosequencing was used to detect JAK2V617F mutational status and measure allele burden, while MPL (exon 10) mutations were analysed by PCR assay and then by direct sequencing. CALR exon 9 mutations were first screened for length changes by GeneScan followed by sequencing. The allele burden of the mutated CALR gene was measured by pyrosequencing. The EEC assay was conducted using a serum-free culture system. RESULTS: The frequencies of the three driver mutations and triple-negative status were similarly distributed between pre-PMF and overt PMF patients, except that pre-PMF patients had a higher incidence of CALR type 2/type-2 like mutations and a lower JAK2V617F allele burden. EEC growth and CALR mutations conferred favourable overall survival (OS). A lower JAK2V617F allele burden and grade 3 bone marrow fibrosis were associated with shorter OS and decreased leukaemia-free survival (LFS). Type 2/type 2-like CAL mutations were associated with better LFS compared with type1/type 1-like mutations. Patients with triple-negative mutation status had significantly worse OS and LFS. The allele burden of CALR mutations remained unchanged, while some JAK2V617F mutations showed clonal expansion in patients during secondary acute myeloid leukaemia transformation. CONCLUSIONS: Our study showed that EEC growth, a higher JAK2V617F allele burden and CALR mutations, especially type 2, were independent predictors for better outcomes in PMF. The allele burden of CALR mutations remained stable, but the allele burden of JAK2V617Fmutations was variable during leukaemia transformation.

Mechanistic Evaluation for Mixed-field Agglutination in the K562 Cell Study Model with Exon 3 Deletion of A1 Gene.

In the case of blood type B3 with typical mixed-field agglutination of RBCs in the presence of anti-B or anti-AB antibody, a number of genetic alternations have been reported. It is well known that the IVS3+5G→A mutation in the B gene destroys the consensus of the splice donor site leading to exon 3 skipping during mRNA splicing. The lack of exon 3 likely causes a short stem region, producing an unstable B3 protein, and is concomitant with a decrease in B3 protein expression. Whether the phenomenon also appears in the type A blood group is of question. In this study, we evaluate whether exon 3 deletion in the blood type A gene also results in mixed-field phenotype. Site-directed mutagenesis was used to generate cDNA encoding A1 gene with exon 3 deletion. The cDNA was stably expressed in K562 cells. The expression of A antigen was compared with expression in parental K562 cells that did not express A antigen and in the stable K562 cell line expressing A(1) cDNA by flow cytometry analyses. The expression of A antigen in A1 stable cells and parental K562 cells was set as 100% and 0%, respectively. The mean relative percentage of A antigen expression for the cells of A1 with exon 3 deletion was 59.9% of A1 stable cells. Consistent with the observations of B3, which is B gene with exon 3 deletion, mixed field agglutination was observed for the cells expressing A1 with exon 3 deletion. Exon 3 deletion results in mixed field phenotype in both type A and B RBCs. However, the degree of antigen expression change for exon 3 deletion in A gene was less severe when compared with the deletion occurred in B gene.

Plasma chromogranin A levels predict survival and tumor response in patients with advanced gastroenteropancreatic neuroendocrine tumors.

AIM: To correlate the baseline and change of chromogranin A (CgA) levels with patient survival and tumor response in Asian patients with advanced gastro-enteropancreatic neuroendocrine tumors (GEP-NETs). PATIENTS AND METHODS: Sixty patients with advanced GEP-NET treated in a medical center between April 2010 and April 2013 were enrolled retrospectively. Plasma CgA level was analyzed for correlation with the patient's clinical outcome and tumor response. RESULTS: Multivariate analysis showed that independent favorable prognostic factors for overall survival were: Eastern Cooperative Oncology Groups performance score 0-1, World Health Organization tumor grade 1-2, single organ metastasis and less than twice the upper normal range of baseline CgA levels. Percentage changes in paired CgA tests (ΔCgA) of more than 17% can predict partial response or stable disease from progressive disease with 91.2% sensitivity and 82.9% specificity. CONCLUSION: Baseline plasma CgA levels predicted overall survival and ΔCgA predicted treatment response in Asian patients with GEP-NETs.

MNSs blood group glycophorin variants in Taiwan: a genotype-serotype correlation study of 'Mi(a)' and St(a) with report of two new alleles for St(a).

BACKGROUND: Glycophorin variants of the MNSs blood group are important in Taiwan. For more than 20 years, screening for the most frequent irregular antibody, anti-''Mi(a)', has been conducted by using 'Mi(a)'(+) RBCs, with a significant success. However, the sensitivity and the specificity of this screening strategy have never been validated, and the true incidences of different glycophorin variants in Taiwan have been in controversy. Also, the significance of another less frequent and usually separately reported variant, St(a), has never been evaluated. METHODOLOGY/PRINCIPAL FINDINGS: We ran a population-based screening (from unselected patients in our hospital) for MNSs blood group glycophorin variants by PCR-sequencing method. GP.Mur (Mil.III) was confirmed by sequence from 57 out of 1027 samples (5.6%), and there was no other Miltenberger subtype glycophorin variant found. Glycophorin variant St(a) was found from 35 out of 1027 samples (3.4%). In contrast to anti-'Mi(a)', which is the most frequently identified irregular antibody in Taiwan, the prevalence of anti-St(a) was only 0.13% as determined by serologic method. In addition, two new alleles for St(a) were found and reported. CONCLUSION/SIGNIFICANCE: We confirm the long-standing assumption that GP.Mur is the only prevalent Miltenberger subtype in Taiwan. The current anti-'Mi(a)' screening method used in Taiwan, although neither sensitive nor specific, is still a suitable practice. Although St(a) antigen has a high prevalence in Taiwan, routine screening for anti-St(a) is not warranted based on current evidence.

Real-time amplification of glyceraldehyde-3-phosphate dehydrogenase gene for quality control of leukopoor platelets.

BACKGROUND: Leukoreduction of blood products is crucial to prevent white blood cell (WBC)-associated complications during transfusion. Of the widely accepted methods for quantifying WBCs in blood components, Nageotte hemocytometry is time-consuming and laborious whereas a specialized instrument is required for flow cytometry. A reliable and affordable method to assess WBC count in blood products is of particular interest. STUDY DESIGN AND METHODS: Real-time polymerase chain reaction (PCR) of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was developed for quantifying WBCs in leukopoor platelets (LPPs). After normalization by the cell-free prefiltrated and postfiltrated plasma DNA, the relative copy number of GAPDH gene in the platelet (PLT) concentrate and its corresponding LPPs was calculated according to the equation of 2(-ΔΔCt) of which Ct is defined as the threshold cycle. The percentage and the number of WBCs that remained in LPPs were consequently determined. This method was compared to Nageotte hemocytometry and was validated by using serially diluted PLT concentrate and 10 pairs of PLT concentrate-LPP samples. RESULTS: Consistent with the removal of WBCs after filtration, the Ct values for the LPP samples were increased when compared to their corresponding PLT concentrate. As revealed by real-time PCR of GAPDH gene, there is a correlation between the calculated and theoretical WBC count in the serially diluted PLT concentrate (correlation coefficient, 0.9532). The WBC counts for the 10 LPP samples were comparable between Nageotte and real-time PCR method and were all below 3.3 × 10(6) WBCs/L. CONCLUSION: The real-time PCR method we report in this study is applicable for routine quality assurance during leukoreduction process.

Genetic and mechanistic evaluation for the mixed-field agglutination in B3 blood type with IVS3+5G>A ABO gene mutation.

BACKGROUND: The ABO blood type B(3) is the most common B subtype in the Chinese population with a frequency of 1/900. Although IVS3+5G>A (rs55852701) mutation of B gene has been shown to associate with the development of B(3) blood type, genetic and mechanistic evaluation for the unique mixed-field agglutination phenotype has not yet been completely addressed. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we analyzed 16 cases of confirmed B(3) individuals and found that IVS3+5G>A attributes to all cases of B(3). RT-PCR analyses revealed the presence of at least 7 types of aberrant B(3) splicing transcripts with most of the transcripts causing early termination and producing non-functional protein during translation. The splicing transcript without exon 3 that was predicted to generate functional B(3) glycosyltransferase lacking 19 amino acids at the N-terminal segment constituted only 0.9% of the splicing transcripts. Expression of the B(3) cDNA with exon 3 deletion in the K562 erythroleukemia cells revealed that the B(3) glycosyltransferase had only 40% of B(1) activity in converting H antigen to B antigen. Notably, the typical mixed-field agglutination of B(3)-RBCs can be mimicked by adding anti-B antibody to the K562-B(3) cells. CONCLUSIONS/SIGNIFICANCE: This study thereby demonstrates that both aberrant splicing of B transcripts and the reduced B(3) glycosyltransferase activity contribute to weak B expression and the mixed-field agglutination of B(3), adding to the complexity for the regulatory mechanisms of ABO gene expression.

Concurrent acute myeloid leukemia and T lymphoblastic lymphoma in a patient with rearranged PDGFRB genes.

Concurrent hematologic malignancies are relatively rare. We encountered a case of concurrent acute myeloid leukemia (AML) and T lymphoblastic lymphoma. The bone marrow chromosome analysis showed the karyotype 46, XY, t(5;12)(q33;p13), which indicated presence of PDGFRB gene translocations. Therefore, this disease belongs to the new WHO category of myeloid and lymphoid neoplasms with abnormalities in PDGFRA, PDGFRB and FGFR1 genes. Although such genetic mutations are prone to multi-lineage differentiation, the present case is in fact the first report of concurrent AML and T lymphoblastic lymphoma involving PDGFRB mutations. The patient was treated with cytarabine and daunomycin in combination with high dose dexamethasone. Allogeneic stem cell transplantation was performed after successful remission induction for both entities. The patient eventually died of chronic graft-versus-host-disease related infection. Based on such an experience, we suggest the decision of stem cell transplantation should be weighed carefully against the risks, especially when tyrosine kinase inhibitors are safe and potentially effective in dealing with such entities.

Chromogranin A is a reliable biomarker for gastroenteropancreatic neuroendocrine tumors in an Asian population of patients.

PURPOSE: To evaluate the significance of plasma chromogranin A (CgA) levels in patients with gastroenteropancreatic neuroendocrine tumors (GEP-NET) in terms of disease status and treatment responses. MATERIALS AND METHODS: Forty-four GEP-NET patients comprising 15 disease-free patients and 29 patients with active disease, as well as 26 healthy participants were enrolled in this study between April 2010 and April 2011. Clinicopathological factors were collected and serial plasma CgA levels were measured. RESULTS: Plasma CgA levels were significantly higher in GEP-NET patients with active disease than in disease-free patients (p = 0.011) or healthy participants (p = 0.001). No difference in CgA levels was observed in terms of primary tumor location, tumor grade, and functional status in patients with active disease. CgA values at 94 U/l distinguished healthy individuals or disease-free patients from patients with active disease. Sensitivity and specificity rates were 86 and 88%, respectively. CgA levels at 110 U/l differentiated patients without recurrence from those with recurrence, with a sensitivity rate of 100% and a specificity rate of 80%. Patients (5/5, 100%) with stable disease and who showed partial response after treatment had a more than 20% decrease in CgA levels compared with the baseline values. Patients (6/6, 100%) with progressive disease showed a less than 20% decrease or increase in CgA levels. CONCLUSION: The plasma CgA level is a reliable biomarker for GEP-NET. We conclude that changes in CgA levels are associated with disease status and treatment responses.

Use of cell study models to confirm the weak ABO phenotypes caused by point mutations among Taiwanese.

Numerous phenotypes with serologically weak expression of A or B antigen have been found on the surface of red blood cells. Some of these alleles have mutation(s) in the ABO gene coding sequence; most of the mutations are single-nucleotide substitutions leading to an amino acid change. However, these mutated ABO alleles leading to attenuated antigen expression are not well defined. In this study, the cDNAs for the A(1v) and B(1) alleles were first obtained from healthy volunteers and were used as the templates for site-directed mutagenesis to generate expression constructs corresponding to various ABO subtypes including A(1), A(2) (539G>C), A(3) (838C>T), A(1) (829G>A), A(1v) (829G>A), and B(el) (502C>T). K562 erythroleukemia cells were used as the cell study model and were transfected with individual ABO expression constructs. Flow cytometry analysis was then performed to determine the levels of surface antigen expression. Using the relative percentage of antigen-expressing cells as an index for comparison, we found that the levels of A antigen expression for A(1v), A(2) (539G>C), A(3) (838C>T), A(1) (829G>A), and A(1v) (829G>A) were 65.71%, 29.78%, 45.04%, 45.2% and 33.9% of A(1), respectively. Similarly, B antigen expression for B(el) was 90.71% of B(1). The mean fluorescence index (MFI) - defined as the product of percent antigen-expressing cells and the mean cell fluorescence intensity of the population with antigen expression - was also used as an index for comparing the antigen expression level. All ABO subtypes we examined herein displayed a decrease in MFI when compared to A(1) or B(1), respectively. These data thus demonstrate that the point mutation in the coding region of ABO subtypes is fundamental to the weak antigen expression, adding to the complexity of the regulatory mechanisms of ABO gene expression.

High-resolution melting analysis is a more effective approach for screening TSC genes mutations.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with a prevalence of 1 in 95,136 in Taiwan. TSC is characterized by hamartomatous lesions in multiple organ systems. Genetic defects in TSC1 and TSC2 genes are the main causes of TSC. A molecular screening protocol using denaturing high-performance liquid chromatography (dHPLC) followed by DNA sequencing is currently performed to locate the genetic lesions in many clinical laboratories. The current screening approach is time consuming and inefficient. In this study, we analyzed all coding exons of TSC1 and TSC2 genes of 30 TSC patients and 47 unaffected family members using the traditional dHPLC protocol and our recently developed diagnostic platform based on high-resolution melting analysis (HRM) followed by bidirectional DNA sequencing. Data indicated that 20 mutations, including 5 mutations in TSC1 (2 sporadic, 1 familial mutation, and 2 of uncertain origin) and 15 mutations in TSC2 (14 sporadic and 1 familial mutation), 8 single-nucleotide polymorphisms (SNPs, including 3 SNPs found in irrelevant individuals without TSC phenotypes studied in the control group), and 3 variants with undetermined significance were identified, including 4 novel mutations. The sensitivities of HRM and dHPLC for TSC mutation screening were estimated as 95% and 75%, respectively. The specificities of HRM and dHPLC for TSC mutation screening were evaluated as 91% and 98%. In addition, results suggested our novel HRM screening protocol to be more economical. In conclusion, we successfully developed a superior approach for TSC genes mutation screening for clinical application.

Real-time biallelic polymorphism-polymerase chain reaction for chimerism monitoring of hematopoietic stem cell transplantation relapsed patients.

BACKGROUND: An accurate analysis of chimerism kinetics permits early detection of hematopoietic stem cell transplantation (HSCT) in patients with high risks of graft-versus-host disease or those liable to relapse. Although short tandem repeats-PCR (STR-PCR) is the golden standard for quantitative chimerism analysis in most of the clinical laboratories, it has a relatively low sensitivity of 5% and the detection of low percentage in mixed chimerism is usually delayed. In this study, we developed a real-time PCR for chimerism analysis based on the informative biallelic polymorphisms (BP). METHODS: The allele frequencies of 19 selective biallelic polymorphic markers were analyzed using the genomic DNA from 100 healthy Taiwanese volunteers. The informative biallelic polymorphic markers with high discrimination power in the Taiwanese population were identified. The TaqMan probe-based real-time BP-PCR for amplification of the informative loci was designed and the detection sensitivity was determined. Clinical application of real-time BP-PCR in chimerism monitoring was evaluated and was compared with the conventional STR-PCR by analyzing the DNA samples obtained at different time points post-HSCT from 4 relapsed and 10 non-relapsed patients. RESULTS: Allele distribution analysis revealed that the loci of S01a, S03, S04a, S05b, S06, S07b, S08b, S09b, S10b and S11a had a relatively high discrimination power and were the informative BP for chimerism monitoring in the Taiwanese population. Real-time BP-PCRs for these 10 BP loci were set up with the detection sensitivity equivalent to 0.003-0.006%. Real-time BP-PCR of the 4 HSCT patients revealed the presence of recipient-specific DNA at early time point than STR-PCR for 3 of the patients, whereas real-time BP-PCR was as effective as STR-PCR in uncovering the sign of relapse for one of the patients. In addition, the baseline value for the patients with no sign of relapse was 0.127 ± 0.193% of recipient DNA. CONCLUSION: We conclude that real-time BP-PCR is a sensitive and reliable method for chimerism monitoring and is superior to the STR-PCR in identifying patients who are at high risk for relapse after transplantation.

Prevalence of human papillomavirus genotypes in northern Taiwanese women.

The prevalence of Human Papillomavirus (HPV) in the general population of northern Taiwan is described. A total of 343 consecutive cervical swabs from women visiting the medical center for routine gynecologic care were included. Cervical cell cytology was examined by the Papanicolaou (Pap) test, and a PCR-based hybridization gene chip analysis was used to identify HPV genotypes. The HPV prevalence in the overall population was 32.4%. When divided into two groups according to cytology, 20.9% of women with normal cytology were HPV positive while 75.3% of women with abnormal cytology were HPV positive. Among positive samples, 68.5% were single type infections while 31.5% harbored multiple HPV types. A total of 32 types of HPV were identified; the leading five were HPV16 (5.8%), HPV58 (5.3%), HPV53 (4.1%), HPV52 (3.8%), and HPV18 (2.3%). Our results constitute baseline data and may provide important implications for future prophylactic programs. The relatively high prevalence of HPV 58, 53, and 52 among northern Taiwanese women has important implications for vaccine development.

Preoperative serum C-reactive protein and gastric cancer; clinical-pathological correlation and prognostic significance.

BACKGROUND: C-reactive protein (CRP) is a widely-used systemic biomarker for inflammation. Serum CRP is elevated in many malignancies, and is also a prognostic indicator of malignant potential. However, the prognostic significance for survival from gastric cancer has not yet been clarified. We studied the clinical- pathologic association and prognostic significance of preoperative serum CRP in gastric cancer patients. METHODS: A total of 170 gastric cancer patients were included in this study. The mean age of the patients was 65.1 years (range, 29-89), and 112 were men. All gastric cancer patients had undergone gastric resection. The serum CRP levels of patients before the operation along with those from 405 healthy controls were measured by a high sensitivity CRP test. RESULTS: The 95th percentile value (=3.0 mg/L) of the serum CRP data in 405 healthy controls was set as the upper cut-off value of the normal range. Abnormally high levels of serum CRP were observed in 65 (38.2%) of our 170 patients in contrast to only 20 (4.9%) of the 405 healthy controls (p<0.001). Elevated CRP was associated with older age (p=0.009), grossly infiltrative type (p=0.001), larger tumors (p<0.001), serosal invasion (p=0.001), lymph node metastasis (p<0.001), distant metastasis (p=0.017), and lymphatic invasion (p=0.002). Overall, a higher CRP level was strongly parallel to a pathologically more advanced stage (p=0.001). The 5-yr survival rate of patients with an elevated (>3.0 mg/L) CRP was significantly worse than those without (

Phosphorylation status of transcription factor C/EBPalpha determines cell-surface poly-LacNAc branching (I antigen) formation in erythropoiesis and granulopoiesis.

The cell-surface straight and branched repeats of N-acetyllactosamine (LacNAc) units, called poly-LacNAc chains, characterize the histo-blood group i and I antigens, respectively. The transition of straight to branched poly-LacNAc chain (i to I) is determined by the I locus, which expresses 3 IGnT transcripts, IGnTA, IGnTB, and IGnTC. Our previous investigation demonstrated that the i-to-I transition in erythroid differentiation is regulated by the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha). In the present investigation, the K-562 cell line was used as a model to show that the i-to-I transition is determined by the phosphorylation status of the C/EBPalpha Ser-21 residue, with dephosphorylated C/EBPalpha Ser-21 stimulating the transcription of the IGnTC gene, consequently resulting in I branching. Results from studies using adult erythropoietic and granulopoietic progenitor cells agreed with those derived using the K-562 cell model, with lentiviral expression of C/EBPalpha in CD34(+) hematopoietic cells demonstrating that the dephosphorylated form of C/EBPalpha Ser-21 induced the expression of I antigen, granulocytic CD15, and also erythroid CD71 antigens. Taken together, these results demonstrate that the regulation of poly-LacNAc branching (I antigen) formation in erythropoiesis and granulopoiesis share a common mechanism, with dephosphorylation of the Ser-21 residue on C/EBPalpha playing the critical role.

C-reactive protein and malignancy: clinico-pathological association and therapeutic implication.

C-reactive protein (CRP) is a widely used systemic biomarker for diagnosing acute and chronic inflammation. During the past decade, serum CRP has been re-emphasized by extending its clinical use to the prediction or diagnosis of cardiovascular diseases and other conditions, particularly malignancies. Serum CRP has also been found to be elevated in patients with many malignancies, implying a close linkage between inflammation and malignancy. Prospective studies have shown a higher risk of developing cancer in those with elevated serum CRP. CRP is produced by hepatocytes in response to inflammatory cytokines, particularly, interleukin-6 from the tumor microenvironment. Preoperative CRP levels are parallel to the progression or pathological stages of malignancies, including gastric cancer in patients in our series. Elevated CRP is a determinant predictor of lower survival rates in patients with several cancers, including esophageal, colorectal, hepatocellular, pancreatic, urinary bladder, renal,ovarian and cervical cancer, after surgical resection. The measurement of serum CRP is simple, cheap, and available in daily practice. It can serve as an additional prognostic predictor for survival and post-treatment monitoring in cancer patients. In the future, CRP-lowering agents might offer a promising benefit in the prevention and therapy of many different types of cancer.

Single-Center Experience: immunosuppressive therapy as frontline treatment for 33 children with acquired severe aplastic anemia.

The authors retrospectively analyzed the records of 33 children with acquired severe aplastic anemia (SAA) diagnosed from July 1998 to October 2007 and first treated by immunosuppressive therapy (IST). Serial hematologic parameters, complications, transfusion requirements, and time to response were assessed. Allogeneic hematopoietic stem cell transplantation (HSCT) was attempted in 7 patients after failure of IST (n = 6) or relapse following an initial response to IST (n = 1). One child died of post-transplant lymphoproliferative disorder. Thirty of the 33 patients are alive and well after a median follow-up of 45 months (range, 7-116 months). Overall (transfusion-independent) response to IST was 73% (24/33). The actuarial 5 years survival rate was 89.4%. In this study, all patients with SAA received IST as standard front-line therapy. Approximately three-fourths of patients with SAA have durable recovery and excellent overall survival.

Use of X-linked short tandem repeats loci to confirm mutations in parentage caseworks.

BACKGROUND: Analysis of short tandem repeats (STRs) has become wide-spread in routine for parentage test. However, the accuracy of STR is sometimes interfered by the presence of microsatellite mutations. Analysis of other DNA markers such as the HV1 and HV2 hypervariable regions of mitochondrial DNA or the Y-STR becomes essential to settle the noncongruence. Owing to the time-consuming nature of these tests, we explored here the use of X-chromosome STR (X-STR) to resolve the paternity and maternity disputes. METHODS: At first, the autosomal STR mutation frequencies among 4758 Taiwanese were analyzed. Population data were obtained from randomly selected 99 females and 101 males to setup the X-STR database. Two families with a mismatch of one allele in autosomal STR analysis were subjected to the X-STR test to explore its clinical application. RESULTS: The STR mutations occurred in all 15 autosomal STR loci with the exception of TH01 and TPOX. The mutation rates could reach as high as 0.106% for the loci of D8S1179 and D18S51. As to the X-STR frequencies, the probability values of exact tests for Hardy-Weinberg equilibrium were 0.1471, 0.0019, 0.0025, 0.1427, and 0.1167 for the loci of DXS7132, DXS981, DXS6789, DXS101, and HPRTB, respectively. In addition, 33 and 34 different haplotypes were revealed for DXS101-DXS6789 and DXS7132-DXS981, respectively. Furthermore, two cases with one allele mismatch in routine parentage test were resolved by performing X-STR analysis. CONCLUSION: Typing of X-STR markers is recommended for parentage test when 1 or 2 alleles mismatch is present or when the samples are difficult to be analyzed.

Second transplant with two unrelated cord blood units for early graft failure after cord blood transplantation for thalassemia.

Early GF is a frequent complication following hematopoietic stem cell transplantation for patients with thalassemia. We report the outcome of double-unit CBT in three patients who developed early GF after CBT. The initial conditioning regimen consisted of i.v. Bu 14 mg/kg (day -9 to -6), i.v. Cy 200 mg/kg (day -5 to -2) and ATG at 120 mg/kg (day -4 to -1). They received GVHD prophylaxis with cyclosporine-A from day -3 and a short course of methylprednisolone (1 mg/kg i.v., every 12 h on days 5-19 with a taper, thereafter 25% decrease every other day). The interval between two transplants was seven and 10 months. The retransplant recipients were preconditioned with i.v. Bu 14 mg/kg (day -7 to -4), i.v. Cy 120 mg/kg (day -3 to -2) and ATG at 150 mg/kg (day -5 to -1 and +1 to +5). GVHD prophylaxis regimen was the same as the first transplant. Neutrophil engraftment were observed in all patients between day +15 and +26. All are alive, between nine and 11 months after retransplant. Our group reported successful utilization of double umblical cord blood grafts in thalassemia patients with early GF.

Use of real time PCR for rapid detection of Del phenotype in Taiwan.

During routine serologic procedures, Rh D(el) blood is often not identified and subsequently labeled as RhD-negative. Recently, several reports have shown the ability of D(el) blood to induce anti-D in RhD-negative recipients. Among Korean, Japanese, and Chinese, almost all individuals with D(el) blood have a nucleotide change in the RHD gene of 1227G>A (RHDK407K). Thus, nucleotide 1227A at RHD exon 9 can be used as a marker for the D(el) phenotype in Asians. Real-time PCR for single nucleotide polymorphisms has been useful in biallelic discrimination of genomic sequence. Use of this methodology to identify 1227A will facilitate identification of blood units with the D(el) blood type and thus prevent potential sensitization of the RhD-negative recipients. In this study, real-time PCR-melting curve analysis at nucleotide 1227 of RHD exon 9 was performed on 990 leftover blood samples. PCR analysis identified 22 samples with the 1227G+A pattern, 965 samples with the 1227G pattern, and 3 with negative real-time results. The RHDEL allele frequency is 0.0116 (22/1980) among Taiwanese. These real-time PCR patterns were validated through DNA sequencing analyses of RHD exon 9 on 22 samples with the 1227G+A pattern and on 50 randomly selected samples from 1227G individuals. The real-time PCR test was then analyzed in 118 apparently RhD-negative Taiwanese donors, including 38 D(el) and 80 true RhD-negative donors, for efficiency studies. All of the D(el) samples (38, 100%) were found to have the 1227A pattern. Among the 80 serologic true RhD-negative samples, 77 were negative for real-time PCR results [1227A(-) /1227G(-)], 2 had the 1227G pattern [1227A(-)/1227G(+)], and one had the 1227A pattern [1227A(+)/ 1227G(-)]. Results of the melting curve analysis of RHD 1227A for the detection of D(el) among apparent RhD-negative individuals in Taiwan had the following characteristics: 100% sensitivity; 98.75% specificity, positive predictive value of 97.44%; negative predictive value of 100%; and an efficiency of 99.15%. Melting curve analysis using RHD 1227A for detection of D(el) phenotype can be efficiently applied in eastern Asian countries, since almost all the D(el) type in eastern Asians have the characteristic 1227A mutation.

Systematic analysis of stutters to enhance the accuracy of chimerism testing.

Post-transplantation chimerism testing is important to monitor the engraftment of donor stem cells and for the diagnosis of relapse. Detecting the presence of donor/recipient-specific short tandem repeats (STRs) is a frequently used method for engraftment study. Unfortunately, the interpretation of the STR-based chimerism tests is often subject to interference by the presence of a stutter peak, which is one 4-base repeat unit smaller than an authentic allele. The aim of this study was to systematically analyze and resolve the effect of stutter peaks on the interpretation of STR-based chimerism tests. The AmpFlSTR Identifiler Amplification kit (Applied Biosystems)was used to amplify 15 STR loci using genomic DNA from 30 randomly selected, healthy donors. We found that the stutter peaks had locus-specific characteristics. The stutter percentage was defined as the percentage of the stutter peak area/main STR peak area. Based on mean values for the 30 DNA samples, the stutter percentage varied from locus to locus and ranged from 3.12% to 10.71% for 15 STR loci. The locus-specific stutter effect can be eliminated through appropriately adjusted equations. The usefulness of these equations in the prediction of relapse was confirmed by the 5% sensitivity test. Hence, this report offers a valuable scheme to enhance the accuracy of chimerism testing.