An induced pluripotent stem cell line (MUSIi019-A) generated from a patient with distal renal tubular acidosis carrying a compound heterozygous mutation in solute carrier family 4 member 1 (SLC4A1) gene.

Distal renal tubular acidosis (dRTA), a disease characterized by the failure of the distal nephron to secrete acid into the urine, can be caused by mutations in SLC4A1 gene encoding erythroid and kidney anion exchanger 1 (AE1). Here, an induced pluripotent stem cell (iPSC) line was generated from a patient with dRTA and hemolytic anemia carrying compound heterozygous SLC4A1 mutations containing c.1199_1225del (p.Ala400_Ala408del), resulting in Southeast Asian ovalocytosis (SAO), and c.1331C>A (p.Thr444Asn). Peripheral blood mononuclear cells (PBMCs) were reprogrammed using Sendai viral reprogramming. The established iPSC line, MUSIi019-A, exhibited pluripotent property and retained the same mutations observed in the patients.

Impaired trafficking and instability of mutant kidney anion exchanger 1 proteins associated with autosomal recessive distal renal tubular acidosis.

BACKGROUND: Mutations in solute carrier family 4 member 1 (SLC4A1) encoding anion exchanger 1 (AE1) are the most common cause of autosomal recessive distal renal tubular acidosis (AR dRTA) in Southeast Asians. To explain the molecular mechanism of this disease with hematological abnormalities in an affected family, we conducted a genetic analysis of SLC4A1 and studied wild-type and mutant AE1 proteins expressed in human embryonic kidney 293T (HEK293T) cells. METHODS: SLC4A1 mutations in the patient and family members were analyzed by molecular genetic techniques. Protein structure modeling was initially conducted to evaluate the effects of mutations on the three-dimensional structure of the AE1 protein. The mutant kidney anion exchanger 1 (kAE1) plasmid construct was created to study protein expression, localization, and stability in HEK293T cells. RESULTS: We discovered that the patient who had AR dRTA coexisting with mild hemolytic anemia carried a novel compound heterozygous SLC4A1 mutations containing c.1199_1225del (p.Ala400_Ala408del), resulting in Southeast Asian ovalocytosis (SAO), and c.1331C > A (p.Thr444Asn). Homologous modeling and in silico mutagenesis indicated that these two mutations affected the protein structure in the transmembrane regions of kAE1. We found the wild-type and mutant kAE1 T444N to be localized at the cell surface, whereas the mutants kAE1 SAO and SAO/T444N were intracellularly retained. The half-life of the kAE1 SAO, T444N, and SAO/T444N mutants was shorter than that of the wild-type protein. CONCLUSION: These results suggest impaired trafficking and instability of kAE1 SAO/T444N as the likely underlying molecular mechanism explaining the pathogenesis of the novel SLC4A1 compound heterozygous mutation identified in this patient.

Cut-off values for diagnosis of G6PD deficiency by flow cytometry in Thai population.

In heterozygous females, X-inactivation causes a change in glucose-6-phosphate dehydrogenase (G6PD) activity from normal to deficient. Most G6PD screening tests are used to accurately diagnose hemizygous males, but they are less reliable for diagnosing heterozygous females. This study established flow cytometric cut-off values for screening of G6PD deficiency in hemizygous males and heterozygous or homozygous females. We studied 205 (125 females, 80 males) leftover blood samples from quantitative methemoglobin reduction (MR) screening. G6PD gene mutations determined by multiplex amplification refractory mutation system-polymerase chain reaction and direct DNA sequencing were used as the gold standard reference. Accuracy of the test, including the sensitivity, specificity, and positive and negative predictive values, was analyzed using MedCalc software. The optimal cut-off values for classification of %red blood cells with normal G6PD activity or %bright cells into homozygous normal, heterozygous, and homozygous deficiency in females were 85.4-100%, 6.3-85.3%, and 0-6.2%, respectively (sensitivity 93.2%, specificity 100%). The cut-offs for classification into hemizygous normal and hemizygous deficiency in males were 76.5-100% and 0-76.4%, respectively (sensitivity 100%, specificity 96.5%). Flow cytometry can be used to differentiate heterozygous females with intermediate phenotype from homozygous females, but cannot distinguish between heterozygous females with extreme phenotype and homozygous females. By flow cytometry, heterozygous and homozygous deficiency was detected in 29.6% and 3.2% of females, respectively. Among males, hemizygous deficiency was found in 31.3%. Flow cytometry can be used to screen patients with G6PD deficiency, and reliably and efficiently identify heterozygous and homozygous females, and hemizygous males based on cellular G6PD activity.

Adult aplastic anemia in Thailand: incidence and treatment outcome from a prospective nationwide population-based study.

The incidence and outcomes of aplastic anemia (AA) in Asia remain limited. This study aimed to explore the incidence and outcomes of patients with adult AA across the country of Thailand. This is a prospective multi-center nationwide population-based observational study of AA patients aged at least 15 years old, diagnosed from August 2014 to July 2016, with a longitudinal follow-up period over 2 years. There were 348 newly diagnosed adult AA patients during the enrollment period, giving an annual incidence of 4.6 per million. The incidence of severe (SAA) and very severe aplastic anemia (VSAA) (3.8 per million) was higher than non-severe AA (NSAA, 0.8 per million). The peak incidence was observed in the patients aged from 80 to 89 years old (14.4 per million). The 2-year overall survival (OS) in NSAA, SAA, and VSAA were 65.5%, 49.3%, and 20.1%, respectively (P < 0.001). With regard to the response to immunosuppressive therapy, the overall response rate (ORR) in SAA/VSAA treated with rabbit anti-thymocyte globulin with/without cyclosporin A (rATG ± CsA) were significantly superior to those treated with CsA alone, or anabolic steroids (44.4% vs 36.4% and 31.2%, respectively, P < 0.001). The 2-year OS in SAA/VSAA treated with rATG ± CsA, CsA, and anabolic steroids were 54.8%, 54.5%, and 37.6% (P = 0.037), respectively. The incidence of adult AA in Thailand is higher than those in Western countries, and the peak incidence is in the elderly. rATG ± CsA provided a better response than anabolic steroids, translating to the superior survival in SAA/VSAA treated with rATG ± CsA.

Defective cytokine production from monocytes/macrophages of E-beta thalassemia patients in response to Pythium insidiosum infection.

BACKGROUND: Pythium insidiosum has been mainly reported to cause morbidity and mortality in thalassemia patients. P. insidiosum zoospores can germinate to be hyphae within a few hours; therefore, it is difficult to study the initial immune response that P. insidiosum zoospores induce. The present study aims to compare immune responses against P. insidiosum zoospore infection by comparing monocytes/macrophages from thalassemia patients with those from non-thalassemia controls. METHODS: In order to keepP. insidiosum in the zoospore stage in vitro for inoculation, the P. insidiosum zoospores were preserved without germination by treatment with inorganic hypochlorite solution. CD14+ cells were isolated from peripheral blood mononuclear cells of thalassemia and non-thalassemia donors and then left to transition to macrophages. Monocytes/macrophage culture was infected with P. insidiosum zoospores and culture supernatants were subjected to Th1/Th2 multiplex cytokine detection. RESULTS: Our study of cytokine production revealed that the basal level of GM-CSF produced by thalassemia monocytes/macrophages was lower than that observed in monocytes/macrophages of non-thalassemia individuals. Higher GM-CSF and IFN-γ response was also found when cells from non-thalassemia people were stimulated with P. insidiosum zoospores compared to thalassemia cells. It was also found that TNF-α, GM-CSF and IFN-γ productions from monocytes/macrophages of thalassemia patients who received iron chelator treatment were significantly higher than those produced from thalassemia patients without iron chelator treatment. CONCLUSION: For the first time, the present study demonstrates defective immune responses in monocytes/macrophages derived from thalassemia patients in response toP. insidiosum zoospore infection. The results also show an inverse correlation between iron overload and cytokine production in monocytes/macrophages of thalassemia patients. This finding could explain why thalassemia patients are susceptible to P. insidiosum infection.

Prevalence and clinical significances of red cell alloimmunization and red cell bound immunoglobulin G in polytransfused patients with thalassemias.

The study was to determine the prevalence and clinical significances of red blood cell (RBC)-bound IgG as detected by flow cytometry in polytransfused patients with thalassemias. Relationship of the presence of RBC-bound IgG with RBC alloimmunization was also evaluated. This study included 59 polytransfused patients with ß-thalassemia disease. We studied the frequency of RBC autoantibodies and alloimmunization. Direct Coombs test and flow cytometry were performed to detect the presence of RBC autoantibodies while RBC alloantibodies were detected by antibody screening and identification assays. Eight (13.6%) and 34 (57.6%) patients were found a positive direct Coombs test and flow cytometry, respectively. Twenty (33.9%) patients developed RBC alloantibodies. The four most frequent RBC alloantibodies were anti-E (55%), anti-Mia (40%), anti-Di(a) (25%) and anti-c (15%), respectively. There was no significant difference in the presence of RBC-bound IgG between polytransfused with thalassemia patients who developed RBC alloimmunization (13 of 20; 65%) and those without RBC alloantibodies (21 of 39; 53.8%), p = 0.412. Splenectomy and increased transfusion requirement were significantly associated with the presence of RBC-bound IgG but not with RBC alloantibody formation. The overall frequency of RBC alloantibody formation in polytransfused patients with thalassemias was 33.9%. The most common RBC alloantibody was anti-E. RBC autoantibody formation was more frequently detected by flow cytometry (57.6%) than by direct Coombs test (13.6%). Splenectomy was significantly associated with the development of autoreactive RBC-bound IgG antibodies in the polytransfused patients with thalassemias. The presence of the anti-RBC autoantibodies may cause an increase of transfusion requirement.

Labile iron pool as a parameter to monitor iron overload and oxidative stress status in ß-thalassemic erythrocytes.

BACKGROUND: Labile iron pool (LIP) is intracellular nonprotein bound iron that can generate oxygen radicals via the Fenton reaction resulting in oxidative cell damage. Therefore, quantitative measurement of LIP will be helpful for detecting and monitoring the toxic iron status in iron overloaded patients. This study demonstrated LIP level and its correlation to oxidative stress status in ß-thalassemic erythrocytes. METHODS: LIP and reactive oxygen species (ROS) level, numbers of erythrocyte vesicles and apoptosis were assayed by flow cytometric methods in 30 blood samples from ß-thalassemia/hemoglobin E patients and 17 blood samples from healthy volunteers with normal hemoglobin type. RESULTS: ß-thalassemic erythrocytes showed higher LIP level, defined as the difference in calcein fluorescent intensity of the cells treated with or without deferiprone, than normal erythrocytes (mean ± 2SD as 62.39 ± 39.58 versus 44.65 ± 35.86, P = 0.003). The LIP level above 67, a cutoff value of LIP level obtained from receiver operating characteristic curve analysis, had a significant positive correlation with oxidative stress status for ROS level (r = 0.90, P < 0.001) and also the amount of erythrocyte vesicles (r = 0.79, P = 0.002). In contrast, the LIP level showed a significant negative correlation with the patients' hemoglobin level (r = -0.66, P = 0.028). CONCLUSIONS: The LIP assay is suggested as an alternative test to monitor the magnitude of iron overload and its consequent oxidative stress in ß-thalassemia. LIP level may also be used as a marker for therapeutic response to iron chelation treatment. © 2018 International Clinical Cytometry Society.

Comparison of Nested PCR and Culture Identification of Pythium insidiosum in Patients with Pythium Keratitis.

Background: Delayed diagnosis can lead to the development of endophthalmitis and blindness, which is difficult to manage because of the lack of an effective antimicrobial agent. Objective: Comparing the nested polymerase chain reaction (PCR) technique with the standard diagnostic culture method for Pythium insidiosum. Material and Method: Eighty-three patients with suspected fungal keratitis were enrolled in this observational, crosssectional study from the Faculty of Medicine Siriraj Hospital between February 2011 and February 2014. Patient symptoms, associated diseases, duration of ulcers, precipitating causes, best-corrected visual acuity, intraocular pressure, and other clinical findings were recorded. Corneal scrapings were taken for Gram staining, bacterial and fungal cultures, staining with potassium hydroxide preparation, and DNA extraction for nested PCR. The sensitivity, specificity, accuracy, and agreement of the nested PCR analysis and culture diagnosis of P. insidiosum were compared. Results: Five patients had a positive result for nested PCR amplification of P. insidiosum, while only one of these was also positive for culture growth of Pythium. Nested PCR sensitivity was 50% (95% confidence interval [95% CI] 1.3-98.7), specificity 94.7% (95% CI 86.9-98.5), and accuracy 93.5% (95% CI 85.7-97.2) with a fair agreement (kappa 0.258, p = 0.011). Conclusion: Therefore, nested PCR may be an appropriate test for P. insidiosum in diagnosing Pythium keratitis with high accuracy, despite small amounts of corneal specimen.

Ribosomal protein L11- and retinol dehydrogenase 11-induced erythroid proliferation without erythropoietin in UT-7/Epo erythroleukemic cells.

Erythropoiesis is the process of proliferation, differentiation, and maturation of erythroid cells. Understanding these steps will help to elucidate the basis of specific diseases associated with abnormal production of red blood cells. In this study, we continued our efforts to identify genes involved in erythroid proliferation. Lentivirally transduced UT-7/Epo erythroleukemic cells expressing ribosomal protein L11 (RPL11) or retinol dehydrogenase 11 (RDH11) could proliferate in the absence of erythropoietin, and their cell-cycle profiles revealed G0/G1 prolongation and low percentages of apoptosis. RPL11-expressing cells proliferated more rapidly than the RDH11-expressing cells. The antiapoptotic proteins BCL-XL and BCL-2 were expressed in both cell lines. Unlike the parental UT-7/Epo cells, the expression of hemoglobins (Hbs) in the transduced cells had switched from adult to fetal type. Several signal transduction pathways, including STAT5, were highly activated in transduced cells; furthermore, expression of the downstream target genes of STAT5, such as CCND1, was upregulated in the transduced cells. Taken together, the data indicate that RPL11 and RDH11 accelerate erythroid cell proliferation by upregulating the STAT5 signaling pathway with phosphorylation of Lyn and cyclic AMP response element-binding protein (CREB).

Acquired somatic mutations of isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) in preleukemic disorders.

Mutations of isocitrate dehydrogenase isoform 1 and 2 (IDH1 and IDH2) genes have been identified in glioblastoma and acute myeloid leukemia (AML). However, little is known about the molecular alterations of IDH genes in preleukemic disorders with a propensity to transform to AML. We performed polymerase chain reaction-denaturing high performance liquid chromatography (PCR-DHPLC) followed by direct sequencing to detect IDH mutations in 237 patients with myeloproliferative neoplasms (MPNs; n=108), myelodysplastic syndrome (MDS; n=22), paroxysmal nocturnal hemoglobinuria (PNH; n=41), and aplastic anemia (AA; n=66). No IDH1 R132 and IDH2 R172 mutations were identified in the entire cohort, whereas IDH1 G105G allele was detected in 4/108 MPN (3.70%), 2/22 MDS (9.09%), and 2/41 PNH (4.88%) patients. Three IDH2 R140Q mutations were found in 2/108 MPN (1.85%) and 1/22 MDS (4.54%) patients, while one IDH2 G145G allele was found in 0.92% (1/108) of MPN patients. Overall, our data suggest that IDH mutations are rare in the preleukemic disorders and may not be the major initial step in AML leukemogenesis.

Evaluation of nested pcr technique for detection of Pythium insidiosum in pathological specimens from patients with suspected fungal keratitis.

Diagnosis of Pythium keratitis is problematic due to the difficulty in obtaining a culture report resulting in unnecessarily prolonged usage of antimicrobial medication due to misdiagnosis. This study evaluated and compared nested PCR technique with culture and immunoperoxidase staining assays of Pythium insidiosum in paraffin-embedded corneal tissues from patients with suspected fungal keratitis. Six of 51 pathological reports compatible with fungal infection and 6 of 48 culture-proven fungal keratitis were identified as Pythium. Twenty-seven specimens were PCR-positive for Pythium insidiosum. In comparison with fungal culture for P. insidiosum, PCR had 83% sensitivity and 77% specificity with fair agreement (Kappa score of 0.227, p = 0.001). The mean age of PCR-positive is younger than PCR-negative group and there is a female preponderance in Pythium-infected group (p = 0.002 and p = 0.004, respectively). Nineteen specimens had positive results using immunoperoxidase staining assay with fair agreement to culture method (Kappa 0.340, p < 0.001), and 83% sensitivity, 85% specificity and 85% accuracy (95% CI: 76.7-90.7). PCR-based technique compared with culture and/or immunoperoxidase staining assay had 91.7% sensitivity, 81.8% specificity and 83% accuracy (95% CI: 74.5-89.1) with moderate agreement (Kappa 0.477, p < 0.001). Thus nested PCR detection of P. insidiosum should be employed in preliminary diagnosis of Pythium keratitis in order to initiate proper management.

Mechanism of paroxysmal nocturnal hemoglobinuria clonal dominance: possible roles of different apoptosis and CD8+ lymphocytes in the selection of paroxysmal nocturnal hemoglobinuria clones.

BACKGROUND AND OBJECTIVES: Paroxysmal nocturnal hemoglobinuria (PNH), a clonal hematopoietic stem cell disorder, manifests when the PNH clone populates in the hematopoietic compartment. We explored the roles of different apoptosis of GPI+ and GPI- (glycosylphosphatidylinositol) cells and CD8+ lymphocytes in a selection of PNH clones. PATIENTS AND METHODS: Granulocytes from PNH patients and normal controls were subjected to an apoptosis assay using annexin V. Hematopoietic cell in semisolid media were cultured with or without CD8+ lymphocytes. RESULTS: In PNH, CD59+ granulocytes exhibited more apoptosis than their CD59- counterparts, after 0 or 4 hours in liquid growth culture system (mean [standard error of mean]: 2.1 (0.5) vs 1.2 (0.2), P=.01 at 0 hour and 3.4 [0.7] vs 1.8 [0.3], P=.03 at 4 hour, respectively). The presence of mononuclear cells (MNCs) rendered a greater difference in apoptosis. The percentages of apoptotic CD59+ granulocytes measured at 4 hours with or without MNC fraction were correlated with the sizes of PNH clones (r=0.633, P=.011; and r=0.648, P=.009; respectively). The autologous CD8+ lymphocytes inhibited CFU-GM and BFU-E colony formation in PNH patients when compared with normal controls (mean [SEM] of percentages of inhibition: 61.7 (10.4) vs 11.9 (2.0), P=.008 for CFU-GM and 26.1 (6.9) vs 4.9 (1.0), P=.037 for BFU-E). CONCLUSIONS: Increased apoptosis of GPI+ blood cells is likely to be responsible in selection and expansion of PNH clones. MNCs or possibly CD8+ lymphocytes may play a role in this phenomenon.

Vascular pythiosis in a thalassemic patient presenting as bilateral leg ulcers.

Human pythiosis is an emerging disease caused by Pythium insidiosum, a fungus-like aquatic organism. Clinical presentations can be classified into four types: (i) cutaneous/subcutaneous, (ii) ocular, (iii) vascular, and (iv) disseminated pythiosis. Serological tests such as immunodiffusion and immunochromotographic test are useful to make rapid diagnosis in cutaneous and vascular pythiosis. We report a case of 35 year-old male with vascular pythiosis of both legs, diagnosed by serology and molecular techniques.

The efficacy of 3-mg warfarin initiating dose in adult Thai patients, who required long-term anticoagulant therapy.

BACKGROUND: Warfarin anticoagulation is the standard treatment for patients with thromboembolic diseases. Prior studies recommended commencing warfarin with the initial doses between 5 mg and 10 mg for the first 1 or 2 days. However, lower warfarin loading dose is advised for the elderly and patients with co-morbid diseases. Moreover, warfarin requirement is also affected by several genetic factors, which differ among various ethnic populations. Currently, the optimal initiating dose of warfarin in Thai patients is unknown. However, based on the observation of the clinical practice at Siriraj hospital, a lower starting dose (3 mg/day) of warfarin was commonly given to patients who required long-term anticoagulant therapy. OBJECTIVE: To investigate the efficacy and safety of 3-mg warfarin initiating dose. MATERIAL AND METHOD: A retrospective study of inpatients who received warfarin 3 mg/day for the first two days of oral anticoagulation therapy with the target INR of 2.0-3.0 at Siriraj hospital from January 2004-December 2007 was performed. The efficacy of 3-mg warfarin loading dose was determined by assessing the proportion of patients who achieved the target INR of 2.0-3.0 between day 3 and day 5 of warfarin treatment. RESULTS: Total of 164 patients was included in the study. Eighty-six patients (52.4%) were males. The mean age was 55.1 + 16.8 years (range 16-88 years). The mean body weight and serum albumin were 61.5 +/- 12.2 kg and 3.7 +/- 0.7 g/dl, respectively. Prosthetic heart valve replacement was the most common indication for warfarin anticoagulation therapy (36%), followed by deep vein thrombosis (32.3%). The mean cumulative weekly dose of warfarin was 22.3 +/- 5.8 mg. The median time to therapeutic INR (2.0-3.0) was 6 days. Forty-seven patients (29%) achieved therapeutic INR between day 3 and day 5 of warfarin treatment. Time to therapeutic INR was not affected by age, gender, body weight, serum albumin, or concomitant medication use. Interestingly, patients who received warfarin due to prosthetic heart valve replacement were more likely to achieve therapeutic INR between day 3 and day 5 when compared to those with other indications with adjusted OR 16.25 (95% CI 5.13-51.44, p < 0.001). Bleeding complication was rare (0.6%) and was not associated with excessive anticoagulation. CONCLUSION: 3-mg warfarin initiating dose appeared to be safe in adult Thai patients. However, the efficacy of 3-mg starting dose as determined by the proportion of patients who achieved the target INR between day 3 and day 5 of warfarin treatment was relatively less efficient when compared with that previously reported with a 5-mg loading dose. Further randomized, prospective study is required to examine the efficacy of 3-mg versus higher warfarin starting dose in Thai patients.

Chronic nonspherocytic hemolytic anemia due to glucose-6-phosphate dehydrogenase deficiency: report of two families with novel mutations causing G6PD Bangkok and G6PD Bangkok Noi.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common hereditary enzymopathies worldwide. Mostly G6PD deficient cases are asymptomatic though they may have the risk of neonatal jaundice (NNJ) and acute intravascular hemolysis during oxidative stress. Chronic nonspherocytic hemolytic anemia (CNSHA) due to G6PD deficiency is rare. In Thailand, one case was reported 40 years ago and by biochemical study this G6PD was reported to be a new variant G6PD Bangkok. We, herein, report two families with CNSHA due to G6PD deficiency. In the first family, we have been following up the clinical course of the patient with G6PD Bangkok. In addition to chronic hemolysis, he had three acute hemolytic episodes requiring blood transfusions during childhood period. Multiple gallstones were detected at the age of 27. His two daughters who inherited G6PD Bangkok from him and G6PD Vanua Lava from his wife are asymptomatic. Both of them had NNJ and persistent evidences of compensated hemolysis. Molecular analysis revealed a novel missense mutation 825 G→C predicting 275 Lys→Asn causing G6PD Bangkok. In the second family, two male siblings are affected. They had NNJ and several hemolytic episodes which required blood transfusions. On follow-up they have been diagnosed with chronic hemolysis as evidenced by reticulocytosis and indirect hyperbilirubinemia. Molecular analysis revealed combined missense mutations in exons 12 and 13. The first mutation was 1376 G→T predicting 459 Arg→Leu (known as G6PD Canton) and the second one was 1502 T→G predicting 501 Phe→Cys. We designated the resulting novel G6PD variant, G6PD Bangkok Noi.

Development of flow cytometry for detection and quantitation of red cell bound immunoglobulin G in autoimmune hemolytic anemia with negative direct Coombs test.

About 2-10% of patients with warm-antibody autoimmune hemolytic anemia (WAIHA) exhibit a negative direct Coombs test (DAT), requiring more sensitive tests, including detection of RBC-bound immunoglobulins by flow cytometry, for diagnosis. In this study, the optimal conditions for detection and quantitation of RBC-bound IgG by flow cytometry were studied using blood samples from six patients with AIHA and two healthy individuals. Quantitation of RBC-bound IgG was performed using quantum simply cellular (QSC) beads coated with goat anti-mouse IgG antibodies. For detection of RBC bound IgG, a 60-minute incubation of all blood samples with 40 microl of 1:10 dilution of FITC-conjugated mouse anti-human IgG gave mean fluorescent intensity (MFI) values comparable to experiments using larger amounts or higher concentrations of the anti-human IgG. The acquired antibody binding capacity (ABC) values (or IgG molecules) for each QSC bead level, at 40 microl of 1:5 and 1:10 dilution of anti-human IgG for 60 minutes were close to the manufacturer-assigned ABC values. The IgG molecules per RBC in all six patients with positive DAT of 4+, 3+, 2+, 1+, trace and negative DAT were 31,725, 3,823, 1,753, 524, 260 and 88 respectively and in two healthy individuals with negative DAT they were 104 and 78.

Incidence and risk factors of relapses in idiopathic autoimmune hemolytic anemia.

BACKGROUND: Patients with idiopathic autoimmune hemolytic anemia (AIHA) of warm antibody type usually respond to corticosteroid therapy. However a proportion of patients will have disease relapse after steroid-induced remission. OBJECTIVE: To assess the incidence and the possible risk factors of the relapse in a cohort of patients with idiopathic AIHA. MATERIAL AND METHOD: We conducted a retrospective and prospective study of 34 idiopathic AIHA patients regularly followed at the Division of Hematology during January 1973 to December 2006. The medical records were reviewed for active hemolytic events and relapses, episodes of infections, pattern of corticosteroids administration and tapering. Types and subtypes of autoantibodies were studied by column agglutination test (the "gel test"). RESULTS: One patient with cold agglutinin disease was excluded, leaving a total of 33 patients (24 with warm type, 9 with mixed warm and cold type AIHA) in the study. The incidence of relapse was 1.157 episodes/person/year. The mean duration of relapse after remission was 23 months. Episodes of recurrent hemolysis were more frequent when corticosteroid administration was tapered from high to low dose (10 mg/day of prednisolone) within two months compared with a longer than two-month tapering (38 vs. 11 episodes; p < 0.01). In addition patients receiving continuing low dose of corticosteroids (< or = 10 mg/day of prednisolone) for > 6 months had lower incidence of relapse and longer duration of remission than those with discontinuing the medication within 6 months (0.443 vs. 1.911 episodes/person/year, p < 0.01; 37.4 vs. 10.6 months, p < 0.01). Episodes of recurrent hemolysis were more frequently observed in patients with events of infection than those without infections (mean 7.69 vs. 2.81, p = 0.032). Types and subtypes of autoantibodies did not seem to influence relapse in AIHA. CONCLUSION: Short duration of maintenance and rapid tapering of corticosteroids and infections are possible risk factors of relapses/recurrent hemolysis in idiopathic AIHA.

Random amplified polymorphic DNA typing and phylogeny of Pythium insidiosum clinical isolates in Thailand.

Forty-three Pythium insidiosum clinical isolates recovered from human pythiosis cases in Thailand were characterized by random amplified polymorphic DNA (RAPD) analysis. Three random oligonucleotide primers, OPW11, OPW12 and OPX13 generated 39, 34 and 35 DNA patterns with high value of typeability (100%), reproducibility (98.5, 88.8 and 93.3%) and discriminatory power (0.83, 0.82 and 0.77), respectively. Using GelCompar software based on band similarity, the 43 clinical isolates of P. insidiosum could be arranged into 9, 13 and 11 clades using OPW11, OPW12 and OPX13, respectively and the combination of all three primers revealed 36 RAPD patterns. Members in each RAPD pattern varied in both clinical forms and/or geographical locations. RAPD pattern 15 was found in 6 isolates, half of which were found in central region of Thailand. Isolates MCC15 and MCC16 isolated from different patients exhibited identical pattern with all three primers. Our results revealed high genetic heterogeneity among Pythium insidiosum isolates in Thailand. RAPD method should be appropriate for future epidemiological studies of P. insidiosum strains from patients and from natural habitats.

Clinical and epidemiological analyses of human pythiosis in Thailand.

BACKGROUND: Pythiosis is an emerging and life-threatening infectious disease in humans and animals that is caused by the pathogenic oomycete Pythium insidiosum. Human pythiosis is found mostly in Thailand, although disease in animals has been increasingly reported worldwide. Clinical information on human pythiosis is limited, and health care professionals are unfamiliar with the disease, leading to underdiagnosis, delayed treatment, and poor prognosis. METHODS: To retrospectively study the clinical and epidemiological features of human pythiosis, we analyzed clinical data from patients with pythiosis diagnosed during the period of January 1985 through June 2003 at 9 tertiary care hospitals throughout Thailand. RESULTS: A total of 102 cases of human pythiosis were documented nationwide. A substantial proportion (40%) of cases occurred in the last 4 years of the 18-year study interval. Clinical presentations fell into 4 groups: cutaneous/subcutaneous cases (5% of cases), vascular cases (59%), ocular cases (33%), and disseminated cases (3%). Almost all patients with cutaneous/subcutaneous, vascular, and disseminated pythiosis (85%) had underlying thalassemia-hemoglobinopathy syndrome. Most ocular cases (84%) were associated with no underlying disease. A majority of the patients were male (71%), were aged 20-60 years (86%), and reported an agricultural occupation (75%). Regarding treatment outcomes, all patients with disseminated infection died; 78% of patients with vascular disease required limb amputation, and 40% of these patients died; and 79% of patients with ocular pythiosis required enucleation/evisceration. CONCLUSIONS: Here, we report, to our knowledge, the largest case study of human pythiosis. The disease has high rates of morbidity and mortality. Early diagnosis and effective treatment are urgently needed to improve clinical outcomes. Because P. insidiosum is distributed worldwide and can infect healthy individuals, an awareness of human pythiosis should be promoted in Thailand and in other countries.

A cohort study of the nature of paroxysmal nocturnal hemoglobinuria clones and PIG-A mutations in patients with aplastic anemia.

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the clonal expansion of blood cells, which are deficient in glycosylphosphatidylinositol anchored proteins (GPI-APs). As PNH frequently occurs during the clinical course of acquired aplastic anemia (AA), it is likely that a process inducing bone marrow failure in AA is responsible for the selection of GPI-AP deficient blood cells or PNH clone. OBJECTIVE: To explore the nature and mutation of a PNH clone in AA. METHODS: We performed regular repeated flow cytometric analyses of CD59 expression on peripheral blood cells from a cohort of 32 patients with AA. Mutation of phosphatidylinositol glycan class A (PIG-A) was also studied. RESULTS: Fifty-one episodes of occurrences of CD59 negative granulocytes out of a total cohort 167 flow cytometric analyses (31%) were observed in 22 patients (69%). CD59 negative erythrocytes were less apparent than the granulocytes. Repeated occurrences of PNH clones were observed in 16 patients. Most of the emerging PNH clones were transient in nature. They were more frequently detected during episodes of lower white blood cell and platelet counts. Persistence and expansion of the GPI-AP deficient blood cell populations to the level of clinical PNH were seen in only four patients (12.5%). Analysis of PIG-A gene demonstrated eight mutations among the four patients, with two and four independent mutations in two patients. CONCLUSIONS: Our study indicates that PIG-A mutations of hematopoietic stem cells with resultant PNH clones, are relatively common among AA patients. It also supports the hypothesis of selection of the PNH clone by a process or condition associated with or responsible for bone marrow failure in AA. However, there must be an additional factor favoring expansion or growth of the clone to the level of clinical or florid PNH.