Thrombopoietin-independent generation of platelet-like particles from megakaryoblastic cells.

The use of megakaryoblastic leukemia MEG-01 cells can help reveal the mechanisms of thrombopoiesis. However, conventional in vitro activation of platelet release from MEG-01 cells requires thrombopoietin, which is costly. Here, we aim to develop a more straightforward and affordable method. Synchronization of the MEG-01 cells was initially performed using serum-free culture, followed by spontaneous cell differentiation in the presence of serum. Different stages of megakaryoblast differentiation were classified based on cell morphology, DNA content, and cell cycle. The MEG-01 cells released platelet-like particles at a level comparable to that of the thrombopoietin-activated MEG-01 cells. The platelet-like particles were distinguishable from PLP-derived extracellular vesicles and could express P-selectin following ADP activation. Importantly, the platelet-like particles induced fibrin clotting in vitro using platelet-poor plasma. Therefore, this thrombopoietin-independent cell synchronization method is an effective and straightforward method for studying megakaryopoiesis and thrombopoiesis.

Diagnostic Performance of 10 Mathematical Formulae for Identifying Blood Donors with Thalassemia Trait.

OBJECTIVE: To compare the diagnostic performance of 10 mathematical formulae for identifying thalassemia trait in blood donors. METHODS: Compete blood counts were conducted on peripheral blood specimens using the UniCel DxH 800 hematology analyzer. Receiver operating characteristic curves were used to evaluate the diagnostic performance of each mathematical formula. RESULTS: In the 66 donors with thalassemia and 288 subjects with no thalassemia analyzed, donors with thalassemia trait had lower values for mean corpuscular volume and mean corpuscular hemoglobin than subjects without thalassemia donors (77 fL vs 86 fL [P < .001]; 25 pg vs 28 pg [P < .001]). The formula developed by Shine and Lal in 1977 showed the highest area under the curve value, namely, 0.9. At the cutoff value of <1812, this formula had maximum specificity of 82.35% and sensitivity of 89.58%. CONCLUSIONS: Our data indicate that the Shine and Lal formula has remarkable diagnostic performance in identifying donors with underlying thalassemia trait.

Glucose-6-phosphate dehydrogenase is dispensable for human erythroid cell differentiation in vitro.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency impairs cellular processes under oxidative stress. Individuals with severe G6PD deficiency still produce sufficient numbers of erythrocytes. Nevertheless, the G6PD independence of erythropoiesis remains questionable. This study elucidates the effects of G6PD deficiency on the generation of human erythrocytes. Peripheral blood-derived CD34-positive hematopoietic stem and progenitor cells (HSPCs) of human subjects with normal, moderate, and severe G6PD activities were cultured in two distinct phases: erythroid commitment and terminal differentiation. Regardless of G6PD deficiency, HSPCs were able to proliferate and differentiate into mature erythrocytes. There was no impairment in erythroid enucleation among the subjects with G6PD deficiency. To our knowledge, this study is the first report of effective erythropoiesis independent of G6PD deficiency. The evidence firmly indicates that the population with the G6PD variant could produce erythrocytes to an extent similar to that in healthy individuals.

Urinary red blood cell-derived microparticles and phosphatidylserine-exposing red blood cells in glomerular and non-glomerular hematuria patients.

BACKGROUND: Distinguishing glomerular hematuria (GH) from non-glomerular hematuria (NGH) is important for treating the cause of hematuria. We aimed to determine red blood cell-derived microparticles (RMPs) and phosphatidylserine (PS)-exposing red blood cells (RBCs) and evaluate their use for diagnosing GH and NGH patients. METHODS: All patients received a physical assessment and urological examination. Dysmorphic RBCs (dRBCs) and acanthocytes were examined using a light microscope. The urinary RMPs and PS-exposing RBCs were determined using flow cytometry. RESULTS: The ratio of RMPs to RBCs was higher in GH patients (n = 29) than in NGH patients (n = 29) (1.06 vs. 0.18). The value of the sum of the PS-exposing RBCs plus RMPs divided by the number of RBCs was higher in GH patients than in NGH patients (48.3% vs. 19.4%). The percentage of RBCs was higher in GH patients than in NGH patients (54.5% vs. 21.8%). Similarly, both the percentages of acanthocytes and of non-acanthocytes were higher in GH patients than in NGH patients (29% vs. 7.7% and 25.4% vs. 14.2%, respectively). The ROC-AUC of the number of PS-exposing RBCs plus RMPs divided by the number of RBCs was 0.9 (95% CI, 0.82-0.97), and the RMPs:RBCs ratio was 0.88 (95% CI, 0.79-0.98). The ROC-AUCs of the dRBCs and acanthocytes were 0.85 (95% CI, 0.78-0.95) and 0.88 (95% CI, 0.8-0.97), respectively. CONCLUSIONS: Patients with GH have higher numbers of urinary RMPs and PS-exposing RBCs. These parameters have the potential to be predictive tools for classifying GH in the future.

Impairment of Invasion and Maturation and Decreased Selectivity of Plasmodium falciparum in G6PD Viangchan and Mahidol Variants.

BACKGROUND: Protection against Plasmodium falciparum is observed in a population deficient in glucose-6-phosphate dehydrogenase (G6PD), particularly in African and Mediterranean regions. However, such protection remains unknown among G6PD-deficient individuals in Southeast Asia. METHODS: In this study, we assessed the invasion and maturation of P falciparum K1 in a culture of erythrocytes isolated from Thai subjects carrying Viangchan (871G > A) and Mahidol (487G > A). RESULTS: We found that the parasites lost their ability to invade hemizygous and homozygous G6PD-deficient erythrocytes of Viangchan and Mahidol variants in the second and third cycles of intraerythrocytic development. It is interesting to note that P falciparum parasites selectively grew in erythrocytes from hemi- and homozygous genotypes with normal G6PD activity. Moreover, externalization of phosphatidylserine upon P falciparum infection was significantly increased only in Viangchan hemizygous variant cells. CONCLUSIONS: This study is the first to show that blockage of invasion in long-term culture and potentially enhanced removal of parasitized erythrocytes were observed for the first time in erythrocytes from Viangchan and Mahidol G6PD-deficient individuals.

Storage Duration and Red Blood Cell-Derived Microparticles in Packed Red Blood Cells Obtained from Donors with Thalassemia.

OBJECTIVE: To address the effects of storage duration on red blood cell (RBC)-derived microparticles (RMPs) in packed RBCs from donors who have thalassemia. MATERIALS AND METHODS: Packed RBCs were prepared according to laboratory routine. The quantity of RMPs was determined using FACSCalibur and counting beads. RESULTS: Across durations of storage, the packed RBCs from donors with thalassemia (n = 28) and healthy volunteers (n = 104) showed average RMPs to be 47,426 (10,139‒127,785) particles/µL vs 49,021 (13,033‒126,749) particles/µL, respectively (P = .63). The peak RMP levels in donors with thalassemia and healthy volunteers, respectively, were shown in products from storage days 34 and 38. Both groups showed a trend toward a positive association between RMP concentration and the duration of storage in packed RBC bags stored under blood bank conditions. CONCLUSION: Our results suggest that storage-induced RMP release has similar effects in stored packed RBCs obtained from both donors with thalassemia and healthy volunteers.

G6PD diaxBox: Digital image-based quantification of G6PD deficiency.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in humans. More than 400 million people worldwide are affected by this genetic condition. Testing for G6PD deficiency before drug administration is essential for patient safety. Rapidly ascertaining the G6PD status of a person is desirable for proper treatment. The device described in this study, the G6PD diaxBOX, was developed to quantify G6PD deficiency using paper-based analytical devices (PADs) and a colorimetric assay. The G6PD diaxBOX is a straightforward, affordable, portable, and instrument-free analytical system. The major components of the G6PD diaxBox are a banknote-checking UV fluorescent lamp and camera that are easy to access and analysis software. When NADPH is generated, it absorbs at UV 340 nm and emits colored light that is detected with the camera. The determined Pearson's coefficient shows that the color intensity measured from the G6PD diaxBOX correlated with G6PD activity level. Also, a Bland-Altman analysis indicated that more than 95% of the measurement error was in the upper and lower boundaries (±2 SD) and the error from the severe and moderate deficiency group was less than ± 1 SD. Therefore, the error from G6PD diaxBOX was within the limit boundary and the overall accuracy was more than 80%. The G6PD diaxBOX facilitates the effective and efficient quantification of G6PD deficiency and as such represents a clinically well-suited, rapid point-of-care test.

Cell-Derived Microparticles in Blood Products from Blood Donors Deficient in Glucose-6-Phosphate Dehydrogenase.

OBJECTIVE: To quantitate the microparticles (MPs) in whole blood and blood products obtained from blood donors who are deficient in glucose-6-phosphate dehydrogenase (G6PD). METHODS: The current study analyzed whole blood and blood components prepared from 49 blood donors with G6PD deficiencies and 98 with G6PD-normal results. Packed red blood cells (PRBCs), platelet concentrate (PC), and plasma were prepared according to transfusion laboratory procedures. MP concentrations were determined using a flow cytometer. RESULTS: Blood components prepared from donors with G6PD deficiency were characterized by higher red blood cell-derived MP (RMP) concentration in PRBCs (25,526 vs 18,738 particles/µL) but lower concentrations of platelet-derived MPs (PMPs; in whole blood and PC), leukocyte-derived MPs (LMP; in whole blood and plasma) and total MP (in PC), compared with those from donors with G6PD-normal test results. CONCLUSIONS: These results suggest that differences in G6PD status may account for variation in RMP levels during processing.

Cell-Derived Microparticles in Blood Products from Thalassemic Blood Donors.

OBJECTIVE: To determine the number of cell-derived microparticles (MPs) in blood products obtained from donors who have thalassemia. METHODS: Packed red blood cells (PRBCs), plasma, and platelet concentrate (PC) were prepared according to routine procedures. We used flow cytometry to quantitate the concentration of MPs. RESULTS: The results of a comparison of MP levels in unprocessed whole blood showed that the concentration of all MPs in the donors without thalassemia trait (n = 255) was higher than in donors with thalassemia trait (n = 70). After processing, increased concentrations of MPs were documented in both groups. Among the blood components, PRBC showed higher platelet-derived MP concentrations in donors with thalassemia than in donors without thalassemia. However, PC showed higher concentrations of total MPs in donors without thalassemia than in donors with that condition. CONCLUSIONS: Our results suggest little influence of thalassemia-trait status on changes in MP concentrations in blood components.

Prevalence and Subtype Distribution of Blastocystis Infection in Patients with Diabetes Mellitus in Thailand.

Diabetes mellitus (DM) is a major global public health problem with an increasing prevalence. DM increases the risk of infections caused by bacteria, fungi, viruses, and parasites. We examined the prevalence, subtypes, and risk factors of Blastocystis infection in patients with and without DM in central Thailand. Stool samples and questionnaires were obtained from 130 people in the DM group and 100 people in the non-DM group. Blastocystis infection was identified via a nested polymerase chain reaction and subtyped via sequencing of the partial small-subunit ribosomal RNA (SSU rRNA) gene. Analysis of potential risk factors was conducted via binary logistic regression. The overall prevalence of Blastocystis infection was 10.8%, including rates of 9% and 12.3% in the non-DM and DM groups, respectively. The most prevalent subtype was ST3, followed by ST1, and ST4. Factors that potentially increased the risk of Blastocystis infection include patients being >65 years old, the presence of DM, a DM duration of ≥10 years, a low level of education, and animal ownership. In conclusion, this is the first study of Blastocystis infection in DM, and a high prevalence was found among this population. Therefore, health education promoting sanitation and hygiene is necessary to reduce and prevent infection in the community.

Quantitation of phosphatidylserine-exposing platelets in platelet concentrate prepared in routine blood transfusion laboratory.

BACKGROUND: Phosphatidylserine (PS) plays important roles in platelets' pro-coagulant function. However, little is known about assessing this molecule in platelet concentrates (PCs) prepared for routine blood transfusion service. AIM: To quantitate the number of PS-exposing platelets in PCs prepared in a routine transfusion laboratory. METHODS: PC products were prepared according to routine laboratory procedure. The numbers of PS-exposing platelets in the PCs and in unprocessed whole blood were determined using flow cytometry. RESULTS: A cross-sectional study of 253 PCs found that they had significantly increased numbers of PS-exposing platelets compared to unprocessed whole blood (47,439 ±â€¯26,500 cells/µL; 5903‒166,156 cells/µL) vs. 30,058 ±â€¯12,958 cells/µL; 8,154-86,606 cells/µL). A heterogeneity study demonstrated that 6% and 2% of the measured PCs and of unprocessed donor whole blood, respectively, showed an increase in the number of PS-exposing platelets that was greater than 2 fold. CONCLUSIONS: The study suggested that the number PS-exposing platelets in PC prepared in a routine transfusion laboratory differs. However, assessment of the number of PS-exposing platelets in platelet products could be a valid measure to use in managing the quality of platelet processing in routine laboratories.

Identification of Enterocytozoon bieneusi in goats and cattle in Thailand.

BACKGROUND: Enterocytozoon bieneusi has been increasingly reported to infect domestic animals and humans, with human infections primarily reported as zoonotic in origin. The aim of the present study was to determine the presence and genotype of E. bieneusi in humans and domestic animals in central Thailand by testing stool samples of 200 apparently healthy humans, 73 goats, 60 cattle and 65 pigs using nested-PCR/ sequence analysis based on the ITS region of SSU rRNA genes. RESULTS: E. bieneusi tested positive in 2 (1%) of the 200 stool samples collected from humans and 56 (28.3%) of the 198 stool samples collected from domestic animals. The highest prevalence of E. bieneusi was observed in pigs (39/65, 60%), followed by goats (14/73, 19.2%) and cattle (3/60, 5%). Seven novel E. bieneusi genotypes were identified, which were named GoatAYE1-4 and PigAYE1-3 and clustered in either zoonotic Group 1 or Group 2. Moreover, eleven previously described E. bieneusi genotypes were also identified (O, D, H, SX1, CHC8, CHG3, CS-10, SHZC1, LW1, WildBoar5, and EbpC). All novel genotypes exhibited zoonotic potential from a phylogenetic analysis of ITS region. CONCLUSION: Our data showed that the prevalence of E. bieneusi is low in apparently healthy individuals and higher in pigs than cattle and goats. This study provides baseline data useful for controlling and preventing E. bieneusi infection in farm communities, where pigs and goats appear to be the major reservoir of E. bieneusi. The results of our study support the view that E. bieneusi is a zoonotic pathogen that should be considered a potential public health threat.

Thalassemia trait and G6PD deficiency in Thai blood donors.

BACKGROUND: Thalassemia trait and G6PD deficiency are asymptomatic and volunteers with these variants are eligible for blood donation. AIMS: This study aimed to investigate prevalence and hematologic profiles of blood donors with thalassemia trait and G6PD deficiency and the influence of these abnormalities have on donor retention and blood component preparation. METHODS: Prospectively recruited blood donors were investigated for thalassemia and G6PD deficiency. Characteristic data, hematologic profiles, proportions of prepared blood components, donor return rate within 12 months and adverse reactions in patients receiving red cell transfusions were compared among thalassemia trait, G6PD deficiency, and normal donors. RESULTS: In Thai blood donors, thalassemia trait prevalence was 21.1% and G6PD deficiency prevalence based on G6PD activity was 7.7%. Blood donors with thalassemia trait had significantly lower hemoglobin, MCV, and MCH than blood donors without thalassemia trait (Hb 13.55 ± 1.00 vs. 13.96 ± 1.25 g/dL, MCV 76.70 ± 6.69 vs. 87.01 ± 5.10 fL, and MCH 25.06 ± 2.17 vs. 28.67 ± 1.91 pg, all respectively and all p < 0.01). However, the hematologic profiles of blood donors with G6PD deficiency were not significantly different from the hematologic profiles of blood donors with normal G6PD activity. No significant difference was observed among thalassemia trait, G6PD deficiency, and normal donors relative to donor retention and blood component preparation. CONCLUSION: The high prevalence of thalassemia trait and G6PD deficiency in Thai blood donors observed in this study does not adversely affect donor retention and blood component preparation.

Effects of donor age, donor sex, blood-component processing, and storage on cell-derived microparticle concentrations in routine blood-component preparation.

BACKGROUND: A number of factors cause increases in the number of cell-derived microparticles (MPs) in blood components. However, the overall effects of these factors on the concentration of MPs during routine blood-component preparation have not fully been elucidated. AIM: To evaluate the effects of donor age, donor sex, blood-component preparation, and storage on MP concentrations. METHODS: Flow cytometry was used to quantitate the number of whole blood-derived MPs. RESULTS: The total MP concentration was similar in male and female donors (26,044 ± 1254 particles/µL vs. 27,696 ± 1584 particles/µL). The total MP concentration did not differ significantly among the different age groups: 18-30 years (28,730 ± 1600 particles/µL), 31-40 years (24,972 ± 5947 particles/µL), and 41-58 years (25,195 ± 1727 particles/µL). However, the total number of MPs in fresh plasma (152,110 ± 46,716 particles/µL) was significantly higher (p < 0.05) than that in unprocessed whole blood (26,752 ± 985 particles/µL), fresh packed red blood cells (PRBCs) (28,574 ± 1028 particles/µL), and platelet concentrate (PC) (33,072 ± 1858 particles/µL). Furthermore, the total numbers of MPs in stored PRBCs and fresh-frozen plasma (FFP) were significantly higher (p < 0.05) than those in fresh PRBCs and fresh plasma, respectively. CONCLUSIONS: The study suggests that donor factors, blood-component processing and storage contribute to the MP concentration in routine blood-product preparation. The findings can improve quality control and management of blood-product manufacturing in routine transfusion laboratories.

Flow rate calibration to determine cell-derived microparticles and homogeneity of blood components.

BACKGROUND: Cell-derived microparticles (MPs) are currently of great interest to screening transfusion donors and blood components. However, the current approach to counting MPs is not affordable for routine laboratory use due to its high cost. AIM: The current study aimed to investigate the potential use of flow-rate calibration for counting MPs in whole blood, packed red blood cells (PRBCs), and platelet concentrates (PCs). METHODS: The accuracy of flow-rate calibration was investigated by comparing the platelet counts of an automated counter and a flow-rate calibrator. The concentration of MPs and their origins in whole blood (n=100), PRBCs (n=100), and PCs (n=92) were determined using a FACSCalibur. The MPs' fold-changes were calculated to assess the homogeneity of the blood components. RESULTS: Comparing the platelet counts conducted by automated counting and flow-rate calibration showed an r2 of 0.6 (y=0.69x+97,620). The CVs of the within-run and between-run variations of flow-rate calibration were 8.2% and 12.1%, respectively. The Bland-Altman plot showed a mean bias of -31,142platelets/µl. MP enumeration revealed both the difference in MP levels and their origins in whole blood, PRBCs, and PCs. Screening the blood components demonstrated high heterogeneity of the MP levels in PCs when compared to whole blood and PRBCs. CONCLUSIONS: The results of the present study suggest the accuracy and precision of flow-rate calibration for enumerating MPs. This flow-rate approach is affordable for assessing the homogeneity of MPs in blood components in routine laboratory practice.

A new paper-based analytical device for detection of Glucose-6-phosphate dehydrogenase deficiency.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a genetic haemolytic disorder. Most persons with G6PD deficiency are asymptomatic, but exposure to oxidant drugs, such as the anti-malarial drug primaquine, may induce haemolysis, which is commonly found in Asian countries. A reliable test is necessary for diagnosing the deficiency to prevent an acute haemolytic crisis. This study proposes a novel quantitative method to detect G6PD deficiency using paper-based analytical devices (G6PDD-PAD). Wax printing was utilized for fabricating circular reaction zone patterns in paper. The colorimetric assay is based on the formation of formazan via a reduction of tetra-nitro blue tetrazolium (TNBT) by the G6PD enzyme on G6PDD-PAD. Detection was achieved by capturing the colour using a desktop scanner and the colour intensity was analysed with Adobe Photoshop C56. The results showed that the G6PD activity analysed by G6PDD-PAD was highly correlated with the standard biochemical assay (SBA) (r2=0.87, p<0.01). Moreover, good agreement by Bland-Altman bias plot was demonstrated between G6PDD-PAD and the SBA (mean bias 1.4 IU/gHb). The detection limit was 0 IU/gHb of G6PD activity. This study demonstrates the feasibility of using G6PDD-PAD. This simple, low-cost test ($0.1/test) should be useful for diagnosing G6PD deficiency in resource-limited settings.

Subtype Distribution of Blastocystis in Communities along the Chao Phraya River, Thailand.

Blastocystis is a common zoonotic enteric protozoan that has been classified into 17 distinct subtypes (STs). A cross-sectional study was conducted to determine the prevalence and subtype distributions of Blastocystis in villagers living along the Chao Phraya River, Ayutthaya Province, Thailand, and to assess the risk of zoonotic infection. In total, 220 stool samples were collected, and DNA was extracted. PCR and sequencing were performed with primers targeting the small-subunit ribosomal RNA (SSU rRNA) genes. Blastocystis was present in 5.9% (13/220) of samples, and ST3 (5.0%; 11/220) was the predominant subtype, followed by ST2 (0.45%; 1/220) and ST6 (0.45%; 1/220). Phylogenetic trees were constructed with the maximum-likelihood method based on the Hasegawa-Kishino-Yano + G + I model, neighbor-joining, and maximum parsimony methods. The percentage of bootstrapped trees in which the associated taxa clustered together was relatively high. All the sequences of the Blastocystis-positive samples (KU051524-KU051536) were closely related to those from animals (pig, cattle, and chicken), indicating a zoonotic risk. Therefore, the villagers require proper health education, especially regarding the prevention of parasitic infection, to improve their personal hygiene and community health. Further studies are required to investigate the Blastocystis STs in the animals living in these villages.

Antioxidant status in haemoglobin E carriers after acute and chronic strenuous exercises.

Haemoglobin E (HbE), an unstable haemoglobin, is highly susceptible to oxidative damages. We examined how acute or chronic physiological challenge induced by exercise affects antioxidant response in HbE carriers. Two independent studies were conducted in individuals with HbE trait and paired normal Hb. In study 1, sedentary participants were tested in a graded maximal exercise and blood samples were collected before, immediately after, and 45 minutes after an acute exercise. Our data showed that erythrocyte glutathione peroxidase (GPx) activity failed to recover in HbE carriers after 45 minutes of rest. In study 2, athletes were trained in a 10-week strenuous training and blood samples were collected before and after training period. We found that athletes with HbE carriers showed a larger increase in plasma GPx activity compared to those with normal Hb. These data suggest that HbE carriers could cope with exercise-induced oxidative stress by adjusting endogenous antioxidant markers.

Subtype distribution of Blastocystis in Thai-Myanmar border, Thailand.

Blastocystis sp. is a common zoonotic intestinal protozoa which has been classified into 17 subtypes (STs). A cross-sectional study was conducted to determine the prevalence and subtype distribution of Blastocystis in villagers living on the Thai-Myanmar border, where the risk of parasitic infection is high. A total of 207 stool samples were collected and DNA was extracted. PCR and sequencing using primers targeting small-subunit ribosomal RNA (SSU rRNA) gene were performed. The prevalence of Blastocystis infection was 37.2% (77/207). ST3 (19.8%; 41/207) was the predominant subtype, followed by ST1 (11.6%; 24/207), ST2 (5.3%; 11/207), and ST4 (0.5%; 1/207). A phylogenetic tree was reconstructed using the maximum likelihood (ML) method based on the Hasegawa-Kishino-Yano + G + I model. The percentage of bootstrapped trees in which the associated taxa clustered together was relatively high. Some sequences of Blastocystis positive samples (TK18, 39, 46, 71, and 90) were closely related to animals (pig and cattle) indicating zoonotic risks. Therefore, proper health education in parasitic prevention for the villagers should be promoted to improve their personal hygiene. Further longitudinal studies are required to monitor the prevalence of parasitic infections after providing health education and to investigate Blastocystis ST in animals living in these villages.