Immune reconstitution in children after haploidentical haematopoietic stem cell transplantation.

INTRODUCTION: Immune reconstitution (IR) kinetics of paediatric patients underwent haploidentical haematopoietic stem cell transplantation (HSCT) with post-transplant cyclophosphamide (PTCy) have not been extensively studied. We compared IR patterns of children receiving HSCT from haploidentical (n = 92) and HLA-matched donors (n = 36), and analysed risk factors for viral infection in these patients. METHODS: We prospectively measured lymphocyte subset numbers before HSCT and at 1, 3, 6 and 12 months after HSCT. Blood cytomegalovirus (CMV), Epstein-Barr virus, adenovirus, BK virus (BKV) and urine adenovirus and BKV viral loads were measured at designated time points. RESULTS: The median numbers of total T and T helper cells at 1 month were significantly lower in the haploidentical group compared with the HLA-matched group. Haploidentical HSCT recipients had significantly lower median numbers of several T cell subsets and B cells for 1 year after HSCT. The median NK cell count of the haploidentical group was lower at 1 month. BKV haemorrhagic cystitis, blood CMV and urine adenovirus reactivation were more frequently found in the haploidentical group. Post-haploidentical HSCT patients receiving anti-T lymphocyte globulin (ATG) had significantly lower median numbers of total T cells (at 1 month) and T helper cells (at 6 and 12 months) and higher rate of blood BKV reactivation compared with those without ATG. CONCLUSION: Paediatric patients who undergo haploidentical HSCT with PTCy are likely to have delayed IR and an increased risk of viral reactivation/infection compared with HLA-matched HSCT. The addition of ATG to PTCy delayed T cell recovery and increased risk of BKV reactivation.

White blood cell differentials performance of a new automated digital cell morphology analyzer: Mindray MC-80.

INTRODUCTION: The manual differential count has been recognized for its disadvantages, including large interobserver variability and labor intensiveness. In this light, automated digital cell morphology analyzers have been increasingly adopted in hematology laboratories for their robustness and convenience. This study aims to evaluate the white blood cell differential performance of the Mindray MC-80, the new automated digital cell morphology analyzer. METHODS: The cell identification performance of Mindray MC-80 was evaluated for sensitivity and specificity using pre-classification and post-classification of each cell class. The method comparison study used manual differentials as the gold standard for calculating Pearson correlation, Passing-Bablok regression, and Bland-Altman analysis. In addition, the precision study was performed and evaluated. RESULTS: The precision was within the acceptable limit for all cell classes. Overall, the specificity of cell identification was higher than 95% for all cell classes. The sensitivity was greater for 95% for most cell classes, except for myelocytes (94.9%), metamyelocytes (90.9%), reactive lymphocytes (89.7%), and plasma cells (60%). Pre-classification and post-classification results correlated well with the manual differential results for all the cell types investigated. The regression coefficients were greater than 0.9 for most cell classes except for promyelocytes, metamyelocytes, basophils, and reactive lymphocytes. CONCLUSION: The performance of Mindray MC-80 for white blood cell differentials is reliable and seems to be acceptable even in abnormal samples. However, the sensitivity is less than 95% for certain abnormal cell types, so the user should be aware of this limitation where such cells are suspected.

Retracted: White blood cell differentials performance of a new automated digital cell morphology analyzer: Mindray MC-80.

White blood cell differentials performance of a new automated digital cell morphology analyzer: Mindray MC-80, K. Paisooksantivatana; N. Khongjaroensakun; P. Chinudomwong; N. Chaothai; L. Chamchomdao; K. Suriyachand, International Journal of Laboratory Hematology, 10.1111/ijlh.13995 The above article, published online on 18 November 2022, in Wiley Online Library (wileyonlinelibrary.com), had been retracted by agreement between the authors, the journal's Editors-in-Chief, Giuseppe D'Onofrio and Ian Mackie, and John Wiley & Sons. The authors contacted the journal after publication to propose extensive changes to the data presented in the accepted article such that it would no longer reflect the version that was peer reviewed. As a result, this retraction has been undertaken.

A Simple and Applicable Method for Human Platelet Lysate Preparation Using Citrate Blood.

OBJECTIVES: To determine and compare the platelet growth factors in human platelet lysate (HPL) prepared from citrated whole blood, with final centrifugations at 4oC and 25oC. METHODS: We collected specimens of citrated whole blood from 27 healthy volunteers. The platelet-rich plasma (PRP) was separated to prepare the HPL, which was further divided into 2 portions for the final centrifugation, at 4oC and 25oC, respectively. Platelet growth factors were measured and compared between the 2 groups. RESULTS: All platelet growth factors were higher than those in PRP prepared from citrated whole blood. Moreover, the final centrifugation at 25oC resulted in noninferiority of platelet-growth-factor level. CONCLUSION: This study provided a simple method for small-volume of HPL preparation using only 10-15 mL of citrated whole blood. Further, the entire process of centrifugation can be performed at room temperature of 25oC, which is more applicable than lower temperatures for other laboratories.

Production and characterization of haploidentical CD19 CAR T cells: Validated to induce a continuous complete remission in a patient with relapsed refractory B-cell ALL.

AIMS: The purpose of this study was to design and manufacture CD19 chimeric antigen receptor (CAR)-modified T cells for clinical use in Thailand, as a model for how this technology can be directly applied at individual institutions treating high-risk leukemia patients. METHODS: We constructed second-generation CAR T cells expressing CD19 scFV-CD28-CD3ζ with different lengths of the spacer region: full, intermediate, and short length, by using a lentiviral vector. We wanted to determine whether the difference in length of the spacer would affect the cytotoxic potential of the CD19 CAR T cells against the leukemic cells. RESULTS: We found that all constructs of CD19 CAR T cells exhibited a similar level of cytotoxicity against several human lymphoma and leukemia cell lines. For the clinical application, we chose the intermediate length spacer construct CD19 CAR T cells, hypothesizing that the highest transduction efficiency coupled with a slower initial proliferation in vitro might lead to effective leukemic cell kill, yet a lower probability for serious clinical side effects. We then tested the clinical efficacy of our CD19 CAR T cells in one patient with refractory/relapsed acute B-cell lymphoblastic leukemia. This patient indeed had minimal clinical side effects after the CAR T-cell infusion, and he remains in an unmaintained, ongoing complete remission 10+ months after his T-cell treatment. CONCLUSION: Our CD19 CAR T cells demonstrated efficacies in acute lymphoblastic B-cell leukemia, and will be used to establish an immunotherapeutic program for high-risk B-cell acute lymphoblastic leukemia in Thailand. We propose that this approach can be used as a model for how this new exciting technology can be applied directly at individual institutions that treat (a large number of) patients with high-risk leukemia.

Targeting UCHL1 Induces Cell Cycle Arrest in High-Risk Multiple Myeloma with t(4;14).

Multiple myeloma (MM) patients considered to be at high cytogenetic risk commonly fail to respond to standard treatment. A thorough understanding of the molecular mechanism of MM development is, therefore, needed. We endeavored to explore the transcriptional signature among different subgroups of newly diagnosed MM using gene chip-based expression microarray. Bone marrow samples of 15 newly diagnosed Thai MM patients were included. The chromosomal translocation t(4;14) was the most frequently identified genetic alteration in the high-risk subgroup. Cluster analysis from expression profiling demonstrated that high-risk MM have a distinctly different expression pattern compared to standard-risk patients. The most significant differentially expressed gene was UCHL1. Functional enrichment analysis by Gene Set Enrichment Analysis, FUNRICH, and Gene Ontology Panther pathway revealed the gene sets involved in cell cycle control to be enriched in the t(4;14) high-risk group. Interestingly, among the well-established downstream targets of UCHL1, only CCND2 was significantly expressed in the t(4;14) high-risk group. Suppression of UCHL1 protein level by LDN-5744 inhibitor could arrest the cell cycle in G1 phase in cell lines. These findings shed light on the molecular mechanism of UCHL1 in t(4;14) high-risk MM and support the evidence that alteration of the UCHL1 pathway may play a role in the pathogenesis of high-risk MM.

Improving the efficiency of the autoverification workflow for nucleated red blood cell reporting in the hematology laboratory.

OBJECTIVE: Although the microscopic manual count is considered the standard method for NRBC enumeration, modern hematology analyzers can perform this task automatically with reliable accuracy and efficiency. This study aims to evaluate the diagnostic performance of the Sysmex XN hematology analyzer and to construct the optimal workflow for accurate and efficient NRBC reporting. METHODS: Specimens containing different levels of NRBC were included. Analytical performance was evaluated via method comparison with flow cytometry (FCM) and manual count (MC). Clinical sensitivity was analyzed by ROC analysis using manual count as the standard method. RESULTS: Correlation study of %NRBC with FCM and MC demonstrated an r value of 0.925 (95% CI 0.905 to 0.942) and 0.990 (95% CI 0.987 to 0.992) with a mean difference of -0.8 (95%CI: -6.7 to +5.0) and +0.50 (95% CI: -6.7 to +7.7), respectively. When the automated NRBC count was equal to zero and >0.07 × 109 /L, the false-negative rate and false-positive rate were 100%, respectively; hence, manual slide review could be omitted. A false-positive rate of 72.7% was noted in specimens containing NRBC count less than 0.07 × 109 /L. CONCLUSION: The Sysmex XN can help improve the efficiency of NRBC enumeration owing to its accuracy, rapidity, and automation. However, further studies are required to improve the accuracy of detection in specimens containing a very low level of NRBC.

Inhibitory activity of FOXP3+ regulatory T cells reveals high specificity for displaying immune tolerance in remission state rheumatoid arthritis.

Immune regulation status may indicate immunological remission in rheumatoid arthritis (RA). This cross-sectional study aimed to determine the Regulatory T cell (Treg) properties, together with 14 plasma cytokines levels between active RA and clinical remission patients. Peripheral blood (PB) Foxp3+ Treg was collected from RA patients for determination of Treg inhibitory activity using a co-culture system. Other PB T cell types and plasma cytokines were determined by flow-cytometry. The Treg results were analyzed according to the disease activity score-28 (DAS28). Then sensitivity and specificity were calculated for the indication of the remission status. The number and inhibitory activity of Treg are higher in the clinical remission as compared to the active RA (p value < 0.0001). Also, Treg: CD4+CD25+CD127+ cell ratio demonstrates the similar result (p value < 0.05). Treg inhibitory activity is inversely correlated with the DAS28. Specificity and positive likelihood ratio of inhibitory activity for indicating remission status are 92.31% (95% CI 63.97-99.81) and 11.14 (95% CI 1.67-74.14), respectively. Treg inhibitory activity is a promising prognostic marker and probably represents the immunological remission status in RA.

Regulatory T Cell Suppressive Activity Predicts Disease Relapse During Disease-Modifying Anti-rheumatic Drug Dose Reduction in Rheumatoid Arthritis: A Prospective Cohort Study.

When the dose of conventional disease-modifying anti-rheumatic drugs (cDMARDs) is tapered in rheumatoid arthritis (RA) patients who achieve sustained remission, biomarkers for predicting disease relapse may be needed. A prospective, unblinded cohort study was conducted in nine RA patients with remission. Peripheral blood samples were collected at baseline and at 6, 12, and 24 weeks after cDMARD dose reduction (dose of combination regimens reduced to 50%) to determine the number of regulatory Foxp3+T cells (Tregs) and other T cell subpopulations as well as Treg suppressive activity. Additionally, plasma levels of 14 cytokines at each time-point were measured via flow cytometry. Univariate and multivariate analyses were performed to identify the factor(s) associated with RA relapse during the observational period. In univariate analysis, Treg suppression and DAS28 and VAS scores were associated with RA relapse after cDMARD dose tapering. However, in multivariate analysis, only Treg suppressive activity (<42%) was found to be an independent factor associated with RA relapse after cDMARD dose reduction to 50%. Of all patients who had ≥42% Treg suppressive activity during cDMAD reduction, three-fourth patients remained in the remission stage for 24 weeks. Treg suppressive activity (<42%) in RA patients with remission could be a potential biomarker for predicting RA relapse after cDMARD dose reduction, especially over a short-term period (24 weeks).

Diagnostic performance of reticulocyte hemoglobin equivalent in assessing the iron status.

BACKGROUND: Measurement of reticulocyte hemoglobin equivalent (RET-He) is rapid, convenient, and cost-effective. Yet, researches on its performance in diagnosing iron deficiency with concurrent inflammation are limited. Hence, this study investigated RET-He value in various states, including inflammation, and evaluated its diagnostic performance in iron status assessment. METHODS: Retrospectively, 953 clinical data and laboratory results-complete blood count, reticulocyte count, RET-He, and serum ferritin-were reviewed. Patients on iron therapy were excluded. Iron status was defined by serum ferritin as the reference method. RET-He among populations was investigated. Its diagnostic performance and optimal cutoff were determined by ROC analysis. RESULTS: Three population groups were classified: healthy control, iron deficiency anemia (IDA), and non-ID anemia. Significantly, RET-He value in IDA was lower than that of healthy control, anemia of inflammation, and chronic kidney disease (P < .0001). Low RET-He was also observed in IDA with concomitant inflammation despite normal-to-high serum ferritin levels. No significant difference was observed between RET-He values in pure IDA and thalassemia (P = .57). ROC curve analysis revealed AUC of 0.876 (P < .0001) at cutoff 30 pg, by which IDA was discriminated with 74.2% sensitivity and 97.4% specificity. Applying cutoff ≤30 pg, IDA can be diagnosed with 96% sensitivity, 97.4% specificity, 80% PPV, and 99.6% NPV. Hence, RET-He >30 pg signifies a non-IDA state. CONCLUSION: In addition to convenience and cost-effectiveness, RET-He cutoff >30 pg can be potentially used to exclude IDA due to its excellent diagnostic sensitivity and specificity.

The use of immature platelet fraction to predict time to platelet recovery in patients with dengue infection.

Background: In dengue infection, knowing time to platelet recovery is essential for optimal management.Aims: To determine a predictor for platelet recovery in patients with dengue infection.Methods: Platelet count and immature platelet fraction (IPF) from daily blood samples of patients with dengue infection during hospitalisation and 1-4 weeks after discharge were retrospectively analysed. The levels of patients' IPF were compared with normal controls recruited from healthy children with normal platelet counts.Results: A total of 244 EDTA blood samples were collected daily from 64 patients (45 males) with dengue infection (36 dengue fever, 28 dengue haemorrhagic fever) during hospitalisation and after discharge from the hospital. They did not receive any platelet concentrate transfusion. The median IPF among normal children was 3.6% with a 95 percentile of 9.9%. In dengue patients, an IPF of ≥10.0% after defervescence was associated with a subsequent platelet count of ≥60 × 109/L within 72 hours.Conclusion: In patients with dengue infection, IPF ≥10.0% after defervescence is a predictor of subsequent platelet recovery to a haemostatic level ≥60 × 109/L within 72 hours.

Efficient short-term expansion of human peripheral blood regulatory T cells for co-culture suppression assay.

Regulatory T cells (Tregs) are a small population of CD4+ lymphocytes and play a key role as suppressors of the immune system, a role that can be identified by employing a co-culture suppression assay. Conventional protocol requires a long period of in vitro expansion of Treg numbers; hence, this study describes an establishment of a co-culture suppression assay using a short-term expansion of peripheral blood (PB) Tregs and autologous T cells (Tconvs) IL-2-pre-cultured in parallel for the same length of time, thereby obviating the need of freeze/thawed autologous Tconvs. Tregs and Tconvs were isolated from PB mononuclear cells employing magnetic bead-aided depletion of CD8+ cells followed by cell sorting of CD4+ CD25high+CD127low- (Treg) and CD4+ CD25-CD127+ (Tconv) cell populations. Following a 3-day co-cultivation period under optimized conditions, Treg suppression activity was monitored by comparing using flow cytometry the number of carboxyfluorescein succinimidyl ester-labeled Tconvs to that of Treg-minus control. The assay allowed significant differentiation between Treg suppression activity of patients with active rheumatoid arthritis and those in remission. This method should be more convenient and time-saving than the conventional Treg suppression assay in current use.

Reticulocyte hemoglobin equivalent in a thalassemia-prevalent area.

BACKGROUND: Reticulocyte hemoglobin equivalent (Ret-He), a direct measure of the hemoglobin (Hb) in the young red blood cells, has been reported to be useful in the diagnosis of iron deficiency anemia (IDA) but may have some limitations in thalassemia trait. This study evaluated the differences in Ret-He in school-aged children, and assessed the diagnostic value of Ret-He in identifying IDA in a thalassemia-prevalent area. METHODS: Blood samples underwent complete blood count analysis, including Ret-He, ferritin, serum iron and total iron binding capacity. Blood samples also underwent Hb typing and a molecular study for α-thalassemia. Receiver operating characteristic analysis was performed to determine the predictive capacity of Ret-He in the diagnosis of IDA. ID was defined as serum ferritin <30 ng/mL and/or transferrin saturation (TSAT) <16%; IDA was defined as serum ferritin <12 ng/mL and/or TSAT <16% with low Hb for age. Normal healthy children (normal controls: NC) had normal iron study, without the thalassemia trait. RESULTS: Ninety-eight children with a mean age of 12.9 ± 0.6 years were included. Ret-He in the thalassemia trait group (26.7 ± 2.4 pg), ID group (29.0 ± 2.9 pg), IDA group (25.4 ± 2.7 pg), ID + thalassemia trait group (26.6 ± 2.8 pg), and the IDA + thalassemia trait group (24.6 ± 2.3 pg) was significantly lower than in the NC group (30.8 ± 1.7 pg; P < 0.001, 0.01, 0.006, 0.002 and <0.001, respectively). Ret-He had an area under the curve of 0.904 in diagnostic ability for IDA, while a cut-off ≤27 pg had a sensitivity of 91.7% and a specificity of 81%. CONCLUSION: Ret-He was lowest in subjects with IDA + thalassemia trait. A Ret-He cut-off ≤27 pg was suggestive of IDA in the present study.

Mutation Screening and Association Study of the Folylpolyglutamate Synthetase (FPGS) Gene with Susceptibility to Childhood Acute Lymphoblastic Leukemia.

BACKGROUND: Folylpolyglutamate synthetase (FPGS), an important enzyme in the folate metabolic pathway, plays a central role in intracellular accumulation of folate and antifolate in several mammalian cell types. Loss of FPGS activity results in decreased cellular levels of antifolates and consequently to polyglutamatable antifolates in acute lymphoblastic leukemia (ALL). MATERIALS AND METHODS: During May 1997 and December 2003, 134 children diagnosed with ALL were recruited from one hospital in Thailand. We performed a mutation analysis in the coding regions of the FPGS gene and the association between single nucleotide polymorphisms (SNPs) within FPGS in a case-control sample of childhood ALL patients. Mutation screening was conducted by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and subsequently with direct sequencing (n=72). Association analysis between common FPGS variants and ALL risk was done in 98 childhood ALL cases and 95 healthy volunteers recruited as controls. RESULTS: Seven SNPs in the FPGS coding region were identified by mutation analysis, 3 of which (IVS13+55C>T, g.1297T>G, and g.1508C>T) were recognized as novel SNPs. Association analysis revealed 3 of 6 SNPs to confer significant increase in ALL risk these being rs7039798 (p= 0.014, OR=2.14), rs1544105 (p=0.010, OR= 2.24), and rs10106 (p=0.026, OR= 1.99). CONCLUSIONS: These findings suggested that common genetic polymorphisms in the FPGS coding region including rs7039789, rs1544105, and rs10106 are significantly associated with increased ALL risk in Thai children.

A study of correlation of osteoblasts from peripheral blood with related bone turnover markers.

The bone remodeling process called osteoblasts has an important role in bone formation working together with osteoclasts of which the cells are responsible for bone resorption. In addition, these bone turnover markers are used to follow-up the conditions of bone remodeling in the patients. Recently, osteoblastic lineage cells have been found that they exist in the human peripheral blood. However there has been no report about the amount of circulating osteoblastic lineage cells that have the relationship with the samples of bone turnover markers showing the bone remodeling condition. In the present study, circulating osteoblasts were quantified in 43 subjects aged between 25-90 years. They were classified by age into 3 groups: A) lower than 60 years old (n = 9), B) from 60 to 79 years old (n = 22) and C) equal and over 80 years old (n = 12). All were studied by the flow cytometry method using an antibody to osteocalcin and bone turnover markers beta-CrossLab (betaCTx), PINP and NMID. These markers including parathyroid hormone were analyzed. The result showed the best positive correlation between the percentage of circulating osteoblasts and bone turnover markers of the equal and over 80-year-old group. While another result exhibited the negative correlation of circulating osteocalcin positive cells with the bone turnover markers in the group of lower than 60 years old. As circulating osteoblasts had the correlation with bone turnover markers in the group aged 80 years old, this could be used as the markers to follow up the bone turnover situation of the patients in this age group. However, this is a pilot study. Further analysis of more amounts of subjects should be done for a better result.

Effect of genetic alterations of cytarabine- metabolizing enzymes in childhood acute lymphoblastic leukemia.

BACKGROUND: Single nucleotide polymorphisms (SNPs) of deoxycytidine kinase (dCK) and cytidine deaminase (CDA) are known to alter their enzymatic activities, which affect the metabolism of cytarabine. Currently, treatment of childhood acute lymphoblastic leukemia (ALL) includes cytarabine, especially in high-risk patients. Therefore, we hypothesized that a genetic variation of dCK and CDA genes may influence the risk of cytarabine-related toxicities and early response to treatment. PATIENTS AND METHODS: We included children diagnosed with ALL and lymphoblastic lymphoma (LL) stage III and IV. The patients received a modified St Jude Total Therapy Study XV protocol. Cytarabine was used during induction remission (low-dose cytarabine) and reinduction II (high-dose cytarabine) phases. Genotyping of dCK-360C>G and -201C>t and CDA 79A>C and 208G>A was performed. Minimal residual disease (MRD) at the end of the induction phase was measured using flow cytometry. RESULTS: Ninety-four children with ALL (n=90) and LL (n=4) were analyzed. The median age at diagnosis was 5.8 years (range, 0.4-15 years). All four SNPs showed predominant wild type alleles. There was no CDA-208A allele in our population. Children with dCK-360G allele were at risk of mucositis after receiving low-dose cytarabine (OR=3.7; 95%CI, 1.2--11.3). neither dCK nor CDA polymorphisms affected the MRD status at the end of the induction phase. CONCLUSION: The dCK-360G allele was found to increase the risk of mucositis after exposure to low-dose cytarabine in childhood ALL therapy.

HemoglobinA1c level in healthy Thai adults: reference interval and fasting plasma glucose.

AIM: To establish reference interval of HbA1c IFCC in Thai. MATERIALS AND METHODS: 699 whole blood samples were used. Samples had fasting plasma glucose >or=126 mg/dl (7.00 mmol/L), renal problem or hemoglobinopathy was excluded. RESULTS: Reference interval of HbA1c IFCC was 2.90-4.90%. CONCLUSION: Effect of age should be determined.