T-cell Subset Profile in Kidney Recipients of Extended or Standard Donors.

INTRODUCTION: The usage of extended-criteria donors (ECD) became a routinely accepted manner in the last decade. ECD is a potential risk factor for antibody-mediated rejection. Analysis of lymphocyte subsets might be a complementary diagnostic toolkit because there is limited knowledge about this term. METHOD: Between May 12, 2016, and September 4, 2019, a total of 130 patients who had undergone kidney transplant were investigated. Patients were divided in ECD and standard criteria donor (SCD) groups. Blood samples were collected before the operation, then in the first week and after 30, 60, 180, and 365 days. Besides routine laboratory tests, multicolor flow cytometry was performed for lymphocyte subsets. RESULTS: ECD grafts were transplanted to older recipients. The number of CD4+ cells increased in the SCDs from the first week to until the end of first month, and then decreased. The number of CD4+ cells decreased from the beginning of the study until the end of first year to 66% of its original value in ECDs. At the first month, the number of CD19+ cells was higher in SCD compared with ECD cases; the number then decreased in both groups. T-regulatory cells had a drop at the first week that lasted until the first month. A bigger increase in SCD and a moderate increase in ECD group were then observed. The kinetics of CD19+ and CD19+ naive cells are similar in the ECD and SCD groups. In the SCD group, cell count decreased in both CD19+ (13%) and CD19+ naive (12%) between third and sixth month. The count of CD19+ cells decreased by 9%, but the count of CD19+ naive cells increased by 11% between the sixth month and first year. DISCUSSION: The prolonged postoperative uremic state caused by the poorer initial function, together with an aging immune system, explains the weaker immune response in ECD patients, which may be the cause of the decreased number of memory and regulatory T cells. Older patients with an ECD graft need a tailored, personalized, and less aggressive immunosuppressive treatment.

Expression Patterns of Coagulation Factor XIII Subunit A on Leukemic Lymphoblasts Correlate with Clinical Outcome and Genetic Subtypes in Childhood B-cell Progenitor Acute Lymphoblastic Leukemia.

BACKGROUND: Based on previous retrospective results, we investigated the association of coagulation FXIII subunit A (FXIII-A) expression pattern on survival and correlations with known prognostic factors of B-cell progenitor (BCP) childhood acute lymphoblastic leukemia (ALL) as a pilot study of the prospective multi-center BFM ALL-IC 2009 clinical trial. METHODS: The study included four national centers (n = 408). Immunophenotyping by flow cytometry and cytogenetic analysis were performed by standard methods. Copy number alteration was studied in a subset of patients (n = 59). Survival rates were estimated by Kaplan-Meier analysis. Correlations between FXIII-A expression patterns and risk factors were investigated with Cox and logistic regression models. RESULTS: Three different patterns of FXIII-A expression were observed: negative (<20%), dim (20-79%), and bright (≥80%). The FXIII-A dim expression group had significantly higher 5-year event-free survival (EFS) (93%) than the FXIII-A negative (70%) and FXIII-A bright (61%) groups. Distribution of intermediate genetic risk categories and the "B-other" genetic subgroup differed significantly between the FXIII-A positive and negative groups. Multivariate logistic regression confirmed independent association between the FXIII-A negative expression characteristics and the prevalence of intermediate genetic risk group. CONCLUSIONS: FXIII-A negativity is associated with dismal survival in children with BCP-ALL and is an indicator for the presence of unfavorable genetic alterations.

Evaluation of Sample Quality As Preanalytical Error in Flow Cytometry Analysis in Childhood Acute Lymphoblastic Leukemia.

INTRODUCTION: Acute lymphoblastic leukemia (ALL) is the most common cancer among children. The intensity of chemotherapy and further therapeutic decisions depend on several prognostic factors, including response to initial treatment by examining peripheral blood (PB), bone marrow (BM) and cerebrospinal fluid (CSF) samples at certain time points. (e.g. day 15 BM). Sample quality is crucial for the correct risk assessment. PATIENTS AND METHODS: We aimed to explore the rate of inadequate samples as a source of preanalytical error. We retrospectively analyzed flow cytometry results of BM (day 15 and day 33) and CSF samples from children with ALL in different cohorts focusing on PB contamination and viable cell ratio among nucleated cells. We also compared viable cell percentages in native and stabilized CSF samples. RESULTS: Due to PB contamination (erythroid precursors < 2%) 12.5% of day 15 and 14% of day 33 BM samples were inadequate for flow cytometry risk stratification. Significantly fewer CSF samples had to be considered inadequate for analysis (defined as viable cells < 30%) in the subgroup of stabilized samples compared to native samples. Four of the CSF samples from children with ALL had identifiable malignant cell population despite the low viable cell percentage. DISCUSSION: Poor sample quality can hamper risk stratification and further therapeutic decision in childhood ALL. Despite low viable cell count malignant cell populations may still be identified in a CSF sample, therefore establishing a certain cutoff point for viable cells is difficult.

T Cell Subset Profile and Appearance of Donor-specific Antibodies in Primary and Retransplanted Kidney Recipients.

INTRODUCTION: Accelerated antibody-mediated rejection is a major challenge after kidney transplantation. While the clinical course, diagnosis, and treatment of cell-mediated acute rejection is agreed upon and has been successfully performed, the antibody-mediated rejection remains a problem. Biopsies cannot be repeated several times, are not always representative, and are refused by many patients. Analysis of T-cell subsets and donor-specific antibodies (DSAs) might be an additive diagnostic tool in the case of kidney transplantation. METHOD: Between 2015 and 2017, 50 kidney transplant patients were enrolled in the study. Patients were divided into 2 clinical groups: primary transplants and regrafted patients. Serum samples were collected right before the operation, then in 1 week; 30, 60, and 180 days; and yearly. Besides routine laboratory, multicolor flow cytometry was performed for T cell subsets, and Luminex Single Antigen Bead assay for the detection on donor-specific anti-HLA antibodies. Medical data were also fixed. RESULTS: The percentage of CD4+ and CD8+ cells (the CD4+/CD8+ rate) did not change much over time in either group. The percentage of CD19+ cells increased until week 1, then decreased back to its original level by day 180. CD56+/3-% was high in both groups and had no characteristic kinetics by the time. The CD4+ naïve absolute cell count increased in first-time transplants and did not decreased back to its original value until the end of year 1. This is in contrast to retransplants, where CD4+ naïve cell count rapidly dropped below its original value and remained low throughout the first year after transplantation. The CD8+ effector memory absolute cell-count was higher in first-time transplants compared to retransplanted patients in all time points. By the end of month 1, the CD19+ naïve absolute cell-count increased in first-time transplants to 170% of its original value; however, it remained or decreased in second transplants. By the end of the first year, the CD19+ naïve absolute cell count diminished to 70% in first-time transplants and 38% in second transplants. DSA was detected in 9 out of the 38 first-time transplants (23.7%) compared to 7 out of 12 (58.3%) in regrafted patients during the observational period (P = .001). It was typical for regrafted patients for DSAs to appear earlier after transplantation, and that more simultaneously different antibodies were detected against more antigens at the first time point compared to first-time transplants. DISCUSSION: The 2 groups were similar in demographics and there were no differences regarding the clinical course, complications, or output data. However, we found statistical differences regarding the dynamics of T cell subsets and DSAs. The parallel measurement of CD subsets and DSAs might be a sensitive and useful additive tool in diagnosing subclinical immunologic changes after transplantation.

Comparative Analysis of Multicolor Flow Cytometry and Immunohistochemistry for the Detection of Disseminated Tumor Cells.

Disseminating cells of a primary solid tumor may represent the origin of metastases and relapses. We aimed at comparing the diagnostic efficacy of multicolor flow cytometry (MFC) and morphology/immunohistochemistry (IHC) in the detection of disseminated tumor cells in the bone marrow (BM) and body fluids of patients with solid tumors, and in pediatric neuroblastoma cases. We investigated 72 samples retrospecively from 50 patients by MFC. Morphology/IHC data were available in 48 cases. In the first cohort, 36 samples derived from 34 patients with various forms of suspected and proven solid tumors and in the second cohort, 36 samples of 16 children with suspected and proven neuroblastoma were analyzed at diagnosis or during follow-up in a 4-color setting by MFC, and the results were compared with those obtained by IHC. In the group of various solid tumors, we found 91% concordance between IHC and MFC, and it was 65% in the neuroblastoma group, and 77% overall. Detection of disseminated tumor cells was found to be more effective by MFC in de novo neuroblastoma samples (100% vs. 86%). The advantage of MFC was even more pronounced when minimal residual disease was evaluated (efficacy, 92% vs. 68%). In contrast, efficacy of IHC was 100% in the group of various solid tumors, whereas it was 91% for MFC. We conclude that MFC and IHC are both essential tools for examining infiltration of BM and body fluids by disseminating solid tumor cells. In the case of neuroblastoma, however, minimal residual disease detection by MFC in a hypoplastic/aplastic BM environment was more effective than IHC, as considerably more cells could be analyzed.

Expression of Coagulation Factor XIII Subunit A Correlates with Outcome in Childhood Acute Lymphoblastic Leukemia.

Previously we identified B-cell lineage leukemic lymphoblasts as a new expression site for subunit A of blood coagulation factor XIII (FXIII-A). On the basis of FXIII-A expression, various subgroups of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) can be identified. Fifty-five children with BCP-ALL were included in the study. Bone marrow samples were obtained by aspiration and the presence of FXIII-A was detected by flow cytometry. G-banding and fluorescent in situ hybridization was performed according to standard procedures. The 10-year event-free survival (EFS) and overall survival (OS) rate of FXIII-A-positive and FXIII-A-negative patients showed significant differences (EFS: 84% vs. 61%, respectively; p = 0.031; OS: 89% vs. 61%; p = 0.008). Of all the parameters examined, there was correlation only between FXIII-A expression and 'B-other' genetic subgroup. Further multivariate Cox regression analysis of FXIII-subtype and genetic group or 'B-other' subgroup identified the FXIII-A negative characteristic as an independent factor associated with poor outcome in BCP-ALL. We found an excellent correlation between long-term survival and the FXIII-A-positive phenotype of BCP lymphoblasts at presentation. The results presented seem to be convincing enough to suggest a possible role for FXIII-A expression in the prognostic grouping of childhood BCP-ALL patients.

Serum thymidine kinase activity: analytical performance, age-related reference ranges and validation in chronic lymphocytic leukemia.

BACKGROUND: To date no age-related reference ranges are available for serum thymidine kinase (TK1) activity. Being a proliferation marker, it may be used as a prognostic marker in malignant diseases, including chronic lymphocytic leukemia (CLL). Our aim was to establish age-specific reference ranges for TK1 and examine its utility as a screening marker in CLL, a disease of the elderly. METHODS: Serum TK1 activity was measured by a competitive chemiluminescent immunoassay in 369 healthy adults and 115 de novo CLL patients. RESULTS: We observed a statistically significant decline in TK1 activity from young (18-35 years) to middle-aged (36-60 years) and further on to elderly (60-86 years) healthy individuals. Age-related reference range was: <30 U/L for young, <25 U/L for middle-aged and <19 U/L for elderly. There was no difference in TK1 activity between the studied healthy men and women. In CLL patients, TK1 activity was the highest in the advanced Rai stages. The area under the receiver operating characteristic curve (ROC-AUC) for TK1 was 0.840 (95% CI: 0.787-0.892), for differentiating CLL patients from age and sex matched healthy controls, with a cut-off value of 10.5 U/L (sensitivity: 80.9%, specificity: 73.4%). TK1 was significantly elevated in CD38+/Zap70+ CLL patients, and showed significant correlation with WBC and absolute B-cell count. CONCLUSION: In the healthy, serum TK1 activity does not differ in the two sexes but declines significantly with age. As such, use of age-related reference ranges is warranted, especially when evaluating CLL patients who generally belong to the elderly age group.

Flow Cytometry in the Diagnosis of Myelodysplastic Syndromes.

Myelodysplastic syndromes are clonal hematopoietic stem cell disorders. Their exact etiology is unknown. Myelodysplastic syndromes cause progressive bone marrow failure resulting in pancytopenia and refractory, transfusion-dependent anemia. One can observe typical morphological alterations in the erythroid, myeloid and/or megakaryocytic cell lineage. Blast counts may also be increased. The pathologic cells are genetically unstable, and a myelodysplastic syndrome might transform into acute myeloid leukemia. The overall survival of these diseases range between few months to around ten years. Correct diagnosis and accurate prognostic classification is essential. In the past decades several scoring systems were established beginning with the French-American-British classification to the most recent Revised International Prognostic Scoring System. In all of these classifications bone marrow morphology is still the most important factor, though nowadays the genetic aberrations and flow cytometry findings are also included. The diagnosis and prognostic classification of myelodysplastic syndromes remain a great challenge for hematologists.

[Interpretation of highly sensitive troponin assays: acute or chronic myocardial damage?]. / Nagy érzékenységu troponintesztek értékelése: akut vagy krónikus szívizom-károsodás?

UNLABELLED: Troponin is the first choice in the diagnosis of acute myocardial infarction. Correct interpretation is challenging, because high sensitive troponin tests used today detect even the smallest cardiac damage. METHODS: High sensitive troponin T (Roche) and troponin I (Mitsubishi Pathfast) and creatine-kinase activity were measured in 20 patients, each having two samples with the time lapse 3-9 hours. RESULTS: In the group without acute myocardial infarction (n = 10) no significant increase in creatine-kinase and creatine-kinase-MB levels were seen, and the mild raise of troponins was due to other cardiovascular problems (atrial fibrillation, paroxysmal supraventricular tachycardia). With acute myocardial infarction (n = 10) a dramatic increase of troponin levels was found in the second samples, and also an increase of creatine-kinase and creatine-kinase-MB activity. According to Fischer-probe a twofold or higher increase of troponin implies 19-times higher risk of acute myocardial infarction in the case of troponin T and 8-times odds ratio at troponin I. CONCLUSIONS: The patient's accompanying diseases should always be considered. If the troponin level is elevated, the measurement should be repeated within 3-6 hours. When troponin shows at least a twofold increase and the patient has chest pain or positive ECG, AMI is likely, and the patient needs special medical care. Although the first troponin level might be elevated if accompanying diseases cause chronic cardiac damage, it can be differentiated by a second troponin measurement.

[Microvolt T-wave alternant: pathomechanism and evaluation of a new marker of arrhythmia risk]. / A fokozott arrhythmiarizikó új markere: a mikrovolt T-hullám-alternáns patomechanizmusa és vizsgálati módszerei.

Microvolt T-wave alternant (microV-TWA) is a beat-to-beat fluctuation in the amplitude of T-wave at a microvolt level. The amount of variation is small, on the order of microvolts, so sensitive digital signal processing techniques are required to detect microV-TWA. The appearance of microV-TWA has been suggested as a predictor of susceptibility to lethal ventricular tachyarrhythmias, and sudden cardiac death in different patients' populations. During the last decade, theoretical, experimental and clinical research efforts have focused primarily on microV-TWA, examining its mechanisms and predictive value using time-invariant cutoff values. The cellular mechanisms involved are not well-defined and are the subject of this investigation. This review discusses the bench-to-bedside literature that, over decades, has linked alternans of repolarization in cellular, whole-heart, and human studies with spatial dispersion of repolarization, alternans of cellular action potential, and fluctuations in ionic currents, intracellular calcium regulation, role of the beta adrenergic receptors and connexins that may lead to ventricular arrhythmias. This review then provides a contemporary framework for the use of microV-TWA methods to enhance risk stratification for sudden cardiac death, identifying populations for whom microV-TWA is best established.

[Atrial fibrillation and the autonomous nervous system]. / A pitvarfibrilláció és a vegetatív idegrendszer.

The autonomic nervous system has a crucial role in the genesis, maintenance and abruption of atrial fibrillation. The substrate and trigger mechanism of atrial fibrillation can be influenced by the changing autonomic tone. The authors summarize the current knowledge on the relationship between autonomic nervous system and atrial fibrillation. The special neuroanatomical status and the role of autonomic reflexes and baroreflex in the initiation, maintenance, and termination of arrhythmia are reviewed. Furthermore, the mechanism and consequences of autonomic effect of the curative radiofrequency catheter ablation of pulmonary vein with atrial vagal neuroablation are discussed. At the end we also summarize the pharmacologic therapy of atrial fibrillation. Classification of atrial fibrillation, as either vagal or adrenergic, has only limited impact on current management.