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OBJECTIVES: Anemia is a severe global public health issue. Testing practices for anemia suggest overuse of screening laboratory tests and misinterpretation of studies even in "easy-to-diagnose" underlying causes, leading to late diagnoses and missed treatment opportunities. We aimed to develop a complete and efficient algorithm for clinical pathologists and laboratory medicine physicians for the differential diagnosis of anemia. METHODS: Comprehensive literature search encompassing original articles, studies, reviews, gold standard books, and other evidence. RESULTS: We created a complex algorithm, primarily for clinical pathology/laboratory use, that explores all major and several rare causes of anemia in an efficient and evidence-based manner. The algorithm includes gold-standard diagnostic laboratory tests available in most clinical laboratories and indices that can be easily calculated to provide an evidence-based differential diagnosis of anemia. CONCLUSIONS: The diagnostic strategy combines previously available diagnostic tests and protocols in an efficient order. Clinical pathologists following the algorithm can independently provide valuable diagnostic support for healthcare providers. Clinical pathologists providing complete differential diagnostic services with the proposed algorithm may create an opportunity for an advanced diagnostic service that supports diagnostic excellence and helps patients receive a timely diagnosis and early treatment opportunities.
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Anemia , Serviços de Laboratório Clínico , Humanos , Diagnóstico Diferencial , Patologistas , Algoritmos , Anemia/diagnósticoRESUMO
Acute promyelocytic leukemia (APL) is generally characterized by t(15;17)(q24;q21). In some cases, the classic translocation cannot be identified by conventional methods, since the PML-RARA fusion protein results from complex, variant, or cryptic translocation. The diagnostic algorithm of APL starts with screening methods, such as flow cytometry (FC), followed by fluorescence in situ hybridization or polymerase chain reaction to confirm the diagnosis. Our aim was to develop a novel protocol for analyzing APL samples based on multidimensional dot-plots that can provide comprehensive information about several markers at the same time. The protocol included four optimized multidimensional dot-plots, which were tested by retrospective reanalysis of FC results in APL (n = 8) and non-APL (n = 12) acute myeloid leukemia (AML) cases. After predicting the potential position of hypergranular- and microgranular-type aberrant promyelocytes, the percentages of blast populations were examined within the gates in all AML cases. The percentage of blasts in each predefined gate was well above the cut-off value (95%) in APL cases in all tubes. In non-APL AML cases, the percentage of blasts in the same gates never reached the cut-off value in all investigated tubes, and even when it did in a single tube, the pattern was markedly different from that observed in APL cases. In conclusion, multidimensional dot-plots can be used for screening APL even in cryptic APL cases, although reproducibility across several laboratories would require standardization of antibodies and fluorochromes. This easy-to-use and quick method can support the diagnosis of APL and the prompt initiation of the appropriate treatment.
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Apresentação de Dados , Detecção Precoce de Câncer/métodos , Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Leucemia Promielocítica Aguda/diagnóstico , Adulto , Idoso , Antígenos CD/análise , Antígenos de Neoplasias/análise , Biomarcadores Tumorais/análise , Medula Óssea/patologia , Bandeamento Cromossômico , Cromossomos Humanos Par 15/genética , Cromossomos Humanos Par 15/ultraestrutura , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 17/ultraestrutura , Fator XIII/análise , Feminino , Citometria de Fluxo/instrumentação , Corantes Fluorescentes , Humanos , Imunofenotipagem/instrumentação , Hibridização in Situ Fluorescente , Leucemia Promielocítica Aguda/sangue , Leucemia Promielocítica Aguda/genética , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/patologia , Proteínas de Fusão Oncogênica/genética , Estudos Retrospectivos , Translocação GenéticaRESUMO
Multicolor flow cytometry (FC) evaluation has a key role in the diagnosis and prognostic stratification of ALL. Our aim was to create new analyzing protocols using multidimensional dot-plots. Seventy-two pediatric patients with ALL were included in this single-center study. Data of a normal BM sample and three BM samples of patients with BCP-ALL were merged, then all B cell populations of the four samples were presented in a single radar dot-plot, and those parameters and locations were selected in which the normal and pathological cell populations differed from each other the most. The integrated profile of immunophenotype resulted in a simple, rapid, and accurate method. There were no significant differences between the percentages of lymphoblasts in the detection of minimal residual disease (MRD) by multidimensional or conventional FC method (p = 0.903 at Day 15 and p = 0.155 at Day 33). Furthermore, we found associations between the position and the number of clusters of blast cells in the radar plots and cytogenetic properties (p = 0.002 and p < 0.0001 by the position and p = 0.02 by the number of subclones). FC analysis based on multidimensional dot-plots is not only a rapid, easy-to-use method, but can also provide additional information to screen cases which require detailed genetic examination.
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Outcome measures of pediatric acute myeloid leukemia (AML) improved considerably between 1990 and 2011 in Hungary. Since 2012, efforts of the Hungarian Pediatric Oncology-Hematology Group (HPOG) included the reduction in the number of treatment centers, contemporary diagnostic procedures, vigorous supportation, enhanced access to hematopoietic stem cell transplantation (HSCT), and to targeted therapies. The major aim of our study was to evaluate AML treatment results of HPOG between 2012 and 2019 with 92 new patients registered (52 males, 40 females, mean age 7.28 years). Two periods were distinguished: 2012-2015 and 2016-2019 (55 and 37 patients, respectively). During these periods, 2 y OS increased from 63.6% to 71.4% (p = 0.057), and the 2 y EFS increased significantly from 56.4% to 68.9% (p = 0.02). HSCT was performed in 37 patients (5 patients received a second HSCT). We demonstrate advances in the diagnosis and treatment of acute promyelocytic leukemia (APL) in two cases. Early diagnosis and follow-up were achieved by multidimensional flow cytometry and advanced molecular methods. Both patients were successfully treated with all-trans retinoic acid and arsenic-trioxide, in addition to chemotherapy. In order to meet international standards of pediatric AML management, HPOG will further centralize treatment centers and diagnostic facilities and join efforts with international study groups.
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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.
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Subpopulações de Linfócitos T/metabolismo , Adulto , Idoso , Antígenos CD19/metabolismo , Linfócitos B/citologia , Linfócitos B/metabolismo , Feminino , Humanos , Transplante de Rim , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Cinética , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Fatores de Tempo , Doadores de Tecidos , TransplantadosRESUMO
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.
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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.
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Background: Leukemic B-cell precursor (BCP) lymphoblasts were identified as a novel expression site for coagulation factor XIII subunit A (FXIII-A). Flow cytometry (FC) revealed three distinct expression patterns, i.e., FXIII-A negative, FXIII-A dim, and FXIII-A bright subgroups. The FXIII-A negative subgroup was significantly associated with the "B-other" genetic category and had an unfavorable disease outcome. Methods: RNA was extracted from bone marrow lymphoblasts of 42 pediatric patients with BCP-acute lymphoblastic leukemia (ALL). FXIII-A expression was determined by multiparameter FC. Genetic diagnosis was based on conventional cytogenetic method and fluorescence in situ hybridization. Affymetrix GeneChip Human Primeview array was used to analyze global expression pattern of 28,869 well-annotated genes. Microarray data were analyzed by Genespring GX14.9.1 software. Gene Ontology analysis was performed using Cytoscape 3.4.0 software with ClueGO application. Selected differentially expressed genes were validated by RT-Q-PCR. Results: We demonstrated, for the first time, the general expression of F13A1 gene in pediatric BCP-ALL samples. The intensity of F13A1 expression corresponded to the FXIII-A protein expression subgroups which defined three characteristic and distinct gene expression signatures detected by Affymetrix oligonucleotide microarrays. Relative gene expression intensity of ANGPTL2, EHMT1 FOXO1, HAP1, NUCKS1, NUP43, PIK3CG, RAPGEF5, SEMA6A, SPIN1, TRH, and WASF2 followed the pattern of change in the intensity of the expression of the F13A1 gene. Common enhancer elements of these genes revealed by in silico analysis suggest that common transcription factors may regulate the expression of these genes in a similar fashion. PLAC8 was downregulated in the FXIII-A bright subgroup. Gene expression signature of the FXIII-A negative subgroup showed an overlap with the signature of "B-other" samples. DFFA, GIGYF1, GIGYF2, and INTS3 were upregulated and CD3G was downregulated in the "B-other" subgroup. Validated genes proved biologically and clinically relevant. We described differential expression of genes not shown previously to be associated with pediatric BCP-ALL. Conclusions: Gene expression signature according to FXIII-A protein expression status defined three novel subgroups of pediatric BCP-ALL. Multiparameter FC appears to be an easy-to-use and affordable method to help in selecting FXIII-A negative patients who require a more elaborate and expensive molecular genetic investigation to design precision treatment.
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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.
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Rejeição de Enxerto/imunologia , Isoanticorpos/imunologia , Transplante de Rim , Reoperação , Subpopulações de Linfócitos T/imunologia , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doadores de Tecidos , Transplantes/imunologiaRESUMO
INTRODUCTION: A growing body of evidence supports the usefulness of dysplastic signs detected by flow cytometry in the diagnosis of myelodysplastic syndromes (MDS). Our aim was to assess the impact of pre-analytical variables (delayed sample handling, type of anticoagulant, and different clones of antibody) in the interpretation of flow cytometric results. MATERIAL AND METHODS: Bone marrow samples were labelled and analysed immediately after aspiration and on two consecutive days. The effect of anticoagulant type was evaluated in 16 bone marrow samples. Thirty-seven different immunophenotypic variables were recorded after eight-colour staining. Furthermore, 8 normal peripheral blood samples collected in K3-EDTA and Na-heparin were examined with different clones of CD11b antibodies and four parameters were recorded with both anticoagulants on two consecutive days. RESULTS: Fourteen significant differences were detected in the initial immunophenotype of fresh samples collected in K3-EDTA and Na-heparin. Regardless of the anticoagulant type, eleven parameters remained stable despite delayed sample handling. Due to delayed sample processing, more alterations were detected in the samples collected in K3-EDTA than in the samples collected in Na-heparin. The type of CD11b clone influenced the reduction of fluorescence intensity only in samples collected in K3-EDTA, where the alterations were contrary to the changes observed in Na-heparin. CONCLUSIONS: Delayed sample processing causes considerable immunohenotypic alterations, which can lead to false interpretation of the results. If delayed sample evaluation is unavoidable, markers that remain more stable over time should be considered with more weight in the diagnosis of MDS.
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Células da Medula Óssea/patologia , Citometria de Fluxo/normas , Imunofenotipagem/normas , Síndromes Mielodisplásicas/diagnóstico , Manejo de Espécimes/normas , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos/análise , Anticoagulantes/química , Biomarcadores/análise , Células da Medula Óssea/imunologia , Antígeno CD11b/imunologia , Erros de Diagnóstico/prevenção & controle , Ácido Edético/química , Feminino , Heparina/química , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/imunologia , Síndromes Mielodisplásicas/patologia , Estudos Prospectivos , Fatores de TempoRESUMO
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.
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Biomarcadores Tumorais/sangue , Fator XIII/análise , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Adolescente , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Humanos , Lactente , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Prognóstico , Estudos Retrospectivos , Adulto JovemRESUMO
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.
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Medula Óssea/patologia , Citometria de Fluxo/métodos , Imuno-Histoquímica/métodos , Recidiva Local de Neoplasia/diagnóstico , Neoplasia Residual/diagnóstico , Neuroblastoma/diagnóstico , Adolescente , Adulto , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sensibilidade e Especificidade , Adulto JovemRESUMO
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.