Diagnostic utility of the lymphoid screening tube supplemented with TRBC1 for the assessment of T-cell clonality.

INTRODUCTION: Flow cytometric panels for the investigation of lymphoproliferative disorders, such as the EuroFlow Lymphoid Screening Tube (LST), often fail to demonstrate T-cell clonality, as a suitable clonality marker was unavailable until recently. Aim of this study was to evaluate the added value of supplementing TRBC1, a flow cytometric T-cell clonality marker, to the LST. METHODS: Flow cytometric analysis was performed on 830 routine samples referred to our lab for suspicion of hematological malignancy. T-cells with monotypic TRBC1-expression were additionally characterized with a 12-color T-cell tube and molecular T-cell receptor gamma gene rearrangement (TRG). RESULTS: LST analysis revealed 97 (11.7%) samples with the presence of a monotypic T-cell population according to TRBC1, including 21 (2.5%) "high-count" (≥500 cells/µL blood or ≥15% of lymphocytes) and 76 (9.2%) "low-count" (<500 cells/µL blood or <15% of lymphocytes) populations. Clinical symptoms indicative for T-CLPD could be correlated to 11/21 "high-count" and 17/76 "low-count" monotypic T-cell populations. Molecular TRG analysis demonstrated a monoclonal result in 76% (16/21) of "high-count" samples and in 64% (42/66; 10 samples not tested) of "low-count" samples, but also in 9/20 samples with polytypic TRBC1 results. CONCLUSION: Analysis of an LST tube supplemented with TRBC1 led to the detection of a high number of monotypic T-cell populations. The detection of numerous small monotypic T-cell populations raises the question of their clinical significance. A possible flowchart for assessment of these populations, based on the available literature, is proposed. Molecular TRG analysis is complementary and cannot be omitted from T-cell clonality assessment.

Parvovirus B19-triggered hemophagocytic lymphohistiocytosis in a patient with Crohn's disease.

Background: Hemophagocytic lymphohistiocytosis (HLH) is a life threatening condition caused by inappropriate immune activity. Infection is often the trigger, both in genetically predisposed and in sporadic cases. Although more commonly seen in the paediatric population, patients of all ages can be affected. Case presentation: A 26-year-old male patient with Crohn's disease, treated with ustekinumab, presented with high fever, epistaxis and anorexia. Laboratory results showed pancytopenia, and a high serum levels of ferritin and LDH. Colonoscopy revealed only mild signs of disease activity. CT-scan showed splenomegaly and multiple lymphadenopathies. Bone marrow aspirate was suggestive for hemophagocytosis. PCR & serology for parvovirus B19 came back positive. Treatment with ustekinumab was temporarily put on hold and supportive care was given. Viral replication decreased and he recovered completely. Conclusion: There is a known association between HLH and Crohn's disease. This is probably because they are more susceptible to infections with CMV, EBV and parvovirus B19, all known as triggers for HLH. The role of ustekinumab is unclear: did it play a role in the pathophysiological evolution of this primo-infection with parvovirus B19? On the other hand, did it contribute to the rather mild course of the disease, acting as a immunomodulator that works on interleukin-12, a cytokine that plays a role in HLH? Further study is warranted to answer these questions.

Evaluation of two automated cell counters for the analysis of hematopoietic progenitor cell apheresis products.

INTRODUCTION: Routine hematology parameters in hematopoietic progenitor cell apheresis products (HPC-A) are usually determined using automated cell counters. These instruments, however, are designed to analyze whole blood samples, that differ considerably from HPC-A in blood cell composition. This study evaluates the performance of two automated cell counters for the analysis of HPC-A. METHODS: Routine hematology parameters [red blood cells (RBC), hematocrit (HCT), mean corpuscular volume (MCV), white blood cells (WBC), WBC differentiation, and platelets (PLT)] were determined on the Unicel DxH 800 instrument (Beckman Coulter) and the XN-350 instrument (Sysmex). Correlations with the reference methods, intrarun precision, and linearity of the analyses were studied. RESULTS: Good correlations were found for almost all parameters. However, RBC count was overestimated by XN-350, using the impedance technique, as was neutrophil percentage using DxH 800. Coefficients of variation for intrarun precision were below 10% on both analyzers for all parameters, except for neutrophil percentage (14.7%) and PLT (10%) on DxH 800. Both instruments showed good linearity for all parameters, except for RBC and HCT on DxH 800. CONCLUSION: With the exception of the measurement of neutrophils on DxH 800 and RBC by the impedance technique on the XN-350, routine hematology parameters in HPC-A can safely be determined using automated cell counters.

A novel approach for BCR-ABL1 standardization to improve International Scale estimation.

INTRODUCTION: Standardization of BCR-ABL1 messenger RNA quantification by real-time PCR on the International Scale (IS) is critical for monitoring therapy response in chronic myelogenous leukaemia. Since 2006, BCR-ABL1 IS standardization is propagated along reference laboratories by calculating a laboratory-specific conversion factor (CF), co-ordinated in Europe through the European Treatment and Outcome Study project. Although this process has proven successful to some extent, it has not been achievable for all laboratories due to the complexity of the process and the stringent requirements in terms of numbers of samples to be exchanged. In addition, several BCR-ABL1 IS quantification methods and secondary reference materials became commercially available. However, it was observed that different IS methods generate consistently different results. METHODS: To overcome these difficulties, we have developed an alternative and simple approach of CF calculation, based on the retrospective analysis of existing external quality assessment (EQA) data. Our approach does not depend on the exchange of samples and is solely based on the mathematical CF calculation using EQA results. RESULTS AND CONCLUSION: We have demonstrated by thorough statistical validation that this approach performs well in converting BCR-ABL1 measurements to improve IS estimation. In expectation of a true golden standard method for BCR-ABL1 IS quantification, the proposed method is a valuable alternative.

Evaluation of the Red Blood Cell Advanced Software Application on the CellaVision DM96.

INTRODUCTION: The CellaVision Advanced Red Blood Cell (RBC) Software Application is a new software for advanced morphological analysis of RBCs on a digital microscopy system. Upon automated precharacterization into 21 categories, the software offers the possibility of reclassification of RBCs by the operator. We aimed to define the optimal cut-off to detect morphological RBC abnormalities and to evaluate the precharacterization performance of this software. METHODS: Thirty-eight blood samples of healthy donors and sixty-eight samples of hospitalized patients were analyzed. Different methodologies to define a cut-off between negativity and positivity were used. Sensitivity and specificity were calculated according to these different cut-offs using the manual microscopic method as the gold standard. Imprecision was assessed by measuring analytical within-run and between-run variability and by measuring between-observer variability. RESULTS: By optimizing the cut-off between negativity and positivity, sensitivities exceeded 80% for 'critical' RBC categories (target cells, tear drop cells, spherocytes, sickle cells, and parasites), while specificities exceeded 80% for the other RBC morphological categories. Results of within-run, between-run, and between-observer variabilities were all clinically acceptable. CONCLUSION: The CellaVision Advanced RBC Software Application is an easy-to-use software that helps to detect most RBC morphological abnormalities in a sensitive and specific way without increasing work load, provided the proper cut-offs are chosen. However, evaluation of the images by an experienced observer remains necessary.

Evaluation of schistocyte analysis by a novel automated digital cell morphology application.

INTRODUCTION: The CellaVision Advanced Red Blood Cell (RBC) Software Application is a new software for advanced morphological analysis of RBC, which automatically performs a preliminary characterization and grouping of RBC into 21 morphological categories, including schistocytes. Upon automated classification, the software offers the possibility of reclassification of RBC by the operator. The aim of this study was to evaluate the schistocyte analysis by the CellaVision Advanced RBC Application. METHODS: Schistocyte counts were evaluated comparing the automated count on a CellaVision DM96, both before and after reclassification, with the reference manual microscopic method according to the ICSH criteria. Thirty-six samples of hospitalized patients and 40 samples of controls were analyzed. RESULTS: Within-run, between-run and between-observer coefficients of variation were lower when counted with the CellaVision compared to the manual microscopic count. The very high sensitivity but rather poor specificity implicates the need for reclassification by the operator, following automated analysis. After reclassification, method comparison studies revealed good agreement with the manual microscopic method, with however slightly higher values of schistocytes for the automated analysis. CONCLUSION: The CellaVision Advanced RBC Software Application provides a sensitive and reproducible measurement of schistocytes in peripheral blood, but still requires manual revision. Furthermore, it is an easy-to-use software and an excellent teaching tool that might contribute to standardization in the investigation of schistocyte-related conditions.

A novel HLA-C null allele, HLA-C*05:99N.

HLA-C*05:99N results from a single nucleotide loss compared with its closest allele HLA-C*05:01:01:01.

Clinical, cytogenetic and molecular characteristics of 14 T-ALL patients carrying the TCRbeta-HOXA rearrangement: a study of the Groupe Francophone de Cytogénétique Hématologique.

Recently, we and others described a new chromosomal rearrangement, that is, inv(7)(p15q34) and t(7;7)(p15;q34) involving the T-cell receptor beta (TCRbeta) (7q34) and the HOXA gene locus (7p15) in 5% of T-cell acute lymphoblastic leukemia (T-ALL) patients leading to transcriptional activation of especially HOXA10. To further address the clinical, immunophenotypical and molecular genetic findings of this chromosomal aberration, we studied 330 additional T-ALLs. This revealed TCRbeta-HOXA rearrangements in five additional patients, which brings the total to 14 cases in 424 patients (3.3%). Real-time quantitative PCR analysis for HOXA10 gene expression was performed in 170 T-ALL patients and detected HOXA10 overexpression in 25.2% of cases including all the cases with a TCRbeta-HOXA rearrangement (8.2%). In contrast, expression of the short HOXA10 transcript, HOXA10b, was almost exclusively found in the TCRbeta-HOXA rearranged cases, suggesting a specific role for the HOXA10b short transcript in TCRbeta-HOXA-mediated oncogenesis. Other molecular and/or cytogenetic aberrations frequently found in subtypes of T-ALL (SIL-TAL1, CALM-AF10, HOX11, HOX11L2) were not detected in the TCRbeta-HOXA rearranged cases except for deletion 9p21 and NOTCH1 activating mutations, which were present in 64 and 67%, respectively. In conclusion, this study defines TCRbeta-HOXA rearranged T-ALLs as a distinct cytogenetic subgroup by clinical, immunophenotypical and molecular genetic characteristics.

Molecular cytogenetic study of 126 unselected T-ALL cases reveals high incidence of TCRbeta locus rearrangements and putative new T-cell oncogenes.

Chromosomal aberrations of T-cell receptor (TCR) gene loci often involve the TCRalphadelta (14q11) locus and affect various known T-cell oncogenes. A systematic fluorescent in situ hybridization (FISH) screening for the detection of chromosomal aberrations involving the TCR loci, TCRalphadelta (14q11), TCRbeta (7q34) and TCRgamma (7p14), has not been conducted so far. Therefore, we initiated a screening of 126 T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma cases and 19 T-ALL cell lines using FISH break-apart assays for the different TCR loci. Genomic rearrangements of the TCRbeta locus were detected in 24/126 cases (19%), most of which (58.3%) were not detected upon banding analysis. Breakpoints in the TCRalphadelta locus were detected in 22/126 cases (17.4%), whereas standard cytogenetics only detected 14 of these 22 cases. Cryptic TCRalphadelta/TCRbeta chromosome aberrations were thus observed in 22 of 126 cases (17.4%). Some of these chromosome aberrations target new putative T-cell oncogenes at chromosome 11q24, 20p12 and 6q22. Five patients and one cell line carried chromosomal rearrangements affecting both TCRbeta and TCRalphadelta loci. In conclusion, this study presents the first inventory of chromosomal rearrangements of TCR loci in T-ALL, revealing an unexpected high number of cryptic chromosomal rearrangements of the TCRbeta locus and further broadening the spectrum of genes putatively implicated in T-cell oncogenesis.

A new recurrent inversion, inv(7)(p15q34), leads to transcriptional activation of HOXA10 and HOXA11 in a subset of T-cell acute lymphoblastic leukemias.

Chromosomal translocations with breakpoints in T-cell receptor (TCR) genes are recurrent in T-cell malignancies. These translocations involve the TCRalphadelta gene (14q11), the TCRbeta gene (7q34) and to a lesser extent the TCRgamma gene at chromosomal band 7p14 and juxtapose T-cell oncogenes next to TCR regulatory sequences leading to deregulated expression of those oncogenes. Here, we describe a new recurrent chromosomal inversion of chromosome 7, inv(7)(p15q34), in a subset of patients with T-cell acute lymphoblastic leukemia characterized by CD2 negative and CD4 positive, CD8 negative blasts. This rearrangement juxtaposes the distal part of the HOXA gene cluster on 7p15 to the TCRbeta locus on 7q34. Real time quantitative PCR analysis for all HOXA genes revealed high levels of HOXA10 and HOXA11 expression in all inv(7) positive cases. This is the first report of a recurrent chromosome rearrangement targeting the HOXA gene cluster in T-cell malignancies resulting in deregulated HOXA gene expression (particularly HOXA10 and HOXA11) and is in keeping with a previous report suggesting HOXA deregulation in MLL-rearranged T- and B cell lymphoblastic leukemia as the key factor in leukaemic transformation. Finally, our observation also supports the previous suggested role of HOXA10 and HOXA11 in normal thymocyte development.

Evaluation of a disk diffusion method with cefoxitin (30 microg) for detection of methicillin-resistant Staphylococcus aureus.

The emergence of heterogeneous populations of methicillin-resistant Staphylococcus aureus (MRSA) causes major problems in routine screening for MRSA. In heterogeneous MRSA populations, a proportion of bacterial cells show low-level resistance to oxacillin, with minimal inhibitory concentrations (MICs) of oxacillin ranging between 1 and 100 mg/l, while in homogeneous MRSA populations, the MIC of oxacillin for all cells is >100 mg/l. Routine oxacillin disk diffusion tests often fail to detect heterogeneous MRSA populations. In the present study, a recently proposed disk diffusion method that employs a cephamycin antibiotic (cefoxitin 30 microg; BD Sensi-disc, Becton Dickinson, Germany) was evaluated using 155 clinical isolates of S. aureus (73 mecA positive and 82 mecA negative). The results were compared with those of other MRSA screening techniques: a disk diffusion test with oxacillin 1 microg and cefoxitin 30 microg (BD Sensi-disc; Becton Dickinson), an MRSA latex agglutination test (Denka Seiken, Japan), and an oxacillin screen agar test (6 microg/ml; Becton Dickinson). Detection of the mecA gene by polymerase chain reaction was considered the gold standard. The performances of the different methods were determined and compared. The results showed that the cefoxitin disk diffusion test is preferable to the oxacillin disk diffusion method for routine screening to detect MRSA.

Simultaneous occurrence of myelodysplastic syndrome and monoclonal B lymphocytes with a different clonal origin.

Bone marrow and peripheral blood from a myelodysplastic syndrome patient with trisomy 13 and monoclonal B lymphocytes (without evidence of systemic lymphoma) were investigated for clonal lymphoid lineage involvement using interphase fluorescence in situ hybridization (FISH) and X-chromosome inactivation assay (HUMARA) on CD19+ and CD34+ sorted cells. Trisomy 13 was detected in 55% of CD34+ cells and in 5.5% of CD19+ cells, the latter being comparable to the negative control specimen. X-chromosome inactivation showed both CD34+ and CD19+ cells to be monoclonal, though their inactivated X-chromosome was different. The results strongly suggested that both populations of CD34+ and CD19+ cells have originated from a different progenitor stem cell.