Reference Values for WBC Differential by Hematoflow Analysis.

Objectives: WBC differentials performed using flow cytometry with monoclonal antibodies have been developed in the last decade and are nowadays integrated into the routine workflow of some laboratories. Definition of reference values for each population is required in order to achieve an automatic validation of the results by laboratory software. Methods: We analyzed 584 samples from three hospitals using the Hematoflow solution to define the reference values. Results: Reference values are presented for five groups according to age (0-5, 6-11, 12-19, 20-69, and >69 years). Conclusions: These normal values will be helpful in the definition of relevant threshold for the automatic validation of samples analyzed by flow cytometry and the flagging of pathologic samples.

Integration of Immature Granulocytes Quantification With the Version 2.0 UniCel DxH 800 in the HematoFlow Strategy.

OBJECTIVES: Our aim was to define whether the early granulocyte cell marker (EGC%_DxH) parameter might replace immature granulocytes counts obtained by HematoFlow (IG%_HF) and/or manual differential count (IG%_manual). METHODS: We conducted a study over a 10-day period in February 2014 whereby 402 samples were analyzed for the IG flag. We correlated the EGC%_DxH vs IG%_HF and IG%_manual, identified any discrepant results and finally looked at the impact on our workflow by incorporation of the EGC% into our WBC differential algorithm. RESULTS: On an initial training set, a receiver operating characteristic (ROC) curve analysis showed a threshold of 0.9% for EGC%_DxH (sensitivity of 91.7%, specificity of 93.5% and an area under the curve of 0.965). Further analysis of the dataset (259 samples) found a correlation of the EGC%_DxH to all our IG% counting methods (r = 0.963). Incorporation of the EGC%_DxH into the WBC HematoFlow differential resulted in a 36% reduction of samples requiring HematoFlow and/or slide review. CONCLUSIONS: The EGC% generated by the DxH 800 can be easily incorporated into existing HematoFlow and slide review algorithms.

Refining the white blood cell differential: the first flow cytometry routine application.

A Complete Blood Count performed by an automated hematology analyzer frequently needs a microscopic slide review. This step is time consuming and requires experienced personnel. Recently, several teams have proposed and validated convenient combinations of monoclonal antibodies for an extended white blood cell (WBC) differential by flow cytometry. The aim of this study was to evaluate the usefulness of this approach in the routine workflow of a hematology laboratory. We compared a workflow chain comprised of a robotic blood preparation system (for antibody labeling), a flow cytometer, and data management software to the standard manual review of a blood film and evaluated the diagnostic quality, the turnaround time, and the labor needed for the two different approaches. The study on 1,973 samples was organized, firstly, to determine analytic thresholds and these settings were then validated. The flow cytometric data management software automatically validated 52% of the samples without significant numbers of false negatives. Of the remaining specimens, an operator validated a further 33% of the samples and 15% needed a manual microscopic review. These results were obtained in a mean timeline similar to the traditional microscopic manual review. Our study demonstrates, for the first time, the efficiency of a flow cytometer integrated into a WBC differential workflow in a routine hematology laboratory.

CD9 expression can be used to predict childhood TEL/AML1-positive acute lymphoblastic leukemia: proposal for an accelerated diagnostic flowchart.

CD9 has been shown to be differentially expressed in childhood TEL/AML1-positive acute lymphoblastic leukemia (ALL). We confirmed this finding in large Affymetrix data sets and in 80 new cases at both RNA and protein levels. Moreover, we showed that mean fluorescence intensity of CD9 by flow cytometry can distinguish TEL/AML1-positive ALL from other BCP-ALL. Using ROC analysis, the most efficient model for predicting TEL/AML1-positive ALL combined CD9 (mean fluorescence intensity 40%). Finally, we propose a faster procedure for optimizing the diagnosis of childhood BCP-ALL subgroups.

Five distinct biological processes and 14 differentially expressed genes characterize TEL/AML1-positive leukemia.

BACKGROUND: The t(12;21)(p13;q22) translocation is found in 20 to 25% of cases of childhood B-lineage acute lymphoblastic leukemia (B-ALL). This rearrangement results in the fusion of ETV6 (TEL) and RUNX1 (AML1) genes and defines a relatively uniform category, although only some patients suffer very late relapse. TEL/AML1-positive patients are thus an interesting subgroup to study, and such studies should elucidate the biological processes underlying TEL/AML1 pathogenesis. We report an analysis of gene expression in 60 children with B-lineage ALL using Agilent whole genome oligo-chips (44K-G4112A) and/or real time RT-PCR. RESULTS: We compared the leukemia cell gene expression profiles of 16 TEL/AML1-positive ALL patients to those of 44 TEL/AML1-negative patients, whose blast cells did not contain any additional recurrent translocation. Microarray analyses of 26 samples allowed the identification of genes differentially expressed between the TEL/AML1-positive and negative ALL groups. Gene enrichment analysis defined five enriched GO categories: cell differentiation, cell proliferation, apoptosis, cell motility and response to wounding, associated with 14 genes -RUNX1, TCFL5, TNFRSF7, CBFA2T3, CD9, SCARB1, TP53INP1, ACVR1C, PIK3C3, EGFL7, SEMA6A, CTGF, LSP1, TFPI - highlighting the biology of the TEL/AML1 sub-group. These results were first confirmed by the analysis of an additional microarray data-set (7 patient samples) and second by real-time RT-PCR quantification and clustering using an independent set (27 patient samples). Over-expression of RUNX1 (AML1) was further investigated and in one third of the patients correlated with cytogenetic findings. CONCLUSION: Gene expression analyses of leukemia cells from 60 children with TEL/AML1-positive and -negative B-lineage ALL led to the identification of five biological processes, associated with 14 validated genes characterizing and highlighting the biology of the TEL/AML1-positive ALL sub-group.

Late ovarian relapse of TEL/AML1 positive ALL confirming that TEL deletion is a secondary event in leukemogenesis.

We describe here a late extramedullary ovarian relapse in an 18-year-old female who was diagnosed with hypotetraploid cell acute lymphoblastic leukaemia (cALL) at the age of 6. At both occurrences of the disease cells were analyzed by morphology, immunophenotyping, cytogenetics and molecular methods. TEL/AML1 was detected by RT-PCR and FISH analysis in both events. We demonstrated, using detection of IGH/TCR rearrangements and TEL/AML1 breakpoints sequencing that the cells were clonally related. Moreover, interphasic FISH using TEL and AML1 probes showed the loss of a second TEL at the time of relapse. This observation confirms that TEL/AML1 alone is not sufficient to trigger ALL and that TEL deletion is a secondary event in leukemogenesis. To our knowledge, it is the first complete description of extramedullary ALL relapse combining all methodologies.

Impact of Topoisomerase II alpha and spermine on the clinical outcome of children with acute lymphoblastic leukemia.

It has been reported in the literature that a leukemic cell may be (or become) resistant to anti-cancer treatment because many mechanisms, such as efflux membrane pump (multi-drug resistance, MDR-P170), intracellular transport (LRP, MRP), or different detoxification systems (glutathione transferases, methallothioneines) may be implicated. Topoisomerase II alpha (TopoII) are also reported as responsible for resistance since their main action is to repair DNA breakage. Polyamines are described as having a protective DNA action by stabilizing the double stranded DNA helix. For these reasons we investigated 65 children with acute lymphoblastic leukemia using an immunocytochemical method to elucidate the potential role of Topoisomerase and polyamines in drug resistance. Most children (60/65) were treated with the French (acute lymphoblastic leukemia, ALL) protocol (FRALLE-93) in which B and C arms include (at least) VP16. Children with cytoplasmic TopoII positivity (18 cases) were more resistant since their overall survival was 34 months compared to more than 110 months for negative cases ( P = 0.0003). Polyamines may be associated with drug resistance since the overall survivals were 51 months and 92 months for positive and negative patients, respectively, but the P-value is only 0.13. We conclude that Topoisomerase and polyamines must be tested at diagnosis as new possible markers for chemo-resistance. Larger series are needed to confirm these preliminary results and to verify if the use of anti epipodophillotoxin agents (as it is the case for FRALLE B or C) should be excluded for positive cases.