[The application of CytoDiff for the differential diagnosis of reactive and tumor lymphocytosis.] / Применение реактива CytoDiff для дифференциальной диагностики реактивного и Ð¾Ð¿ÑƒÑ Ð¾Ð»ÐµÐ²Ð¾Ð³Ð¾ лимфоцитоза.

CytoDiff reagent was developed to optimize the performance of a WBC differential evaluation. It determines the main population of white blood cells by the expression of linear markers. One of the additional possibilities of using CytoDiff is the differential diagnosis of reactive and tumor lymphocytosis. We studied peripheral blood samples of 76 patients of the All-Russian Central Research Center A.M. Nikiforova EMERCOM of Russia with absolute lymphocytosis of more than 3,0x109/l. The control group included 26 practically healthy people. All performed a clinical blood test on a 5Diff hematology analyzer with smear microscopy, and determination of leukocyte populations by phenotype using CytoDiff. Reference intervals were determined for subpopulations of lymphocytes using CytoDiff. An algorithm has been developed for evaluating the results obtained when determining leukocyte populations by phenotype using CytoDiff for differential diagnosis of reactive and tumor lymphoproliferation. To detect B-cell lymphoproliferative diseases, the use of a cut-off value of 13% or more of the number of leukocytes is optimal. At low values of the relative number of B-lymphocytes, it is important to take into account the results of microscopy of blood smears. If atypical mononuclear cells are absent in smears, then additional clinical and laboratory studies are necessary to establish the cause of lymphocytosis, including phenotyping of peripheral blood lymphocytes to exclude T-cell lymphoproliferative diseases. The expediency of using the CytoDiff reagent for the differential diagnosis of the reactive and tumor nature of lymphocytosis is shown. Already at the stage of primary screening studies, the use of CytoDiff makes it possible to efficiently collect blood samples from patients with possible lymphoproliferative diseases, which significantly reduces the time required for a diagnostic search.

Evaluation of the detection of blasts by Sysmex hematology instruments, CellaVision DM96, and manual microscopy using flow cytometry as the confirmatory method.

INTRODUCTION: The aim of the present study was to evaluate the diagnostic ability of blast flags generated by Sysmex instruments (XE/XN) by comparing with immunophenotyping by flow cytometry (IFCM). Additionally, the ability of manual microscopy and CellaVision DM96 (pre- and reclassification) to predict the presence of "true" blasts was investigated. METHODS: Blood samples (n = 240) with suspect pathology flags reported by the XE were collected from the daily workload and examined by the XN, by manual microscopy, by CellaVision DM96 and by IFCM (CytoDiff Panel). RESULTS: The ROC analysis for blasts showed an area under the curve of 0.64 ("Blasts?") (XE), 0.57 ("Blasts/Abn Lympho?") (XN), 0.75 (CellaVision preclassification procedure), 0.78 (CellaVision reclassification procedure), and 0.81 (manual microscopy). The sensitivity of blast detection varied between the methods from 0.41 (XE) to 0.90 (XN), and the specificity varied from 0.17 (XN) to 0.95 (CellaVision reclassification). CONCLUSIONS: The CellaVision reclassification procedure has a diagnostic ability for predicting blasts close to that of manual microscopy. The blood smear methods show a notable number of false negative results. The Sysmex XN reported a higher rate of true positive blast flags than the XE. Taken together, the CytoDiff method could be a useful alternative to smear examination to correctly identify blasts.

Upregulation of CD16- monocyte subsets in systemic lupus erythematous patients.

Monocytes are an important component in the innate immune system. However, studies to date have failed to conclude whether their levels are altered in patients with systemic lupus erythematosus (SLE). We applied the cytodiff counting method and comprehensively measured the circulating levels of distinct white blood cell (WBC) subsets, including CD16+, CD16-, and total monocytes, in 61 SLE patients as well as in 203 age-matched healthy controls (HCs). The absolute number of CD16- monocytes, total monocytes, immature granulocytes, mature neutrophils, total neutrophils, and T cell blasts was significantly higher, that of non-cytotoxic T lymphocytes, cytotoxic T + NK lymphocytes, T + NK lymphocytes, total lymphocytes, basophils, and eosinophils significantly lower (all p < 0.05), but that of CD16+ monocytes, B lymphocytes, B cell blasts, non-B and non-T cell blasts, and total blasts was not statistically different in SLE patients, as compared to HC. Specifically, among all subsets examined, the percentage of CD16- monocytes and total monocytes was the only one that could discriminate active SLE from quiescent SLE (p = 0.033 and 0.026, respectively). SLE patients with lupus nephritis were also associated with higher levels of circulating CD16- monocytes and total monocytes, in comparison with that of controls (both p < 0.0001). This study suggests the significance of distinct WBC subsets, particularly the differential regulations of monocyte subsets, in the pathogenesis and development of SLE.

Flow cytometric white blood differential using CytoDiff™ in the diagnosis of neonatal early-onset infection.

OBJECTIVE: Neonatal early-onset infection is a life-threatening disease, requiring early diagnosis and treatment. Newborns at risk are identified by a combination of risk factors, clinical signs of infection and laboratory parameters such as white blood cell count and C-reactive protein (CRP). This method is labor-intensive, time consuming and has a variable reproducibility. New reliable diagnostic markers are needed to identify neonatal infection. This study presents additional leukocyte differential parameters produced by the automated flow cytometry and processing software using CytoDiff™ reagent (Beckman Coulter) in newborns suspected for early-onset infection. METHODS: An analytic prospective observational case-control study was performed in which 185 newborns were included and retrospectively allocated into two groups, "infection likely" and "infection unlikely". Leukocyte parameters of the CytoDiff™ technique were compared with microscopic slide differentiation and routine tests. RESULTS: We showed significant lower numbers of monocytes, CD16(-) monocytes and lymphocytes (including T+/NK-lymphocytes) in neonates suspected for early-onset infection using CytoDiff™ technique. The manual counting did not demonstrate changes with respect to the number of monocytes in these neonates. CONCLUSIONS: The automated routine CytoDiff™ leukocyte differential provides an interesting additional diagnostic tool, next to routine laboratory diagnostics, in the diagnosis of neonatal early-onset infection.

Prognostic implication of leucocyte subpopulations in diffuse large B-cell lymphoma.

BACKGROUND: Recent studies have suggested that variables related to host adaptive immunity and the tumor microenvironment may predict the outcome in patients with non-Hodgkin's lymphoma. This study was undertaken to determine the prognostic value of peripheral blood leucocyte subpopulations in diffuse large-B-cell lymphoma patients. METHODS: We prospectively analyzed the 16 leukocyte subpopulations using Cytodiff flow cytometric technique in a cohort of 45 diffuse large-B-cell lymphoma patients at a single institution between February and December 2014. The Cox proportional hazards model was used to evaluate prognostic factors for overall survival and progression free survival. RESULTS: Diffuse large-B-cell lymphoma patients had decreased cytotoxic and non-cytotoxic NK&T cells as well as increased CD16+ monocytes, CD16- monocytes and mature neutrophils. The decreased CD16- monocyte/CD16+ monocyte ratio and increased mature neutrophil/cytotoxic NK&T cell ratio were related to poor progression-free and overall survival outcome in single and multivariate analysis. The co-constructed model using International Prognostic Index and mature neutrophil/cytotoxic NK&T cell ratio can also help discriminate the clinical outcome. CONCLUSIONS: The decreased CD16-monocyte/CD16+monocyte ratio and increased mature neutrophil/cytotoxic NK&T cell ratio predict poor prognosis in diffuse large-B-cell lymphoma patients. This finding provides a strong rationale for the study of cellular immunotherapy in B-cell lymphoma.

Flow cytometric white blood cell differential using CytoDiff is excellent for counting blasts.

BACKGROUND: The usefulness of the CytoDiff flow cytometric system (Beckman Coulter, USA) has been studied in various conditions, but its performance including rapidity in detecting and counting blasts, the most significant abnormal cells in the peripheral blood, has not been well evaluated. The objective of this study was to evaluate the performance of the CytoDiff differential counting method in challenging samples with blasts. METHODS: In total, 815 blood samples were analyzed. Samples flagged as "blasts" or "variant lymphocytes" and showing <10% blasts by manual counts were included. In total, 322 samples showed blasts on manual counts, ranging from 0.5% to 99%. The CytoDiff method was performed by flow cytometry (FC500; Beckman Coulter, USA) with a pre-mixed CytoDiff reagent and analyzing software (CytoDiff CXP 2.0; Beckman Coulter). RESULTS: The average time required to analyze 20 samples was approximately 60 min for manual counts, and the hands-on time for the CytoDiff method was 15 min. The correlation between the CytoDiff and manual counts was good (r>0.8) for neutrophils and lymphocytes but poor (r<0.8) for other cells. When the cutoff value of the CytoDiff blast count was set at 1%, the sensitivity was 94.4% (95% CI; 91.2-96.6) and specificity was 91.9% (95% CI; 89.0-94.1). The positive predictive value was 88.4% (95% CI; 84.4-91.5) (304/344 cases) and negative predictive value was 96.2% (95% CI; 93.9-97.7) (453/471 cases). The CytoDiff blast counts correlated well to the manual counts (r=0.9223). CONCLUSIONS: The CytoDiff method is a specific, sensitive, and rapid method for counting blasts. A cutoff value of 1% of at least 1 type of blast is recommended for positive CytoDiff blast counts.

An extended leukocyte differential count (16 types of circulating leukocytes) using the CytoDiff flow cytometric system can provide information for the discrimination of sepsis severity and prediction of outcome in sepsis patients.

BACKGROUND: The Beckman Coulter CytoDiff flow cytometric system (Beckman Coulter, Miami, FL) was recently developed for performing leukocyte differential counts in up to 16 leukocyte subpopulations. We compared these leukocyte subpopulation levels among patients with three stages of sepsis (uncomplicated sepsis, severe sepsis, septic shock), especially focused on the discrimination of complicated sepsis from uncomplicated sepsis. METHODS: We examined a total of 181 samples with sepsis who were admitted to the surgical intensive care unit. In addition, we examined samples obtained from 60 normal healthy volunteers. Both the proportions and absolute numbers of each cell type in the four groups were obtained using the CytoDiff flow cytometric system and compared. RESULTS: Mature neutrophils and immature granulocytes failed to discriminate patients with complicated sepsis from those with uncomplicated sepsis although their absolute numbers were increased compared with normal controls. In contrast, almost all lymphocyte subpopulations and CD16(-) monocytes decreased significantly in patients with complicated sepsis compared with uncomplicated sepsis. Among them, only B lymphocytes showed independent ability to discriminate two groups. Both B lymphocytes and CD16(-) monocytes possessed a significant adverse prognostic impact on overall survival when their absolute numbers decreased. CONCLUSIONS: Almost all lymphocyte subpopulations and CD16(-) monocytes decrease in size with increasing sepsis severity. Among them, only B lymphocytes showed independent ability to discriminate patients with complicated sepsis from those with uncomplicated sepsis. Both B lymphocytes and CD16 (-) monocytes show a significant adverse prognostic impact on overall survival outcomes in sepsis patients when their absolute numbers are decreased.

An extended leukocyte differential count (16 types of circulating leukocytes) using the cytodiff flow cytometric system can provide informations for the discrimination of sepsis severity and prediction of outcome in sepsis patients.

Background: The Beckman Coulter CytoDiff flow cytometric system (Beckman Coulter, Miami, FL, USA) was recently developed for performing leukocyte differential counts in up to 16 leukocyte subpopulations. We compared these leukocyte subpopulation levels among patients with three stages of sepsis (uncomplicated sepsis, severe sepsis, septic shock), especially focused on the discrimination of complicated sepsis from uncomplicated sepsis. Methods: We examined a total of 181 samples with sepsis who were admitted to the surgical intensive care unit. In addition, we examined samples obtained from 60 normal healthy volunteers. Both the proportions and absolute numbers of each cell type in the four groups were obtained using the CytoDiff flow cytometric system and compared. Results: Mature neutrophils and immature granulocytes failed to discriminate patients with complicated sepsis from those with uncomplicated sepsis although their absolute numbers were increased compared with normal controls. In contrast, almost all lymphocyte subpopulations and CD16(-) monocytes decreased significantly in patients with complicated sepsis compared with uncomplicated sepsis. Among them, only B lymphocytes showed independent ability to discriminate two groups. Both B lymphocytes and CD16(-) monocytes possessed a significant adverse prognostic impact on overall survival when their absolute numbers decreased. Conclusions: Almost all lymphocyte subpopulations and CD16(-) monocytes decrease in size with increasing sepsis severity. Among them, only B lymphocytes showed independent ability to discriminate patients with complicated sepsis from those with uncomplicated sepsis. Both B lymphocytes and CD16(-) monocytes show a significant adverse prognostic impact on overall survival outcomes in sepsis patients when their absolute numbers are decreased. © 2013 Clinical Cytometry Society.

Comparison of the Cytodiff flow cytometric leucocyte differential count system with the Sysmex XE-2100 and Beckman Coulter UniCel DxH 800.

INTRODUCTION: Routine automated haematology analysers categorize leucocytes into five types. The Cytodiff (Beckman Coulter) is a 16-part leucocyte differential analysis system that uses six markers and five colours. We compared leucocyte differential counts obtained by the Cytodiff with five-part differential counts obtained by routine automated haematology analysers. METHODS: We collected 477 EDTA blood samples from healthy individuals and patients with malignancies, sepsis and multi-organ failure. Leucocyte differential counts were simultaneously analysed by a Cytodiff multiparametric flow cytometric system and the XE-2100 (Sysmex) and UniCel DxH 800 (Beckman Coulter) automated haematology analysers. Regression and correlation analyses were performed between the different systems. RESULTS: Our Cytodiff results were well correlated with those produced using the DxH 800 and XE-2100 analysers except for monocytes and basophils. The correlations were poorer for leukopenic than for nonleukopenic samples. For most samples, Cytodiff obtained a higher correlation with manual counts according to a case analysis; however, in several samples, the Cytodiff generated false decreases in monocyte levels and false increases in basophil levels. CONCLUSION: The Cytodiff may have an advantage, as it could sensitively detect blasts and immature granulocytes. Additionally, it was less labour-intensive than manual counting, and therefore, the Cytodiff might be useful for differential counts.

Reliable, accurate determination of the leukocyte differential of leukopenic samples by using Hematoflow method.

BACKGROUND: Hematology analyzers may ineffectively recognize abnormal cells, and manual differential counts may be imprecise for leukopenic samples. We evaluated the efficacy of the Hematoflow method for determining the leukocyte differential in leukopenic samples and compared this method with the manual differential method. METHODS: We selected 249 blood samples from 167 patients with leukopenia (WBC counts, 500-2,000/µL) for analysis in this study. The EDTA-anticoagulated blood samples were analyzed using an automatic blood cell counter (DxH800; Beckman Coulter, USA) and flow cytometry (FC 500; Beckman Coulter) by using Cytodiff reagent and analysis software (Beckman Coulter). Hematoflow results were selected or calculated from DxH800 and Cytodiff results. Two trained pathologists performed a manual differential count by counting 50-100 cells. RESULTS: The precision of the Hematoflow method was superior to that of the manual method in counting 5 leukocyte subpopulations, immature granulocytes (IGs), and blasts. Blasts were detected in all 45 cases (100%) by Hematoflow. The correlation of the Cytodiff blast count to the reference count was high (r = 0.8325). For all other cell populations, the correlation of the Hematoflow results with the reference count was stronger than that of the other manual counts with the reference count. CONCLUSIONS: The Hematoflow differential counting method is more reproducible and sensitive than manual counting, and is relatively easy to perform. In particular, this method detected leukemic blasts more sensitively than manual differential counts. The Hematoflow method is a very useful supplement to automated cell counting.

Flow Cytometric White Blood Cell Differential Using CytoDiff is Excellent for Counting Blasts

BACKGROUND: The usefulness of the CytoDiff flow cytometric system (Beckman Coulter, USA) has been studied in various conditions, but its performance including rapidity in detecting and counting blasts, the most significant abnormal cells in the peripheral blood, has not been well evaluated. The objective of this study was to evaluate the performance of the CytoDiff differential counting method in challenging samples with blasts. METHODS: In total, 815 blood samples were analyzed. Samples flagged as "blasts" or "variant lymphocytes" and showing 0.8) for neutrophils and lymphocytes but poor (r<0.8) for other cells. When the cutoff value of the CytoDiff blast count was set at 1%, the sensitivity was 94.4% (95% CI; 91.2-96.6) and specificity was 91.9% (95% CI; 89.0-94.1). The positive predictive value was 88.4% (95% CI; 84.4-91.5) (304/344 cases) and negative predictive value was 96.2% (95% CI; 93.9-97.7) (453/471 cases). The CytoDiff blast counts correlated well to the manual counts (r=0.9223). CONCLUSIONS: The CytoDiff method is a specific, sensitive, and rapid method for counting blasts. A cutoff value of 1% of at least 1 type of blast is recommended for positive CytoDiff blast counts.

Reliable, Accurate Determination of the Leukocyte Differential of Leukopenic Samples by Using Hematoflow Method / 대한진단검사의학회지

BACKGROUND: Hematology analyzers may ineffectively recognize abnormal cells, and manual differential counts may be imprecise for leukopenic samples. We evaluated the efficacy of the Hematoflow method for determining the leukocyte differential in leukopenic samples and compared this method with the manual differential method. METHODS: We selected 249 blood samples from 167 patients with leukopenia (WBC counts, 500-2,000/microL) for analysis in this study. The EDTA-anticoagulated blood samples were analyzed using an automatic blood cell counter (DxH800; Beckman Coulter, USA) and flow cytometry (FC 500; Beckman Coulter) by using Cytodiff reagent and analysis software (Beckman Coulter). Hematoflow results were selected or calculated from DxH800 and Cytodiff results. Two trained pathologists performed a manual differential count by counting 50-100 cells. RESULTS: The precision of the Hematoflow method was superior to that of the manual method in counting 5 leukocyte subpopulations, immature granulocytes (IGs), and blasts. Blasts were detected in all 45 cases (100%) by Hematoflow. The correlation of the Cytodiff blast count to the reference count was high (r = 0.8325). For all other cell populations, the correlation of the Hematoflow results with the reference count was stronger than that of the other manual counts with the reference count. CONCLUSIONS: The Hematoflow differential counting method is more reproducible and sensitive than manual counting, and is relatively easy to perform. In particular, this method detected leukemic blasts more sensitively than manual differential counts. The Hematoflow method is a very useful supplement to automated cell counting.

Reliable, Accurate Determination of the Leukocyte Differential of Leukopenic Samples by Using Hematoflow Method / 대한진단검사의학회지

BACKGROUND: Hematology analyzers may ineffectively recognize abnormal cells, and manual differential counts may be imprecise for leukopenic samples. We evaluated the efficacy of the Hematoflow method for determining the leukocyte differential in leukopenic samples and compared this method with the manual differential method. METHODS: We selected 249 blood samples from 167 patients with leukopenia (WBC counts, 500-2,000/microL) for analysis in this study. The EDTA-anticoagulated blood samples were analyzed using an automatic blood cell counter (DxH800; Beckman Coulter, USA) and flow cytometry (FC 500; Beckman Coulter) by using Cytodiff reagent and analysis software (Beckman Coulter). Hematoflow results were selected or calculated from DxH800 and Cytodiff results. Two trained pathologists performed a manual differential count by counting 50-100 cells. RESULTS: The precision of the Hematoflow method was superior to that of the manual method in counting 5 leukocyte subpopulations, immature granulocytes (IGs), and blasts. Blasts were detected in all 45 cases (100%) by Hematoflow. The correlation of the Cytodiff blast count to the reference count was high (r = 0.8325). For all other cell populations, the correlation of the Hematoflow results with the reference count was stronger than that of the other manual counts with the reference count. CONCLUSIONS: The Hematoflow differential counting method is more reproducible and sensitive than manual counting, and is relatively easy to perform. In particular, this method detected leukemic blasts more sensitively than manual differential counts. The Hematoflow method is a very useful supplement to automated cell counting.