Automated Analysis of Cerebrospinal Fluid Cells Using Commercially Available Blood Cell Analysis Devices-A Critical Appraisal.

The analysis of cells in the cerebrospinal fluid (CSF) is a routine procedure that is usually performed manually using the Fuchs-Rosenthal chamber and cell microscopy for cell counting and differentiation. In order to reduce the requirement for manual assessment, automated analyses by devices mainly used for blood cell analysis have been also used for CSF samples. Here, we summarize the current state of investigations using these automated devices and critically review their limitations. Despite technical improvements, the lower limit for reliable leukocyte counts in the CSF is still at approximately 20 cells/µL, to be validated depending on the device. Since the critical range for clinical decisions is in the range of 5-30 cells/µL this implies that cell numbers < 30/µL require a manual confirmation. Moreover, the lower limit of reliable erythrocyte detection by automated devices is at approximately 1000/µL. However, even low erythrocyte numbers may be of clinical importance. In contrast, heavily hemorrhagic samples from neurosurgery may be counted automatically at an acceptable precision more quickly. Finally, cell differentiation by automated devices provides only a rough orientation for lymphocytes, granulocytes and monocytes. Other diagnostically important cell types such as tumor cells, siderophages, blasts and others are not reliably detected. Thus, although the automation may give a gross estimate sufficient for the emergency room situation, each CSF requires a manual microscopy for cytological evaluation for the final report. In conclusion, although automated analysis of CSF cells may provide a first orientation of the cell profile in an individual sample, an additional manual cell count and a microscopic cytology are still required and represent the gold standard.

Hemocytometric characteristics of COVID-19 patients with and without cytokine storm syndrome on the sysmex XN-10 hematology analyzer.

OBJECTIVES: COVID-19 is an ongoing global pandemic. There is an urgent need for identification and understanding of clinical and laboratory parameters related to progression towards a severe and fatal form of this illness, often preceded by a so-called cytokine-storm syndrome (CSS). Therefore, we explored the hemocytometric characteristics of COVID-19 patients in relation to the deteriorating clinical condition CSS, using the Sysmex XN-10 hematology analyzer. METHODS: From March 1st till May 16th, 2020, all patients admitted to our hospital with respiratory complaints and suspected for COVID-19 were included (n=1,140 of whom n=533 COVID-19 positive). The hemocytometric parameters of immunocompetent cells in peripheral blood (neutrophils [NE], lymphocytes [LY] and monocytes [MO]) obtained upon admission to the emergency department (ED) of COVID-19 positive patients were compared with those of the COVID-19 negative ones. Moreover, patients with CSS (n=169) were compared with COVID-19 positive patients without CSS, as well as with COVID-19 negative ones. RESULTS: In addition to a significant reduction in leukocytes, thrombocytes and absolute neutrophils, it appeared that lymphocytes-forward scatter (LY-FSC), and reactive lymphocytes (RE-LYMPHO)/leukocytes were higher in COVID-19-positive than negative patients. At the moment of presentation, COVID-19 positive patients with CSS had different neutrophils-side fluorescence (NE-SFL), neutrophils-forward scatter (NE-FSC), LY-FSC, RE-LYMPHO/lymphocytes, antibody-synthesizing (AS)-LYMPHOs, high fluorescence lymphocytes (HFLC), MO-SSC, MO-SFL, and Reactive (RE)-MONOs. Finally, absolute eosinophils, basophils, lymphocytes, monocytes and MO-FSC were lower in patients with CSS. CONCLUSIONS: Hemocytometric parameters indicative of changes in immunocompetent peripheral blood cells and measured at admission to the ED were associated with COVID-19 with and without CSS.

Comparison of methods and TAT assessment: Volumetric AQUIOS CL and bead-based FACS CANTO II cytometers.

BACKGROUND: Measurement of lymphocyte subpopulations is a crucial parameter in the diagnosis and monitoring of therapy in a wide variety of clinical conditions. We compared different flow cytometry-based methods to determine lymphocyte subsets counts of routine samples by volumetric AQUIOS CL (Beckman Coulter) and bead-based FACS CANTO II (BD Biosciences) cytometers. We evaluated the possible decrease of the labor intensive technical work using the fully automated AQUIOS CL system in the pre and post analytical steps in comparison with our routine flow cytometer FACS CANTO II, toward the reduction of laboratory analytical turnaround time (TAT). METHODS: The analytical performance of AQUIOS CL flow cytometer compared with FACS CANTO II was evaluated testing 224 routine samples, attending the Immunology and Allergology Laboratory Unit, S. Giovanni di Dio Hospital, (Florence, Italy) between September and October 2015. RESULTS: Bland-Altman plot of the differences between the two methods showed an excellent agreement for absolute and percentage counts. Furthermore, the study showed that automated AQUIOS CL system is simple to be used during all analytical steps with a reduction of TAT. CONCLUSION: In routine conditions, AQUIOS CL flow cytometer could be a suitable tool for subpopulations subset analysis. © 2017 International Clinical Cytometry Society.

Evaluation of the Flagging Performance of the Hematology Analyzer Sysmex XN Series on the Basis of "Q Values".

CONTEXT: - In the XN series of hematology analyzers (Sysmex, Kobe, Japan), the probability of the presence of abnormal cells is indicated by flags based on Q values. OBJECTIVE: - To evaluate the Q value performance of the Sysmex XN-20 modular analyzer. DESIGN: - The interinstrumental concordance, intrainstrumental precision, and diagnostic accuracy of Q values, with tested flags of "blasts/abnormal lymphocytes," "atypical lymphocytes," and "blasts," were investigated. RESULTS: - Absolute concordance rates in flagging between 2 analyzers ranged from 69.8% to 80.8%, and κ values ranged from 0.43 to 0.61. In samples with absolute related cell counts lower than 100/µL, the values ranged from 0.31 to 0.52. For intrainstrumental precision, standard deviations ranged from 4.8 to 23.9 for the blasts/abnormal lymphocytes, from 18.7 to 59.1 for the blasts, and from 11.0 to 23.0 for the atypical lymphocytes. Using a default Q value cutoff, diagnostic accuracy values based on the area under the curve, sensitivity, and specificity were, respectively, 0.910, 90.9%, and 72.2% for blasts/abnormal lymphocytes; 0.927, 84.9%, and 89.8% for blasts; and 0.865, 74.4%, and 84.9% for atypical lymphocytes. The diagnostic accuracy of Q values was much lower in samples with absolute related cell counts lower than 100/µL than in those 100/µL or higher. CONCLUSIONS: - Q values of the Sysmex XN-20 analyzer were found to be imprecise and irreproducible, especially with samples containing a small number of pathologic cells. Adjustments in the Q value threshold may help in the detection of these cells.

Single wall carbon nanotube electrode system capable of quantitative detection of CD4+ T cells.

Development of CNT-based CD4+ T cell imunosensors remains in its infancy due to the poor immobilization efficiency, lack of reproducibility, and difficulty in providing linear quantification. Here, we developed a fully-integrated single wall carbon nanotube (SWCNT)-based immunosensor capable of selective capture and linear quantification of CD4+ T cells with greater dynamic range. By employing repeated two-step oxygen (O2) plasma treatment processes with 35 days of recovery periods, we achieved the enhanced functionalization of the CNT surface and the removal of the byproduct of spray-coated SWCNTs that hinders charge transfer and stable CD4+ T cell sensing. As a result, a linear electrochemical signal was generated in direct proportion to the bound cells. The slope of a SWCNT electrode in a target concentration range (102~106cells/mL) was 4.55×10-2µA per concentration decade, with the lowest detection limit of 1×102cells/mL. Since the reduced number of CD4+ T cell counts in patients' peripheral blood corresponds to the progression of HIV disease, our CD4+ T cell-immunosensor provides a simple and low-cost platform which can fulfill the requirement for the development of point-of-care (POC) diagnostic technologies for human immunodeficiency virus (HIV) patients in resource-limited countries.

Human T cells monitored by impedance spectrometry using field-effect transistor arrays: a novel tool for single-cell adhesion and migration studies.

Cytotoxic T lymphocytes (CTLs) play an important role in the immune system by recognizing and eliminating pathogen-infected and tumorigenic cells. In order to achieve their function, T cells have to migrate throughout the whole body and identify the respective targets. In conventional immunology studies, interactions between CTLs and targets are usually investigated using tedious and time-consuming immunofluorescence imaging. However, there is currently no straightforward measurement tool available to examine the interaction strengths. In the present study, adhesion strengths and migration of single human CD8(+) T cells on pre-coated field-effect transistor (FET) devices (i.e. fibronectin, anti-CD3 antibody, and anti-LFA-1 antibody) were measured using impedance spectroscopy. Adhesion strengths to different protein and antibody coatings were compared. By fitting the data to an electronically equivalent circuit model, cell-related parameters (cell membrane capacitance referring to cell morphology and seal resistance referring to adhesion strength) were obtained. This electronically-assessed adhesion strength provides a novel, fast, and important index describing the interaction efficiency. Furthermore, the size of our detection transistor gates as well as their sensitivity reaches down to single cell resolution. Real-time motions of individually migrating T cells can be traced using our FET devices. The in-house fabricated FETs used in the present study are providing a novel and very efficient insight to individual cell interactions.

Automated quantification of apoptosis in B-cell chronic lymphoproliferative disorders: a prognostic variable obtained with the Cell-Dyn Sapphire (Abbott) automated hematology analyzer.

INTRODUCTION: B-chronic lymphocytic leukemia CLL, a neoplastic clonal disorder with monomorphous small B lymphocytes with scanty cytoplasm and clumped chromatin, can be morphologically differentiated in typical and atypical forms with different prognosis: Smudge cells (Gumprecht's shadows) are one of the well-known features of the typical CLL and are much less inconsistent in other different types CLPD. Abbott Cell-Dyn Sapphire uses the fluorescence after staining with the DNA fluorochrome propidium iodide for the measurement of nucleated red blood cells (NRBCs) and nonviable cells (FL3+ cell fraction): We have studied the possible correlation between presence and number of morphologically identifiable smudge cells on smears and the percentage of nonviable cells produced by Cell-Dyn Sapphire. METHODS: 305 blood samples from 224 patients with B-cell lymphoproliferative disorders and 40 healthy blood donors were analyzed by CBC performed by Cell-Dyn Sapphire, peripheral blood smear, and immunophenotype characterization. RESULT: FL3+ fraction in CLPD directly correlated with the percentage of smudge cells and is significantly increased in patients with typical B-CLL. This phenomenon is much less evident in patients with atypical/mixed B-CLL and B-NHL. CONCLUSION: In small laboratories without FCM and cytogenetic, smudge cells%, can be utilized as a preliminary diagnostic and prognostic tool in differential diagnosis of CLPD.

The lymph index: a potential hematological parameter for viral infection.

OBJECTIVE: An LH750 hematology analyzer with VCS (volume, conductivity, and light scatter) technology can determine morphologic properties of peripheral leukocytes, known as cell population data (CPD). We have previously demonstrated that the lymphocyte CPD exhibit significant changes in acute hepatitis B virus infection. A simplified lymphocyte CPD, the lymph index, was proposed. We conducted the current study to further evaluate the clinical usefulness of the lymph index, and included patients with various viral infections, as well as those with acute bacterial infections. METHODS: Peripheral blood was collected from 72 patients with viral infections, 46 patients with acute bacterial infections, and 204 controls. The lymphocyte CPD included the mean volume (LV) with its standard deviation (LV-SD) and the conductivity (LC). The lymph index was calculated as LV × LV-SD ÷ LC. RESULTS: The lymph index was significantly increased in viral infections and only mildly increased in acute bacterial infections compared to controls. Using a lymph index cutoff value of ≥ 12.92, we achieved 91.67% sensitivity and 97.2% specificity for diagnosing viral infection. CONCLUSIONS: The findings may be clinically useful since these morphological parameters are readily obtained by hematology analyzer during automated leukocyte differentials. They are quantitative, objective, and fast. The lymph index could be a potential hematological parameter for viral infection.

Pertinence of the Sysmex XE-5000™ parameters: rule of slide review in a context of 'normal' lymphocyte count (defined from control and mantle cell lymphoma blood specimens).

INTRODUCTION: As most hematology cell analyzers, the different parameters of Sysmex XE-5000™ are little informative in the qualitative analysis of lymphoid cells, and especially when the lymphocyte count is below 4 × 10(9) /L (i.e., 'normal'). The aim of our study was to investigate whether some parameters and/or 'flags' not routinely provided by this analyzer, but reachable by operator could be reliable to define rules of slide review in absence of common qualitative and quantitative alarms particularly in case of 'normal' lymphocyte count. METHODS: Blood samples from 13 mantle cell lymphoma fully annotated cases, and 180 control specimens were studied with Sysmex XE-5000™ analyzer. All cases did not present any anomalies in common quantitative and structural parameters. RESULTS: Using the method of area under the curve and ROC curve analysis, we described a novel threshold of alarm VAL_ABN LYMPH (≥40 instead of 100 defined by Sysmex), as well as a pertinent LyX threshold (≥89). The combination of these thresholds allowed defining a rule of slide review in context of 'normal' lymphocyte count. CONCLUSION: Among the parameters provided by the Sysmex XE-5000™ analyzer, the combination of the alarm VAL_ABN LYMPH and the LyX value, routinely available on a simple blood analysis appears particularly informative to trigger slide review in a context of 'normal' lymphocyte count with a good sensitivity (85%) to detect circulating lymphoma cells and with <1% of false positive results.

Utilidad del recuento e inmunofenotipaje de los linfocitos intraepiteliales de la mucosa intestinal en el diagnóstico de la enfermedad celiaca / Usefulness of intraepithelial lymphocytes immunophenotyping in intestinal mucosa for the diagnosis of coeliac disease

Introducción. Se han observado cambios característicos en las poblaciones de linfocitos intraepiteliales (LIE) de la mucosa intestinal en pacientes celiacos infantiles y adultos. Objetivos. Determinar el rango normal de las poblaciones de LIE por citometría de flujo y establecer su rentabilidad diagnóstica en la enfermedad celiaca (EC). Material y métodos. Estudio retrospectivo de 246 niños y 461 adultos con sospecha de EC a los que se había realizado estudio de poblaciones de LIE. El grupo de EC (221 niños y 98 adultos) lo forman individuos con serología celiaca positiva e histología con lesión grado Marsh 1 o mayor. El grupo control (25 niños y 363 adultos) lo constituyen individuos sin lesión intestinal y serología celiaca negativa a los que también se había realizado inmunofenotipo de LIE por citometría de flujo. Resultados. En el grupo de pacientes celiacos se observa un aumento significativo de LIE totales y LIE TCRγδ y un descenso significativo de LIE NK-like en comparación con los grupos control. En función de las curvas ROC, los puntos de corte en la población infantil fueron: %LIE > 14,2%, %LIE TCRγδ > 16,5% y %LIE NK-like < 10,1%. Los puntos de corte en la población adulta fueron: %LIE > 14,2%, %LIE TCRγδ > 16,1% y %LIE NK-like < 4,4%. En ambas poblaciones se obtienen una especificidad y VPP cercano o igual al 100%, con unos CP+ > 5 y CP− < 2 o próximos. Conclusiones. En el presente trabajo se han establecido los valores de corte para los tres parámetros analizados de los LIE. Estos parámetros permiten diagnosticar con una especificidad cercana al 100% la EC en el niño y en el adulto. Los valores de CP+ y CP− obtenidos muestran que estos parámetros son muy útiles en el diagnóstico de EC activa infantil y del adulto. Por lo tanto, el análisis de las poblaciones de LIE por medio de la citometría de flujo es una nueva herramienta diagnóstica de la EC que complementa el estudio anatomopatológico clásico aumentando su especificidad (AU)

Introduction: The intestinal mucosa of children and adult with coeliac disease shows characteristic changes in intraepithelial lymphocyte (IEL) populations. Objectives: Determination of the normal range of IEL populations by flow cytometry and its diagnostic usefulness in coeliac disease (CD). Methods: A retrospective study of 246 children and 461 adults with suspected CD with IEL immunophenotype results. The CD group (221 children and 98 adults) are individuals with positive coeliac serology and a histology lesion Marsh grade 1 or greater. The control group included 25 children and 363 adults without bowel lesion, negative serology and with IEL immunophenotype results. Results: The group of coeliac patients, adults and children, shows a significant increase in total IEL and TCRdeltagamma IEL, and a significant decrease in NK-like IEL compared with control groups. Based on ROC curves, the cut-off in coeliac children was: %IEL >14.2%, %TCRdeltagamma IEL>16.5% and %NK-like IEL<10.1%. The cut-off in the adult coeliac population was: %IEL >14.2%, %TCR IEL>16.1% and %NK-like IEL<4.4%. In both populations the specificity and PPV are close or equal to 100%, a CP+ >5 and a CP− <2 or near. Conclusions: The cut-off values of the LIE population analysed has been established in this study. The values of CP+ and CP− show that these parameters are very useful for the diagnosis of celiac disease in children and adults, with a specificity of approximately 100%. The immunophenotyping of LIE is a very useful technique in the diagnosis of CD, and complements the classical pathological study, thus increasing the specificity (AU)

Regulation of lymphocytes by nitric oxide.

Shortly after the identification of nitric oxide (NO) as a product of macrophages, it was discovered that NO generated by inducible NO synthase (iNOS) inhibits the proliferation of T lymphocytes. Since then, it has become clear that iNOS activity also regulates the development, differentiation, and/or function of various types of T cells and B cells and also affects NK cells. The three key mechanisms underlying the iNOS-dependent immunoregulation are (a) the modulation of signaling processes by NO, (b) the depletion of arginine, and (c) the alteration of accessory cell functions. This chapter highlights important principles of iNOS-dependent immunoregulation of lymphocytes and also reviews more recent evidence for an effect of endothelial or neuronal NO synthase in lymphocytes.

Label free detection of CD4+ and CD8+ T cells using the optofluidic ring resonator.

We have demonstrated label free detection of CD4+ and CD8+ T-Lymphocyte whole cells and CD4+ T-Lymphocyte cell lysis using the optofluidic ring resonator (OFRR) sensor. The OFRR sensing platform incorporates microfluidics and photonics in a setup that utilizes small sample volume and achieves a fast detection time. In this work, white blood cells were isolated from healthy blood and the concentrations were adjusted to match T-Lymphocyte levels of individuals infected with HIV. Detection was accomplished by immobilizing CD4 and CD8 antibodies on the inner surface of the OFRR. Sensing results show excellent detection of CD4+ and CD8+ T-Lymphocyte cells at medically significant concentrations with a detection time of approximately 30 minutes. This work will lead to a rapid and low-cost sensing device that can provide a CD4 and CD8 count as a measure of HIV progression.

Rapid automated cell quantification on HIV microfluidic devices.

Lab-chip device analysis often requires high throughput quantification of fluorescent cell images, obtained under different conditions of fluorescent intensity, illumination, focal depth, and optical magnification. Many laboratories still use manual counting--a tedious, expensive process prone to inter-observer variability. The manual counting process can be automated for fast and precise data gathering and reduced manual bias. We present a method to segment and count cells in microfluidic chips that are labeled with a single stain, or multiple stains, using image analysis techniques in Matlab and discuss its advantages over manual counting. Microfluidic based cell capturing devices for HIV monitoring were used to validate our method. Captured CD4(+) CD3(+) T lymphocytes were stained with DAPI, AF488-anti CD4, and AF647-anti CD3 for cell identification. Altogether 4788 (76 x 3 x 21) gray color images were obtained from devices using discarded 10 HIV infected patient whole blood samples (21 devices). We observed that the automatic method performs similarly to manual counting for a small number of cells. However, automated counting is more accurate and more than 100 times faster than manual counting for multiple-color stained cells, especially when large numbers of cells need to be quantified (>500 cells). The algorithm is fully automatic for subsequent microscope images that cover the full device area. It accounts for problems that generally occur in fluorescent lab-chip cell images such as: uneven background, overlapping cell images and cell detection with multiple stains. This method can be used in laboratories to save time and effort, and to increase cell counting accuracy of lab-chip devices for various applications, such as circulating tumor cell detection, cell detection in biosensors, and HIV monitoring devices, i.e. CD4 counts.

[Improvement of the performances of the messages for the identification of the lymphocyte population with the Beckman Coulter LH750 haematology analyser]. / Amélioration des performances des messages d'identification de la population lymphocytaire sur l'analyseur d'hématologie Beckman Coulter LH 750.

We have compared the efficiency of the detection for lymphocyte abnormalities using the LH 750 analyzer according to standard CLSI recommendations (Clinical and Laboratory Standards Institute) and three new rules dedicated to abnormal lymphocyte recognition. These new rules are defined by combining quantitative and qualitative criteria. They improve the detection of lymphocytes with abnormal morphology with an efficiency of 97.7% as compared to 81% with the standard protocol and a sensitivity of 92.8% compared to 23% initially. Regarding the detection of large granular lymphocytes, the new rules demonstrate an efficiency of 97.6%, a specificity of 81.8% and a specificity of 98.2%. These new criterias, which can be easily implemented in the settings of the automatic counter improve the technical efficiency of the validation process with an expected decrease of the number of manual slide reviews.

Comparison of four hematology analyzers, CELL-DYN Sapphire, ADVIA 120, Coulter LH 750, and Sysmex XE-2100, in terms of clinical usefulness.

We evaluated the clinical usefulness (leukocyte distribution classification, morphologic classification, and morphologic flags) of the following four hematology analyzers: CELL-DYN Sapphire (CD-Sapphire) (Abbott Diagnostics, Santa Clara, CA, USA), ADVIA 120 (Bayer Diagnostics, Tarrytown, NY, USA), Beckman Coulter LH 750 (Beckman Coulter, Miami, FL, USA), and Sysmex XE-2100 (TOA Medical Electronics Co., Kobe, Japan). Four hundred thirty samples from patients and 100 samples from healthy individuals were analyzed. For distributional classification, the sensitivity rates of CD-Sapphire, ADVIA 120, LH 750, and XE-2100 were 93.1, 95.9, 94.9, and 94.9%, respectively, and the efficiency rates were 80.7, 81.6, 84.1, and 84.2%, respectively. For morphologic classification, the sensitivity rates of CD-Sapphire, ADVIA 120, LH 750, and XE-2100 were 88.6, 93.2, 77.3, and 94.3%, respectively, and the efficiency rates were 80.9, 73.0, 79.5, and 74.2%, respectively. Comparing the findings in different morphologic flags, XE-2100 showed the highest sensitivity for Blasts flag (90.9%); CD-Sapphire showed the highest sensitivity for Immature granulocytes and/or Left-shift flag (85.5%); ADVIA 120 showed the highest sensitivity for Atypical lymphocytes flag (60.0%); and LH 750 showed the highest sensitivity for Nucleated RBC flag (75.0%). Our results demonstrate that the four analyzers are comparable in overall performance.

Computed CD4 percentage as a low-cost method for determining pediatric antiretroviral treatment eligibility.

BACKGROUND: The performance of the WHO recommendations for pediatric antiretroviral treatment (ART) in resource poor settings is insufficiently documented in routine care. METHODS: We compared clinical and immunological criteria in 366 children aged 0 to 12 years in Kinshasa and evaluated a simple computation to estimate CD4 percent, based on CD4 count, total white blood cell count and percentage lymphocytes. Kappa (kappa) statistic was used to evaluate eligibility criteria and linear regression to determine trends of CD4 percent, count and total lymphocyte count (TLC). RESULTS: Agreement between clinical and immunological eligibility criteria was poor (kappa = 0.26). One third of children clinically eligible for ART were ineligible using immunological criteria; one third of children immunologically eligible were ineligible using clinical criteria. Among children presenting in WHO stage I or II, 54 (32%) were eligible according to immunological criteria. Agreement with CD4 percent was poor for TLC (kappa = 0.04), fair for total CD4 count (kappa = 0.39) and substantial for CD4 percent computational estimate (kappa = 0.71). Among 5 to 12 years old children, total CD4 count was higher in younger age groups (-32 cells/mm3 per year older), CD4 percent was similar across age groups. CONCLUSION: Age-specific thresholds for CD4 percent optimally determine pediatric ART eligibility. The use of CD4 percent computational estimate may increase ART access in settings with limited access to CD4 percent assays.

Optimization of flow cytometric measurement of ZAP-70 in chronic lymphocytic leukemia.

BACKGROUND: The goal of this study was to optimize a cell staining procedure for flow cytometric detection of zeta-chain associated protein-70 (ZAP-70). Our specific objectives were to improve antibody selection criteria, identify a cell permeabilization procedure better tailored to ZAP-70 analysis, as well as to establish objective criteria to control antigen stability. METHODS: Sequentially titrated 2F3.2-FITC, 1E7.2-FITC, and 1E7.2-Alexa Fluor 488 anti-ZAP-70 antibodies were used to stain normal B and T cells and Scatchard analysis was applied to calculate K(d) and B(max) values from saturation curves of specific binding. ZAP-70 staining was compared in cells permeabilized with two commercially available kits, Triton X-100, and a custom saponin procedure. RESULTS: Normal B-cells were found to provide an excellent measure of nonspecific staining while varying ZAP-70 antibodies and concentrations. Comparing Scatchard analyses of specific T-cell binding revealed that 1E7.2-Alexa Fluor 488 had the highest binding affinity of the tested anti-ZAP-70 antibodies and was the best choice. The highest levels of ZAP-70 fluorescence occurred when cells were permeabilized using a noncommercial saponin procedure. Decrease of chronic lymphocytic leukemia cell viability correlated with diminished ZAP-70 expression; when viability was lower than 95% the percentage of bright positive samples was significantly decreased, indicating a possibility of false-negative results. CONCLUSIONS: The efficiency and reliability of flow cytometric detection of ZAP-70 can be optimized by using Scatchard analysis to help select the most effective antibodies and antibody concentrations that maximize specific to nonspecific binding, by using a "custom" ZAP-70 permeabilization procedure, and by better controlling antigen stability by measuring cell viability.

Monoclonal antibody fluorescence for routine lymphocyte subpopulation analysis with the Abbott CELL-DYN Sapphire haematology analyser.

Using previously described procedures, this study quantified T-cell, T-cell subset, B-cell and NK-cell populations with the CD-Sapphire haematology analyser in a series of patients with mild to moderate lymphocytosis. Lymphocyte counts ranged from 6.0 to 14.9 x 10(9)/l, with 86/97 being <10.0 x 10(9)/l. Immunophenotyping (CD3/CD19/HLA-DR, CD4/CD8 and CD16/CD56 combinations) was performed using EDTA-anticoagulated blood, automated CD-Sapphire analysis and subsequent software processing. Of 35 samples from younger (<12 years) patients, 22 (63%) had nonspecific lymphocyte changes, 4 (11%) showed specific increases in nonreactive T-Helper or T-Suppressor cells, and five showed a reactive T-cell lymphocytosis. The remaining four were classified as 'Transient/Persistent NK-associated (NKa) Expansion' (n = 3) and specific B-cell lymphocytosis (n = 1). For older patients (n = 59), 15 (25%) had an increase (>1.5 x 10(9)/l) in B-cells, and seven investigated for surface immunoglobulin expression were all found to be clonal. The remaining samples were categorized as 'Transient/Persistent NK-associated (NKa) Expansion' (13/59), Reactive Lymphocytosis (5/59), 'Reactive Lymphocytosis or Transient/Persistent NKa Expansion' (8/59), specific T-Helper cell (n = 8) or T-Suppressor cell (n = 3) lymphocytosis, and 'Lymphocytosis of Undetermined Significance' (n = 7). This study has demonstrated the feasibility of applying limited immunophenotyping protocols to the investigation of patients with abnormal lymphocyte counts in routine haematology. By using commercially purchased liquid monoclonal reagents to determine lymphocyte subpopulation profiles, haematology laboratories can provide more definitive information of potential clinical importance.