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

Abstract

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.

Immunological screening for tumor cells in serous body fluids has added value with the CELL-DYN Sapphire

“…As described earlier, our results confirm previous publications that it is feasible to use CELL-DYN Sapphire routinely for immunofluorescent applications, not only in whole blood [11,12], but also in body fluids [14]. We obtained satisfactory agreement between the lymphocytes and leukocytes as established with monoclonal antibodies and with the standard optical differential count (using a modified algorithm and the Sapphire's FCS files) [13], demonstrating the utility of the immunophenotyping mode of the analyzer.…”

“…In this mode, the analyzer takes fixed volumes of blood from the sample tube and injects them into two reagent tubes containing predefined antibody mixtures. Red blood cells are lysed and after a timed incubation, measurements of optical scatter and fluorescence are made [12]. For the present study, we adapted this procedure by substituting the two reagent tubes.…”

“…In addition, the analyzer has an embedded three-color flow cytometer for special applications, including immunofluorescence in three open modes enabling for user-defined applications with monoclonal antibodies. The comparability of CELL-DYN Sapphire and flow cytometry measurements is evidenced in recent publications [11,12].…”

Analysis of serous body fluids using the CELL-DYN Sapphire hematology analyzer

“…The instrument has an operational mode in which T-cell subsets are determined using CD3/CD4 and CD3/CD8 antibody mixtures. After a timed incubation period, the multiangle optical scatter and fluorescence signals are measured [ 6 ]. For the present study, we adapted this procedure by substituting the monoclonal antibodies.…”

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

“…These questions were not addressed in this study but we know for instance that activated monocytes (CD14 pos /CD16 pos ) are modulated during atopic eczema, malaria infection, and sepsis, whereas the monitoring of NK cells could give relevant information following a kidney allograft (27)(28)(29)(30)(31). Recently, efforts have been made to improve the differential count in several ways, such as by integrating directly fluorescent antibodies in a cell counter (32). By evaluating B-, T-, and NK-lymphocyte ratios, this study demonstrates the screening potential of such an approach.…”