Desmond A. McCarthy

Expression of functional antigens on neutrophils: Effects of preparation

We have studied how different conditions of cell labelling and isolation affect the expression of five functional antigens on neutrophils from healthy subjects. Fluorescein isothiocyanate conjugated (FITC) antisera specific for the C3bi receptor CR3 (CD11b), aminopeptidase N (CD13), the LPS:LPS binding protein receptor (CD14) and the receptors for human IgG (Fc gamma RII CDw32 and Fc gamma RIII CD16) were incubated with (i) unfixed whole blood at 4 degrees C and at room temperature (RT, approximately 20 degrees C), and the leukocytes prepared for analysis using the Coulter Q-Prep system, (ii) leukocytes which had obtained following the removal of erythrocytes from whole blood by dextran sedimentation and which had been washed or left unwashed at RT, and (iii) leukocytes which had been prepared from whole blood that had been formaldehyde fixed immediately following venesection. The amount of fluorescence associated with the cells was determined by flow cytometry. The expression of CD14 was low under all conditions. However the expression of CD11b, CD16 and CDw32 was significantly higher (p less than 0.05) on neutrophils obtained by dextran sedimentation (n = 4) than on cells which had been fixed with formaldehyde ex vivo; the increase in expression was even greater if the cells had been washed. In contrast, the expression of CD13 on formaldehyde fixed cells was higher than on cells which had been labelled at 4 degrees C or at room temperature and was similar to or slightly lower than that on cells obtained by dextran sedimentation. Increasing the time between 10 and 60 min for which the whole blood was incubated with antisera at RT or at 4 degrees C, resulted in progressive increases in the expression of CD11b and CD13. When neutrophils which had been obtained by dextran sedimentation were incubated with unlabelled antibodies to CD16 or CDw32 and FITC labelled antibodies to CD11b there was a marked increase in the expression of CD11b. Altogether these findings indicate that the analysis of functional molecules on neutrophils (which may be rapidly up-regulated during activation) should be performed under clearly defined and controlled conditions. Dual fluorescence studies may, in some circumstances, produce misleading results.

A simple flow cytometric procedure for the determination of surface antigens on unfixed leucocytes in whole blood

A novel procedure has been developed for the quantitation by flow cytometry of function-associated antigens on neutrophils and monocytes in unlysed, unfixed, peripheral blood samples. Freshly drawn blood anticoagulated with the serine esterase inhibitor, phenylmethylsulphonyl fluoride, is mixed with the vital nucleic acid stain, LDS-751, labelled with monoclonal antibodies for 5 min at 4 degrees C, diluted and analysed in a five-parameter flow cytometer. The three major leucocyte subpopulations (neutrophils, lymphocytes and monocytes) can be resolved in real time on the basis of their side light scattering and staining intensity with LDS-751 in the FL3 channel (erythrocytes and platelets stain very weakly), whilst the fluorescence intensity due to bound fluorescein isothiocyanate- or phycoerythrin-labelled antibody is monitored simultaneously in the FL1 or FL2 channels respectively. This procedure avoids potential artefacts that can occur due to the use of fixatives, erythrocyte lysing agents, or anticoagulants which are also divalent metal ion chelators. It should be widely applicable for the quantitation of those function-associated antigens, such as adhesion molecules and immune complex receptors, whose surface expression can be rapidly upregulated following activation, as well as for the quantitation of those leucocyte surface antigens whose expression is not subject to rapid modulation.

Comparative study of five commercial reagents for preparing normal and leukaemic lymphocytes for immunophenotypic analysis by flow cytometry

The flow cytometric analysis of leucocytes in whole blood is usually performed on samples in which the erythrocytes have been lysed and the leucocytes fixed. Because lysis and fixation reagents have the potential to introduce artefacts, several commercially available reagents were used to prepare normal and leukaemic lymphocytes for immunophenotypic analysis by flow cytometry, and the results were compared with those obtained from live whole blood. The reagents tested were the ImmunoPrep system and OptiLyse C (Coulter), LF-1000-Lyse and Flow (Harlan), Uti-Lyse (Dako) and FACS Lysing Solution (Becton Dickinson). The effect of each reagent on the apparent expression of CD3, CD5, CD11b, CD45, FMC7, kappa and lambda antigens was determined on lymphocytes from six normal controls and from six patients with chronic lymphocytic leukaemia (CLL). The following observations were made: (i) the time in minutes for each procedure varied markedly and was 1.5, 15, 20, 30 and 30 for the ImmunoPrep system, OptiLyse C, Uti-Lyse, FACS Lysing Solution, and LF-1000, respectively, but only 0.5 min for live whole blood. (ii) The forward and side scatter characteristics were affected by all of the lysis and fixation procedures, and this was most marked for LF-1000-Lyse and Flow. (iii) OptiLyse C gave preparations with poor forward and side scatter resolution due to the presence of residual red cell fragments. (iv) Lysis and fixation procedures did not affect the apparent expression of the CD3, CD45, or FMC7 antigens on normal or CLL samples, but gave highly variable results for the expression of the CD5, CD11b, kappa, and lambda antigens on the CLL samples. We conclude that lysis and fixation procedures can introduce different artefacts in the analysis of normal and leukaemic samples that are best avoided by analysing live whole blood.

Human blood dendritic cells: binding to vascular endothelium and expression of adhesion molecules

To investigate the binding properties of dendritic cells (DC) to vascular endothelium, a comparative analysis was undertaken of DC, monocytes and lymphocytes isolated from the blood of 25 healthy subjects using monolayers of human umbilical vein endothelial cells as the adherence substrate. More blood DC (mean 24% adherence) were adherent to endothelial monolayers than monocytes (mean 18%; P < 0.001) and lymphocytes (mean 12%; P < 0.001). When the monolayers were pretreated with tumour necrosis factor‐alpha (TNF‐α) all leucocyte populations exhibited an increased attachment, but there was still greater binding of DC (mean 37% adherence) in comparison with monocytes (mean 23%; P < 0.001) and lymphocytes (mean 18%; P < 0.001). Flow cytometric analysis revealed that in relation to monocytes and lymphocytes the DC had a higher surface expression of the adhesion molecules CD11a (P < 0.05), CD11c (P < 0.05) and CD54 (P < 0.05) but a lower prevalence of cells bearing CD49d (mean 38%; P < 0.05) and the homing receptor CD62L (mean 14%; P < 0.001). CD1a was present on 22% of DC and virtually absent from the surface of monocytes and lymphocytes. The intensity of expression of the β1‐integrins, CD49c, CD49d and CD49e was greater on DC than lymphocytes and monocytes (P < 0.05). Antibody blocking studies demonstrated that DC binding to untreated and TNF‐α‐treated endothelium was dependent upon the expression of CD11a, CD18 and CD49d, and the simultaneous application of anti‐CD18 and anti‐CD49d antibodies produced an approximate 70% inhibition of adhesion (P < 0.001). Thus, the expression of both β1‐ and β2‐integrins contributes to the adhesive interaction between DC and endothelium.

Ethylenediaminetetraacetic acid plus citrate-theophylline-adenosine-dipyridamole (EDTA-CTAD): a novel anticoagulant for the flow cytometric assessment of platelet and neutrophil activation ex vivo in whole blood.

Ethylenediaminetetraacetic acid (EDTA) is the anticoagulant recommended for full blood counts, citrate is recommended for coagulation and platelet studies, and citrate‐theophylline‐adenosine‐dipyridamole (CTAD) inhibits platelet activation. Because the combination of EDTA and CTAD (E/C) is better than EDTA or CTAD alone for measuring platelet parameters on the ADVIA 120 Haematology System, we investigated whether it also offers advantages for the flow cytometric assessment of platelet and/or neutrophil activation and platelet–leucocyte aggregate formation ex vivo. Blood from healthy subjects was collected into E/C or citrate, kept at room temperature or at 4°C, and analysed 0 to 360 min later in the ADVIA 120 and by immunofluorescent flow cytometry. Platelet count, mean platelet volume, number of platelet clumps, mean platelet component, numbers of CD62P+ platelets and platelet–leucocyte aggregates, and expression of CD11b on neutrophils changed little over 360 min in blood with E/C kept at 4°C. In contrast, one or more parameter changed when blood was kept with E/C at ambient temperature or with citrate at either temperature. The use of E/C in in vitro and in vivo studies is illustrated. Platelet and neutrophil activation status ex vivo can be reliably assessed if blood is collected into E/C, held at 4°C, and analysed within 6 h. Cytometry Part B (Clin. Cytometry) 51B:30–40, 2003.

How should CD34+ cells be analysed? A study of three classes of antibody and five leucocyte preparation procedures

For patients undergoing stem cell transplantation after intensive marrow ablative therapy it is important to enumerate the CD34+ stem cells in peripheral blood so that the harvest can be timed in order to maximize the number of cells collected by leucophoresis for subsequent haematopoietic reconstitution. The use of rapid flow cytometric techniques for the determination CD34+ leucocyte numbers has been advocated, although there is no consensus as to the best method. In this study, we have examined the effects of preparation procedures for flow cytometry on the binding of four CD34 antibodies (Immu-133, QBEND-10, HPCA2 and BIRMA-K3) to the three classes of epitopes on leucocytes. Whole blood, bone marrow and leucophoresis samples were analysed either directly after labelling with a vital nuclear dye (LDS-751) and fluorochrome-conjugated antibodies or after additional erythrocyte lysis and leucocyte fixation using four commercially available reagents (Q-Prep, OptiLyse B, OptiLyse C and FACS Lysing Solution). By comparison with the results obtained from viable leucocytes in unmanipulated samples, it was found that the binding of all four antibodies could be affected by lysis and fixation procedures and that the binding of the class I antibody Immu-133 was most markedly decreased. We conclude that CD34+ cells are best analysed using a whole blood procedure in which nucleated cells are identified by their side light scatter and the fluorescence associated with a vital nuclear dye (in this instance LDS-751) and the CD34+ cells are detected with fluorescein isothiocyanate- or phycoerythrin-conjugated antibodies.

Adhesion molecules are upregulated on dendritic cells isolated from human blood

This study investigated whether the high expression of adhesion molecules on enriched preparations of circulating dendritic cells (DCs) was an intrinsic property of the cells or whether it was a consequence of the procedure used to isolate them from blood. Expression of the &bgr;1, &bgr;2 integrins (CD11/CD18 family) and other adhesion molecules on DCs in whole blood was compared with that on isolated DCs. Dendritic cells were identified by flow cytometry as leucocytes that were positive for human leucocyte antigen (HLA)‐DR, but negative for CD3, CD14, CD16, CD19 and CD56. In contrast to a minority of DCs in whole blood, the majority of isolated DCs expressed the &bgr;2 integrins and there were a greater number of cells bearing CD44, CD54 and some of the &bgr;1 integrins (notably CD49b, CD49d, CD49e and CD29). An increase in the proportion of DCs bearing adhesion molecules was generally apparent at the isolation stage when mononuclear cells, which had been incubated overnight, were centrifuged on a metrizamide gradient to enrich for cells of low density. Inclusion of an inhibitor of protein glycosylation and exocytosis (brefeldin A) at all stages of separation partially prevented an increase in the percentage of DCs bearing CD18, C29 and C54 whereas the inclusion of cycloheximide (an inhibitor of polypeptide synthesis) interfered with increases in the percentage of cells bearing CD29 and CD54. Neither of these antagonists had an effect on the intensity of adhesion molecule expression. We suggest that some of the adhesion‐dependent functions of isolated DCs are caused, in part, by an upregulation of surface adhesion molecules induced by the enrichment procedure.

The Q-Prep system: Effects on the apparent expression of leucocyte cell surface antigens

To facilitate the analysis of immunolabelled peripheral blood or bone marrow leucocytes by flow cytometry, a number of reagents are available commercially that lyse erythrocytes and fix leucocytes. This study has investigated the effect on antibody-labelled whole blood of the Q-Prep procedure, in which erythrocytes are lysed with formic acid, and leucocytes are fixed with formaldehyde. Whole blood samples were labelled with the nuclear dye LDS-751 and with antibodies to HLA-DR or belonging to CD2, CD3, CD4, CD7, CD8, CD10, CD13, CD14, CD19, CD20, CD29, CD33, CD45, CD45RA, CD56, and CD62L (TQ-1) that were directly conjugated to either phycoerythrin (PE) and/or fluorescein isothiocyanate (FITC). Leucocytes were analysed by flow cytometry either in unfixed, unlysed whole blood (15) or after preparation using the Q-Prep system. The binding of eight antibodies, CD19-FITC, CD2-PE, CD3-PE, CD4-PE, CD19-PE, CD29-PE, CD45RA-PE, and CD56-PE, to the surface of lymphocytes was reduced, resulting in significant changes (P < 0.05) in the percentages of cells that stained positively and/or their mean molecules of equivalent fluorochrome (MEF). Further analysis revealed that this was due to the formic acid used during the erythrocyte lysis stage.

Rapid flow cytometric identification of putative CD14− and CD64− dendritic cells in whole blood

Blood dendritic cells (DCs) may be identified as mononuclear leucocytes with high expression of HLA-DR, but lacking the antigens CD3, CD14, CD16, CD19, and CD56, which are characteristically expressed by T cell, monocytes, B cells, and natural killer cells. However, some DCs have recently been reported to express the monocyte-associated antigen CD14; also some monocytes may shed CD14 and so appear to be CD14-. It is therefore possible that the expression of CD64, which is absent on blood DCs but which is expressed by both CD14+ and CD14- monocytes may better distinguish DCs from monocytes. DCs were identified by flow cytometry as mononuclear leucocytes with the phenotype HLA-DR+, CD2-, CD16-, CD19-, CD57-, and either CD14- or CD64- and hence are described herein as either CD14- DCs or CD64- DCs, respectively. CD14- DCs and CD64- DCs occurred, respectively, at a concentration of 65 +/- 48 x 10(6) cells 1(-1) and 149 +/- 103 x 10(6) cells 1(-1) (mean +/- S.D.) in samples of peripheral blood (corresponding, respectively, to 3.0 +/- 1.8% and 6.6 +/- 3.8% of the mononuclear cells). The expression of CD14 and CD64 on monocytes in blood was also investigated. Cells with the immunophenotype CD14- CD64+ comprised 12.7 +/- 3.3% of the monocyte population and had high expression of HLA-DR. DCs identified as CD14- or CD64- were isolated by flow cytometric sorting, prepared for electron microscopy, and both were found to have the characteristic morphology of resting DCs. We conclude that mononuclear cells with the phenotype HLA-DR+, CD3-, CD16-, CD19-, CD56-, and CD64- are blood DCs that may be CD14+ or CD14-. The method described therefore provides a more accurate and rapid means of identifying circulating DCs.

Multiparameter flow cytometric analysis of polymorphonuclear leucocytes in whole blood from patients with adult rapidly progressive periodontitis reveals low expression of the adhesion molecule L-selectin (Cd62L)

Numerous studies of polymorphonuclear leucocyte (PMN) function in patients with adult periodontitis, including rapidly progressive periodontitis, have yielded conflicting findings, perhaps because most of the assays were performed on PMNs that had been separated from whole blood by a variety of procedures. To avoid the problems associated with in vitro analysis of isolated cells, PMN function and antigen expression in live whole unmanipulated blood of eight patients with rapidly progressive periodontitis were compared with those of age-, race-, and sex-matched controls. Using multiparameter flow cytometry, a) L-selectin (CD62L) expression, b) cell size, and c) respiratory burst activity were measured in PMNs in whole blood immediately ex vivo and during incubation with Porphyromonas gingivalis and Staphylococcus aureus. By comparison with PMNs from the control group, PMNs from the patient group expressed significantly lower levels of CD62L and had an increased size before stimulation. PMNs from both groups produced respiratory bursts similar to those of the two bacteria, but in both groups the responses to S. aureus were significantly greater than those to P. gingivalis. The significantly reduced expression of the adhesion molecule CD62L on PMNs in the patient group may lead to reduced tethering of neutrophils at sites of inflammation and may partly explain the susceptibility of these individuals to recurrent and severe periodontal infections.