Marion G. Macey

Use of mean platelet component to measure platelet activation on the ADVIA 120 haematology system

Platelet activation results in changes in a number of cell surface molecules including an increase in P-Selectin (CD62P) that may be rapidly and conveniently measured by immunofluorescent flow cytometry. The ADVIA 120 (Bayer) is a new system that facilitates more accurate measurement of platelet volume and in addition provides an approximate measure of the mean refractive index (RI) of the platelets reported as mean platelet component (MPC) concentration. We were interested to determine whether changes in MPC might reflect changes in platelet activation status. To investigate this, the platelet CD62P expression, determined by flow cytometry, and change in MPC, measured on the ADVIA 120 system, was first examined in vitro after stimulation of EDTA anticoagulated whole blood with submaximal concentrations of bovine thrombin in the presence or absence of the thromboxane synthase inhibitor, Ridogrel. Thrombin produced a dose-dependent increase in platelet CD62P expression and a decrease in MPC that could be inhibited by Ridogrel at physiological concentrations. In the second set of experiments, blood from 20 normal controls was collected into both EDTA and sodium citrate (SC) anticoagulants. Within 30 min of venesection and again at 3 h post-venesection after storage at room temperature, the platelet MPC and CD62P expression were determined. Platelets in all samples with both anticoagulants showed very low levels of CD62P expression when first analysed. At 3 h there was a small increase in CD62P expression on platelets in whole blood anticoagulated with SC, but a significant (P < 0.001) increase was observed on platelets anti-coagulated with EDTA. A negative correlation was found between the change in MPC of the platelets and the increase in the mean fluorescence intensity (MFI) (r = -0.69, P < 0.001, n = 20) and the percentage (r = -0.72, P < 0.001, n = 20) of CD62P positive platelets at 3 h in blood anticoagulated with EDTA. We conclude that a reduction in MPC as measured by the ADVIA 120 may be used to detect anticoagulant induced, as well as thrombin stimulated, in vitro platelet activation in blood anticoagulated with EDTA. Further, we conclude that platelet activation is negligible for up to 3 h in sodium citrate anticoagulated whole blood.

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.

Human peripheral blood monocytes express protease receptor-2 and respond to receptor activation by production of IL-6, IL-8, and IL-1β

Protease‐activated receptor‐2 (PAR‐2) belongs to a family of G‐coupled receptors activated by proteolytic cleavage to reveal a tethered ligand. PAR‐2 is activated by trypsin and trypsin‐like serine proteases and experimentally, by receptor‐activating peptides (APs), which mimic the tethered ligand. PAR‐2 has recently been implicated in proinflammatory immune responses. For example, PAR‐2−/− mice exhibit markedly diminished contact hypersensitivity reactions and are completely resistant to adjuvant‐induced arthritis. The present study shows that human blood monocytes express low‐level cell‐surface PAR‐2 ex vivo, which is up‐regulated upon cell purification by the mobilization of intracellular stores of PAR‐2 protein. PAR‐2 expression is also present on monocyte‐derived macrophages, but only a small proportion of monocyte‐derived dendritic cells (DC) is PAR‐2+, and blood DC are PAR–. Freshly isolated monocytes responded to the PAR‐2 AP ASKH 95 (2‐furoyl‐LIGKV‐OH) with the generation of a calcium flux and production of interleukin (IL)‐1β, IL‐6, and IL‐8. The results presented thus suggest that PAR‐2 contributes to inflammatory responses by inducing the production of proinflammatory cytokines in peripheral blood monocytes.

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.

The Effect of Hypnosis on Systemic and Rectal Mucosal Measures of Inflammation in Ulcerative Colitis

OBJECTIVES:Hypnotherapy is effective in several diseases with a psychosomatic component. Our aim was to study the effects of one session of hypnosis on the systemic and rectal mucosal inflammatory responses in patients with active ulcerative colitis (UC).METHODS:In total, 17 patients with active UC underwent a 50-min session of gut-focused hypnotherapy. Before and after each procedure, the systemic inflammatory response was assessed by serum interleukin (IL)-6 and IL-13 concentrations, tumor necrosis factor-alpha (TNF-α) and IL-6 production by lipopolysaccharide (LPS)-stimulated whole blood, leukocyte count, natural killer (NK) cell number, platelet activation, and platelet–leukocyte aggregate formation. Rectal inflammation was assessed by mucosal release of substance P (SP), histamine, IL-13 and TNF-α, reactive oxygen metabolite production, and mucosal blood flow. Eight patients with active UC underwent a control procedure.RESULTS:Hypnosis decreased pulse by a median 7 beats per minute (bpm) (P= 0.0008); it also reduced the median serum IL-6 concentration by 53% (P= 0.001), but had no effect on the other systemic variables assessed. Hypnosis reduced rectal mucosal release of SP by a median 81% (P= 0.001), histamine by 35% (P= 0.002) and IL-13 by 53% (P= 0.003), and also, blood flow by 18% (P= 0.0004). The control protocol had no effect on any of the variables assessed.CONCLUSIONS:Hypnosis reduced several components of the systemic and mucosal inflammatory response in active ulcerative colitis toward levels found previously in the inactive disease. Some of these effects may contribute to the anecdotally reported benefits of hypnotherapy and provide a rationale for controlled trials of hypnotherapy in UC.

Establishment and validation of a standard protocol for the detection of minimal residual disease in B lineage childhood acute lymphoblastic leukemia by flow cytometry in a multi-center setting;

Flow cytometry to detect minimal residual disease is a sensitive and specific means to detect impending relapse in B lineage acute lymphoblastic leukemia (ALL). However as with all relatively sophisticated procedures assurance is needed that the methodology is widely applicable, and reproducible in different laboratories. In this paper, the UK ALL Flow MRD Group and the UK MRD steering group describe a four color flow protocol that was applicable to most patients and showed similarly high levels of concordance both between different laboratories and with MRD as detected molecularly. See related perspective article on page 748. Minimal residual disease detection, used for clinical management of children with acute lymphoblastic leukemia, can be performed by molecular analysis of antigen-receptor gene rearrangements or by flow cytometric analysis of aberrant immunophenotypes. For flow minimal residual disease to be incorporated into larger national and international trials, a quality assured, standardized method is needed which can be performed in a multi-center setting. We report a four color, flow cytometric protocol established and validated by the UK acute lymphoblastic leukemia Flow minimal residual disease group. Quality assurance testing gave high inter-laboratory agreement with no values differing from a median consensus value by more than one point on a logarithmic scale. Prospective screening of B-ALL patients (n=206) showed the method was applicable to 88.3% of patients. The minimal residual disease in bone marrow aspirates was quantified and compared to molecular data. The combined risk category concordance (minimal residual disease levels above or below 0.01%) was 86% (n=134). Thus, this standardized protocol is highly reproducible between laboratories, sensitive, applicable, and shows good concordance with molecular-based analysis.

Formation of platelet-leukocyte aggregates in inflammatory bowel disease

Objectives:Formation of platelet-leukocyte aggregates (PLAs) is increased in several inflammatory and thrombotic conditions. This may result from and enhance platelet and neutrophil activation and could contribute to the inflammatory process in inflammatory bowel disease (IBD). We investigated platelet-leukocyte aggregation in patients with IBD and its relation to treatment, disease activity and platelet and neutrophil activation. Methods:PLAs, platelet activation (P-selectin expression) and neutrophil activation (L-selectin expression) were assessed 30 and 180 minutes after drawing blood into EDTA/citrate-theophylline-adenosine and dipyridamole, a novel anticoagulant, using fluorescent antibodies to CD45 (for leukocytes), CD42a (for platelets), CD62P (P-selectin) and CD62L (L-selectin) and flow cytometry. Platelet activation was also measured using the ADVIA 120 hematology analyser. Results:Samples from 67 patients with IBD measured within 30 minutes had a higher platelet count (P < 0.001), more platelets expressing P-selectin (P = 0.01), and more PLAs (P < 0.01) than from 20 healthy controls and more PLAs (P < 0.05) than from 9 controls with inflammatory arthropathies. IBD patients on thiopurines had fewer PLAs than those not taking them (P < 0.05); corticosteroids and aminosalicylates had no such effects. Incubation for 180 minutes increased the number of platelets expressing P-selectin (P < 0.0001), and the number of PLAs (P < 0.0001). The PLAs correlated with the number of platelets expressing P-selectin before (r = + 0.40, P < 0.001) and after (r = + 0.66, P < 0.0001) incubation. Conclusions:The number of PLAs is higher in patients with IBD than in healthy and inflammatory controls, but their numbers are lowered by thiopurines. Increased PLA formation may in part be due to increased platelet activation and could be pathogenic in IBD.

Platelet activation and endogenous thrombin potential in pre-eclampsia.

INTRODUCTION Platelets and the coagulation system may be involved in the pathogenesis of pre-eclampsia. We investigated whether platelet and coagulation activation markers, are elevated in pre-eclampsia. MATERIALS/METHODS Case-control study in which activated platelets, platelet-monocyte/ neutrophil aggregates, platelet microparticles (measured by flow cytometry) and four markers of thrombin generation capacity (endogenous thrombin potential (ETP), peak height, lag time and time to peak) using the Calibrated Automated Thrombogram system were assessed in pregnant women of similar gestational age with (n=46) and without (n=46) pre-eclampsia, and in healthy non-pregnant women (n=42). RESULTS The percentage of, CD62P+ platelets (p=0.013), CD62P+ platelet microparticles (p=0.029) and platelet-monocyte aggregates (p=0.019) were significantly higher in women with pre-eclampsia than the pregnant controls. Both groups of pregnant women had significantly higher ETP and peak height (p <0.001) than the healthy non pregnant group and the women with pre-eclampsia had significantly higher ETP and peak height (p<0.001) than the normotensive pregnant controls. CONCLUSION In the most comprehensive laboratory analysis to date, we found evidence of both platelet and coagulation activation in women with pre-eclampsia.

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.