Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets.

BACKGROUND: Natural killer (NK) and NK T (NKT) cells are important in innate immune defense. Their unequivocal identification requires at least four antigens. Based on the expression of additional antigens, they can be further divided into functional subsets. For more accurate immunophenotyping and to describe multiple expression patterns of leukocyte subsets, an increased number of measurable colors is necessary. To take advantage of the technologic features offered by slide-based cytometry, repeated analysis was combined with sequential optical-filter changing. METHODS: Human peripheral blood leukocytes from healthy adult volunteers were labeled with antibodies by direct or indirect staining. Tandem dyes of Cy7 (phycoerythrin [PE]-/allophycocyanin [APC]-Cy7), Cy5.5 (PE-/APC-Cy5.5), and PE-Cy5 and the fluorochromes fluorescein isothiocyanate (FITC), PE, and APC were tested alone and in combinations. Optical filters of the laser scanning cytometer were 555 DRLP/BP 530/30 nm for photomultiplier tube (PMT) 1/FITC, 605 DRLP/BP 580/30 nm for PMT 2/PE, 740 DCXR/BP 670/20 nm for PMT 3/Cy5/APC, and BP 810/90 nm for PMT 4/Cy7. Filter PMT 3 was replaced for detection of PE/Cy5.5 and APC/Cy5.5 by 740 LP/BP 710/20 nm and the sample was remeasured. Both data files were merged into one to combine the different information on a single-cell basis. The combination of eight antibodies against CD3, CD4, CD8, CD14, CD16, CD19, CD45, and CD56 was used to characterize NK and NKT cells and their subsets. RESULTS: In this way Cy5.5 is measurable at 488-nm and 633-nm excitation. Further, with the two different filters it is possible to distinguish Cy5 from Cy5.5 in the same detection channel (PMT 3). With this method we identified NK and NKT cells, subsets of NK (CD3-16+56+, CD3-16+56-, CD3-16-56+) and NKT (CD3+16+56+, CD3+16-56+) and their CD4+8-, CD4-8+, CD4-8- and CD4+8+ subsets. CONCLUSION: With our adaptations it is possible to discriminate tandem conjugates of Cy5, Cy5.5, and Cy7 for eight-color immunophenotyping. Using this method, novel rare subsets of NK and NKT cells that are CD4/CD8 double positive are reported for the first time.

Fibrinogen receptor antagonists induce conformational changes of the human platelet glycoprotein IIb.

BACKGROUND: Controversial results have been reported concerning the ability of fibrinogen receptor antagonists (fibans) to induce conformational changes in the fibrinogen receptor after binding to it as the initial step of fibrinogen binding and platelet activation. METHODS: Platelets in citrated whole blood were stained with several pairs of anti-glycoprotein (anti-GP) IIb-directed monoclonal antibodies conjugated to phycoerythrin (PE) or indirectly labeled with Cy5. Pairs of monoclonal antibodies that induced a high-fluorescence resonance energy transfer (FRET) efficiency served as tools to detect activation-dependent changes of GP IIb after addition of adenosine diphosphate and several fibans. RESULTS: Using the combination of the clones 5B12-PE and P2-biotin/SA-Cy5, a concentration-dependent alteration of the GP IIb conformation was observed after addition of tirofiban, eptifibatide, and lotrafiban. Magnitude and kinetics differed among the investigated substances. CONCLUSION: The newly developed FRET assay allows the direct investigation of conformational changes of GP IIb after addition of platelet agonists or receptor ligands, as shown for three fibans.

Differentiation of platelet-leukocyte conjugate formation by short term exercise.

Platelet-leukocyte conjugates are increased in cardiovascular disease, but exercise is also able to trigger platelet-leukocyte formation in healthy subjects. The aim was to investigate the heterogeneity of platelet-leukocyte conjugate formations triggered by short term exercise. 18 healthy non-smokers underwent a 90 second maximal test on a SRM cycle ergometry system and a control experiment. Blood samples were taken after 30 min rest, immediately before and after, 15 min and 1 h after exercise. The different platelet-leukocyte conjugates were detected by flow cytometry via CD45, CD14, CD16, CD41, together with CD62P antibodies for the investigation of platelet activation in the conjugates. In addition, a stimulation of conjugate formation in vitro with 8 microM TRAP-6 was initiated. Immediately after exercise platelet-granulocyte (+24%), and -lymphocyte (+17%) conjugates were increased (p<0.01), while the platelet-monocyte conjugates (+40%) were enhanced (p<0.05) 15 min after exercise. The differentiation after stimulation showed that the regular (CD14(+)16(-); +32%) and mature (CD14(+)16(+); +35%) monocytes were both increased after exercise (p<0.01) but the regular monocytes were preferred (p<0.001) in platelet-monocyte conjugate formation. In addition, these conjugates revealed the highest CD62P expression. Maximal short term exercise is useful for the investigation of platelet-leukocyte formation; e.g., it could be shown, that regular monocytes may be preferred in conjugate formation and that these conjugates revealed the highest CD62P expression.

The platelet function defect of paroxysmal nocturnal haemoglobinuria.

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, acquired stem cell disorder, characterised by an abnormal susceptibility of red blood cells to complement induced lysis, resulting in repeated episodes of intravascular haemolysis and haemoglobinuria, thromboembolic events at atypical locations and, to a much lesser extent, bleeding complications. Platelet function is assumed to be abnormal, however, a defect has not yet been characterised and underlying mechanisms remain elusive. To explore these issues, we investigated platelet function in PNH patients using assays for clot formation under low and high shear force (thrombelastography and PFA100 device), adhesion to glass beads in native whole blood (Hellem method), aggregometry using various agonists (Born method), and flow cytometric assays for baseline and agonist-induced surface expression density of alpha-granule (CD62P) and lysosomal granule proteins (CD63), ligand binding to surface receptors (thrombospondin), and expression density of activation-induced neoepitopes of the fibrinogen receptor complex (PAC-1). Platelet PNH clone size determined by CD55 and CD59 labelling was compared to the clone sizes of granulocytes, monocytes, erythrocytes, and reticulocytes. A profound reduction of platelet reactivity was observed in PNH patients for all "global function" assays (clot formation, adhesion, aggregation). Platelet hyporeactivity was confirmed using flow cytometric assays. Whereas baseline levels of flow cytometrically determined platelet activation markers did not differ significantly between controls and PNH patients, agonist-induced values of all markers were distinctly reduced in the PNH group. Moreover, significantly reduced white blood cell counts (3.1/nl vs. 5.9/nl), haemoglobin values (9.5 vs. 14.3/g per dl), and platelet counts (136 vs. 219/nl) delineate profound tricytopenia in PNH patients. The fraction of particular cell types lacking the surface expression of GPI-anchored glycoproteins is referred to as the respective PNH clone; median PNH clone sizes of cells with short life spans (reticulocytes, platelets, granulocytes) was 50-80% of total cell populations compared to 20% of red blood cells. The results of our laboratory investigations show, that in PNH, reduced platelet counts coincide with reduced platelet reactivity. The foremost clinical complication in PNH, however, is venous thromboembolism, very probably induced by an activated and dysregulated plasmatic coagulation system. From these seemingly contradictory findings we infer, that part of the platelet hyporeactivity is probably due to reactive downregulation of platelet function in response to chronic hyperstimulation. The overall result is thought to be an unsteady balance, associated with thromboembolism in a larger proportion of patients, and with bleeding in a smaller proportion.

Plasmatic coagulation and fibrinolytic system alterations in PNH: relation to clone size.

Paroxysmal nocturnal haemoglobinuria (PNH) is characterized pathophysiologically by intravascular lysis of blood cells and clinically by thromboembolic events, often atypical in localization. In this study, we examined the plasmatic coagulation system of PNH patients to investigate a potential relation between coagulation alterations and disease intensity (PNH clone size). We found evidence for both an increase in procoagulant and in fibrinolytic activity, resulting in increased fibrin generation and turnover. Whereas a positive association of the procoagulant potential with PNH clone size was notable, fibrinolytic activity showed an inverse association with clone size. As a possible cause, a growing impairment of fibrinolytic activation and/or an increasing displacement of fibrinolytic activity is assumed. These mechanisms are most likely caused by the detachment of the glycosyl-phosphatidyl-inositol-anchored urokinase plasminogen activator receptor from cell surfaces, causing a progressive resistance to fibrinolytic stimuli, together with a probable shift of the fibrinolytic potential from cell surfaces to soluble, circulating complexes, resulting in a cellular fibrinolysis-steal phenomenon. Together, these processes are accused of mediating an increased thrombophilic risk in PNH. As hereditary prothrombogenic defects were found more frequently in patients suffering ischaemic complications, genetic thrombophilia seems to confer an additional thromboembolic risk in PNH, and should therefore be screened for.

Effect of 300- and 450-mg clopidogrel loading doses on membrane and soluble P-selectin in patients undergoing coronary stent implantation.

BACKGROUND: After coronary artery stent implantation patients are treated with the adenosine diphosphatase (ADP) receptor antagonist clopidogrel to prevent subacute stent thromboses. Today these patients initially receive a loading dose of 300 mg of clopidogrel to accelerate the complete drug effect. In the current study we investigated whether a higher loading dose can shorten the time until the maximum platelet inhibitory effect of clopidogrel is achieved. METHODS: P-selectin expression of nonstimulated and ADP-stimulated platelets was flow cytometrically measured before the clopidogrel loading dose and on 3 consecutive days in 52 patients with coronary artery disease: 21 patients in group 1 received 300 mg of clopidogrel after stent implantation and 11 patients in group 2 received the higher 450-mg clopidogrel loading dose followed by a daily dose of 75 mg of clopidogrel for both groups. The control group consisted of 20 patients who were monitored over 2 days before coronary intervention. Soluble P-selectin levels in plasma were determined by an enzyme-linked immunosorbent assay. RESULTS: Inducible P-selectin expression on ADP-stimulated platelets was significantly reduced (P =.05) on days 1 and 2 in patient group 2 (450-mg loading dose) compared with group 1 (300-mg loading dose). No influence of clopidogrel on the P-selectin levels in plasma was observed. CONCLUSIONS: In our study the application of 450 mg of clopidogrel as the loading dose in patients undergoing coronary stenting shortens the period until the maximum effect of the ADP receptor antagonist is achieved and thus may lead to a more successful prevention of subacute coronary stent thromboses.