Whole Blood Flow Cytometric Assay Research Articles

A flow cytometric assay to detect platelet-activating antibodies in VITT after ChAdOx1 nCov-19 vaccination

AbstractVaccination is crucial in combatting the severe acute respiratory syndrome coronavirus 2 pandemic. The rare complication of thrombocytopenia and thrombotic complications at unusual sites after ChAdOx1 nCov-19 vaccination is caused by platelet-activating antibodies directed against platelet factor 4 (PF4). We present a widely applicable whole-blood standard flow cytometric assay to identify the pathogenic antibodies associated with vaccine-induced immune-mediated thrombotic thrombocytopenia (VITT) after ChAdOx1 nCov-19 vaccination. This assay will enable rapid diagnosis by many laboratories. This trial was registered at www.clinicaltrials.gov as #NCT04370119.

Host CCL3L1 Gene Copy Number in Relation to HIV-1-Specific CD4+ and CD8+ T-Cell Responses and Viral Load in South African Women

HIV-specific T-cell responses play an important role in control of infection. Because CCL3 has immune modulatory and antiviral activities, we hypothesized that host CCL3 genotype (CCL3L1 gene duplications) would influence the development of effective HIV-specific immune responses. Copy numbers of CCL3L1 were determined for 71 HIV-infected women, and HIV-specific CD4 and CD8 T-cell responses to overlapping peptide pools spanning the HIV-1 subtype C genome were simultaneously measured by an interferon-gamma and interleukin-2 whole-blood flow cytometric assay. Host CCL3L1 copy number correlated negatively with viral load (r=-0.239, P=0.045), as did magnitudes of Gag CD4 (r=-0.362, P=0.002) and CD8 (r=-0.261, P=0.028) T-cell responses. Patients with a Gag CD4 response (P=0.002) or dominant Gag CD8 (P=0.006) response had significantly lower viral loads than those whose dominant response targeted another region of the genome, whereas a dominant Nef-specific CD8 T-cell response was associated with higher HIV viral load. CCL3L1 copy number greater than or equal to the population median of 5 was significantly associated with increased magnitude of CD4 Gag responses (P=0.017), and women who had CD4 and CD8 Gag-specific responses had significantly lower viral loads (P=0.004) and higher CCL3L1 copy number (P=0.015) than those women with only CD8 Gag-specific responses.

Defective neutrophil oxidative burst in preterm newborns on exposure to coagulase-negative staphylococci.

The neutrophil oxidative burst is a product of the regulated assembly of the multicomponent oxidase enzyme. Our aim was to compare the oxidative burst in term (n = 10) and preterm newborns <31 wk gestational age (n = 10) after stimulation with coagulase-negative staphylococci in vitro. Strains of Streptococcus epidermidis with different invasive and slime-producing properties, one strain of S. haemolyticus, and one strain of group B-streptococcus were investigated. A whole-blood flow cytometric assay using the oxidation of hydroethidine to ethidium bromide was used. The oxidative activity in unstimulated neutrophil granulocytes [polymorphonuclear leukocytes (PMNLs)] was similar in term and preterm newborns, but the preterm newborns showed a significantly lower capacity to up-regulate the oxidative burst intensity after bacterial stimulation (p = 0.004). In the term but not in the preterm group, the oxidative burst intensity after bacterial stimulation correlated with the baseline oxidative burst intensity. After bacterial stimulation, there was a trend toward a greater percentage of activated neutrophils in the term group than in the preterm group, but the difference was less pronounced than that in oxidative burst intensity. Significant differences in oxidative burst response to different bacterial strains were observed (p < 0.001), but the differences could not be correlated exclusively to invasive capacity or slime-producing properties. It is concluded that the baseline oxidative activity is similar in term and preterm PMNLs but that preterm PMNLs have a decreased capacity to increase the oxidative burst in response to bacterial stimulation.

Detection of granulocyte antibodies by flow cytometry without the use of pure granulocyte isolates

Established methods used to detect serum antibodies to granulocytes require the isolation of granulocytes. Flow cytometric analysis of granulocytes with monoclonal antibodies eliminates the need for granulocyte isolation. The purpose of this study was to develop a method to evaluate reactions of antibodies to granulocytes without separating granulocytes from other leukocytes. Three screening cell samples for granulocyte antibody detection were prepared from whole-blood samples in which the red blood cells (RBCs) were lysed and remaining leukocytes tested against sera at 4 degrees C. Binding of human alloantibodies to the screening cells was determined by flow cytometric analysis using phycoerythrin-conjugated antibody to human immunoglobulin. Forward and side scatter were used to analyze granulocytes separately from other leukocytes. The assay was validated by testing granulocytes with reference alloantibodies directed to NA1, NA2, 5b, and Mart antigens. Samples from 32 patients were tested, and the results of the assays were compared with the results of testing the samples in a granulocyte immunofluorescence (GIF) assay performed by a reference laboratory. In the whole-blood flow cytometric (WBFC) assay the mean fluorescence intensities of reference antisera with antigen-positive cells, expressed in arbitrary units, were anti-NA1 = 48 to 221, anti-NA2 = 24 to 69, anti-5b = 13 to 57, and anti-Mart = 42 to 72. In contrast, the mean fluorescence intensity of type AB-negative control sera ranged from 3 to 11. Of the 32 patient sera tested, 23 were positive (range = 12 to 56) and 9 were negative (range = 3 to 10). When compared with the results obtained by the reference laboratory, 27 sera were concordant between the WBFC and the GIF assays. Four of the samples were positive in WBFC (range = 11 to 31) and negative in GIF and one sample was negative in WBFC (range = 5 to 6) and positive in GIF. Leukocytes prepared from whole blood after lysis of RBCs can be used in flow cytometric analysis to detect granulocyte alloantibodies. The results of testing for granulocyte antibodies with this assay were similar to results of testing sera in GIF. Further comparative studies are indicated to confirm findings and explain the discordant results.

Morphine inhibits NF-kappaB nuclear binding in human neutrophils and monocytes by a nitric oxide-dependent mechanism.

The transcription factor NF-kappaB plays a pivotal role in gene expression of inflammatory mediators such as cytokines or adhesion molecules. NF-kappaB-mediated transcriptional activation of these genes is inhibited by nitric oxide (NO) in a variety of cells, including monocytes. Morphine mediates NO release in a naloxone antagonizable manner in monocytes and neutrophils. The influence of morphine on NF-kappaB activation was investigated in a whole-blood flow cytometric assay. A specific antibody against the p65 subunit of NF-kappaB was used and detected by fluoresceine-isothiocyanate-labeled anti-immunoglobulin G. Nuclei were stained with propidium iodide. Leukocyte subpopulations were evaluated by gating on neutrophils and monocytes. The median fluorescence channel was determined. Different morphine concentrations (50 nm, 50 microm, 1 mm) and incubation intervals (10-150 min) were used. Morphine inhibits lipopolysaccharide-induced NF-kappaB nuclear binding in human blood neutrophils and monocytes in a time-, concentration-, and naloxone-sensitive-dependent manner. Similar effects were achieved with the NO donor S-nitroso-N-acetyl-pencillamine and the antioxidant N-acetyl-cysteine. The NO synthase inhibitors Nomega-nitro-l-arginine-methyl-esther and Nomega-nitro-l-arginine completely abolished the morphine-induced attenuation of NF-kappaB nuclear binding, demonstrating that the inhibitory action is mediated by NO release. Morphine causes immunosuppression, at least in part, via the NO-stimulated depression of NF-kappaB nuclear binding.

Flow cytometric visualisation of cytokine production by CD3-CD56+ NK cells and CD3+CD56+ NK-T cells in whole blood.

Natural killer (NK) cells produce multiple cytokines with potential immune regulatory roles. We standardised a whole-blood flow cytometry method to visualise intracellular cytokine production by NK cells for the study of NK cell biology and for clinical monitoring. With a three-colour fluorescent labelling technique, specific cytokine production by NK or T cells was visualised directly in whole blood in the same sample after stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin and by electronically gating on the CD3-ve/CD56+ve NK population or on the CD3+/CD56+ NK-T-cell population. Detectable levels of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) but not of interleukin-2 (IL-2) or IL-4 were easily observed in NK cells. The visualisation of the cytokine production by NK cells was dependent on the addition of a Golgi transport inhibitor, Brefeldin A. Other known stimuli for NK cells (IL-2 and CD16 monoclonal antibody and incubation with K562, the NK-sensitive cell line) promoted IFN-gamma and TNF-alpha production in NK cells to a lesser extent than did PMA and ionomycin stimulation. This whole-blood flow cytometric assay appears to be an useful and easy method to examine cytokine production by NK cells and/or by CD3+CD56+ NK-T lymphocytes in patients with relevant diseases.

The importance of sampling site in the measurement of whole-blood platelet flow cytometry

Purpose: Flow cytometry is an emerging technology that may be of use in clarifying the defects of platelet function after cardiopulmonary bypass. However, the technique used for platelet sampling may affect results. The objective of this study was to evaluate the influence of the sampling site on the degree of expression of a variety of platelet-associated proteins.Methods: Whole-blood flow cytometric assays for the detection of platelet glycoprotein (GP) lb, guanosine monophosphate (GMP)-140, thrombospondin, activated GPIIb/llla, and platelet-associated factor (FXllla) were developed. These markers were then measured in samples taken simultaneously from a peripheral vein, radial artery, and the side port of the central venous catheter, in eight patients about to undergo surgery.Results: When multiple samples from individual patients were assessed, the degree of activation with all of the activation assays (GMP-140, thrombospondin, activated GPIIb/IIIa, FXIIIa) was significantly greater in samples taken from the arterial catheter (p < 0.05) compared with the central venous catheter or the peripheral vein. The mean difference between sample sites was calculated in the study patients. Percent activation of FXllla from arterial blood was significantly greater than from the central vein and the peripheral vein (arterial-peripheral venous, 18.7 ± 8.6; central venous-peripheral venous, 3.7 ± 3.6; p = 0.005). There was no site-related difference in the detected expression of platelet GPlb.Conclusion: The site of platelet sampling significantly affects the degree of activation detected by flow cytometry. To approximate results that would be obtained from peripheral blood, samples should be taken from the side port of the central venous catheter and not from the arterial catheter in patients studied during surgery.

Platelet activation results in a redistribution of glycoprotein IV (CD36).

To investigate the possibility that thrombin and/or other platelet activators change the platelet surface expression of glycoprotein IV (GPIV, CD36), we used a panel of five GPIV-specific monoclonal antibodies (OKM5, 5F1, FA6-152, 8A6, and F13) directed against different epitopes. All these antibodies bound to resting platelets in a concentration-dependent and saturable manner, as determined by flow cytometry of washed platelets. Thrombin (1 U/mL) induced an approximately twofold increase in the platelet surface binding of each of these monoclonal antibodies. Immunofluorescence microscopy demonstrated an internal pool of GPIV that, after thrombin stimulation, redistributed to the platelet surface. In a whole-blood flow-cytometric assay, alpha-thrombin and the thromboxane A2 analogue U46619 each resulted in an approximately twofold increase in the platelet surface binding of OKM5, whereas ADP had a more modest effect, and collagen and epinephrine had little effect. The activation-induced up-regulation of the platelet OKM5 epitope occurred in vivo as demonstrated by flow cytometric analysis of whole blood emerging from a standardized skin puncture site. In summary, both in vitro and in vivo platelet activation results in increased platelet surface expression of GPIV, as a result of a redistribution of GPIV from an internal pool.

The Platelet Function Defect of Cardiopulmonary Bypass

AbstractThe use of cardiopulmonary bypass (CPB) during cardiac surgery is associated with a hemostatic defect, the hallmark of which is a markedly prolonged bleeding time. However, the nature of the putative platelet function defect is controversial. In this study, blood was analyzed at 10 time points before, during, and after CPB. We used a whole-blood flow cytometric assay to study platelet surface glycoproteins in (1) peripheral blood, (2) peripheral blood activated in vitro by either phorbol myristate acetate, the thromboxane (TX)A2 analog U46619, or a combination of adenosine diphosphate and epinephrine, and (3) the blood emerging from a bleeding-time wound (shed blood). Activation-dependent changes were detected by monoclonal antibodies directed against the glycoprotein (GP)lb-IX and GPIIb-IIIa complexes and P-selectin. In addition, we measured plasma glycocalicin (a proteolytic fragment of GPIb) and shed-blood TXB2 (a stable breakdown product of TXA2). In shed blood emerging from a bleeding-time wound, the usual time-dependent increase in platelet surface P-selectin was absent during CPB, but returned to normal within 2 hours. This abnormality paralleled both the CPB-induced prolongation of the bleeding time and a CPB-induced marked reduction in shed-blood TXB2 generation. In contrast, there was no loss of platelet reactivity to in vitro agonists during or after CPB. In peripheral blood, platelet surface P-selectin was negligible at every time point, demonstrating that CPB resulted in a minimal number of circulating degranulated platelets. CPB did not change the platelet surface expression of GPIb in peripheral blood, as determined by the platelet binding of a panel of monoclonal antibodies, ristocetin-induced binding of von Willebrand factor, and a lack of increase in plasma glycocalicin. CPB did not change the platelet surface expression of the GPIIb-IIIa complex in peripheral blood, as determined by the platelet binding of fibrinogen and a panel of monoclonal antibodies. In summary, CPB resulted in (1) markedly deficient platelet reactivity in response to an in vivo wound, (2) normal platelet reactivity in vitro, (3) no loss of the platelet surface GPIb-IX and GPIIb-IIIa complexes, and (4) a minimal number of circulating degranulated platelets. These data suggest that the “platelet function defect” of CPB is not a defect intrinsic to the platelet, but is an extrinsic defect such as an in vivo lack of availability of platelet agonists. The near universal use of heparin during CPB is likely to contribute substantially to this defect via its inhibition of thrombin, the preeminent platelet activator.