Particle‐induced myeloperoxidase release in serially diluted whole blood quantifies the number and the phagocytic activity of blood neutrophils and opsonization capacity of plasma

Abstract

Luminol-amplified chemiluminescence (CL) from phagocytes has previously been shown to be almost completely dependent on the release of myeloperoxidase (MPO) from azurophilic granules. We measured the luminol-amplified chemiluminescence response (WBCL) by using serially diluted whole blood. In these experiments, non-opsonized and serum-opsonized zymosan (NWBCL and OWBCL, respectively) were used concurrently as phagocytosable particles. We found two whole-blood dilution ranges with clinical significance: first, <0.04% of whole blood in the reaction volume, where MPO released by the zymosan-activated leukocyte population came almost totally from neutrophils and the OWBCL response could be exploited as a measure of a neutrophil count in a given blood specimen, despite the pathophysiological state of the host. In contrast, the NWBCL response was two-fold in blood samples from bacterial infection patients compared to those of controls and patients with viral infection, suggesting the use of NWBCL for the differential diagnosis of bacterial infections from viral infections; second, 0.16-1.2% of whole blood in the reaction volume, where the opsonization capacity of plasma (OC(50)) can be determined. We also found that at whole blood content >0.04%, erythrocytes quickly start to absorb chemiluminescence light, and that at whole blood content >1.2%, plasma proteins, most probably albumin and fibrinogen, start to inhibit MPO release.

Respiratory burst activity of Atlantic cod (Gadus morhua L.) blood phagocytes differs markedly from that of rainbow trout

“…Interestingly, NOB induced WBCL response never reached the same magnitude of WBCL induced by OB as observed with trout ( Fig. 1D) or human phagocytes [25]. Under similar measurement conditions with trout blood phagocytes, the CL response induced with NOB reaches the magnitude of OB at a final blood dilution of 6.0 ml ml ÿ1 showing that at this blood concentration the amount of plasma factors (w0.3% of plasma concentration) is sufficient to fully opsonise NOB in situ.…”

“…The RB activities of blood phagocytes were stimulated with various bacterial strains (see Section 2) at a final blood dilution of 1:650 in the presence of 0.4% of gelatin and 0.3% of cod serum at 15 C. Heparin always gave at least 25% higher CL response compared to EDTA or citrate indicating that cod blood phagocytes were activated in the heparin (Fig. 4A), as also observed by us with human phagocytes [25]. In addition, the concentration of EDTA in tubes is w6 mM when the tube is filled with blood.…”

Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake

“…The induction of ROS production in leukocytes caused by the A. actinomycetemcomitans D7S wild-type and ΔhofQ mutant cells was studied using a luminol-amplified chemiluminescence experiment [51] with some modifications. Briefly, triplicate reactions were prepared in a white 96-well plate (Greiner Bio-One, #9,502,887) in a total volume of 250 µl: 100 µl of bacterial suspension (1.25 × 10 7 CFU), 10 µl of luminol (10 mM in borate buffer), 3 µl of human plasma/serum, 50 µl of human leukocytes (1/3 dilution) and 87 µl of gHBSS (1 mg/ml gelatin in HBSS: 1 mM CaCl 2 , 5 mM KCl, 0.4 mM KH 2 PO 4 , 0.5 mM MgCl 2 ∙6H 2 O, 0.4 mM MgSO 4 ∙7H 2 O, 140 mM NaCl, 4 mM NaHCO 3 , 0.3 mM Na 2 HPO 4 , and 6 mM glucose).…”

“…A homogenous bacterial suspension was prepared from three-day plate cultures of the A. actinomycetemcomitans D7S wild-type or ΔhofQ mutant strains. Plasma/serum was collected from 3 ml of (EDTA-coagulated) whole blood after centrifugation (1000 × g, 10 min, RT), and leukocytes were freshly isolated from 1 ml of (EDTA-coagulated) whole blood following a protocol by Lilius and Nuutila [51]. Measurement of chemiluminescence (every 2 min for 200 min; Victor [3] multilabel plate reader (Perkin Elmer) or Hidex Sense microplate reader (Hidex)) was started immediately after leukocytes were added to the reaction.…”

Repeated dose 28-day oral toxicity study of moniliformin in rats

“…The resultant CL emission is directly linked to the activity of neutrophils. Previous studies have shown that the signal is due to the release of myeloperoxidase (MPO) from the azurophilic granules of the phagocytes (Lilius and Nuutila, 2006).…”