Effect of pyridoxal 5'-phosphate on human neutrophil aggregation in vitro.

Pyridoxal 5'-phosphate inhibited polymorphonuclear leukocyte aggregation in vitro in a dose-dependent fashion at concentrations ranging from 0.5 to 0.001 mmol/l. At pyridoxal 5'-phosphate concentrations between 0.4 and 0.5 mmol/l mean inhibition of aggregation was about 50%-60%. In this range, and at lower pyridoxal 5'-phosphate concentrations, phorbol myristate acetate-activated polymorphonuclear leukocytes showed a normal increase in volume, whereas swelling was inhibited at higher concentrations of pyridoxal 5'-phosphate. Since pyridoxal 5'-phosphate can be administered as a vitamin to man without any relevant side-effects its role as a physiological anti-aggregant of polymorphonuclear leukocytes warrants further investigation.

Assessment of neutrophil aggregation by Coulter STKR and STKS haematological analysers.

We have studied an alternative method to aggregometry for the assessment of human polymorphonuclear (PMN) leucocyte aggregation. This simple, rapid and reliable procedure counts unaggregated cells on both Coulter STKS and STKR haematological analysers by the impedance principle. Aggregation of PMN was induced by 15 min incubation with fresh autologous serum (FAS) after a 10 min phorbol myristate acetate (PMA) activation of neutrophils in small aliquots (0.25 ml) of suspension containing about 4.0 x 10(9) PMN/1. Differences (x 100) between count of resting and PMA+FAS treated neutrophils/count of resting PMN reflect percent aggregation. By this procedure, PMN aggregation did not occur in autologous plasma from EDTA anticoagulated whole blood; it was partially inhibited by hydrocortisone, whereas inactivated or Zymosan activated sera gave values similar to those from FAS induced aggregation. PMA aggregation was dependent on Ca2+ + Mg2+ concentration. Intra-assay analytical variability did not exceed 4% on either instrument. Reference values (n = 20) of percent PMN aggregation were 50.7 +/- 4.7 on STKS and 47.1 +/- 4.8 on STKR. Most probably, the interindividual variance was due to the physiological variability of Mg2+ and/or Ca2+ concentrations in FAS. Thus, this procedure reflects the true PMN aggregability status in a given subject, and in a given electrolyte environment.

High light scatter by neutrophils in the Bayer-Technicon H*2 analyzer: a screening test of morphologically defective responsiveness to in vitro chemotactic stimulation.

The Bayer-Technicon H*2 haematological analyser provides differential white blood cell count, including the assay of polymorphonuclear leukocytes by light scattering and the absorbance increase following the cytochemical reaction for myeloperoxidase. The mean value of polymorphonuclear leukocytes scatter, which reflects polymorphonuclear leukocytes volume, is printed in a separate report "for laboratory use only" as a ybar value in arbitrary units. In certain patients neutrophils displayed an unreported correlation between polymorphonuclear leukocytes high ybar basal values (> or = 37.00 arbitrary units) (determined on the H*2) and a defective response in vitro to the chemoattractant, formyl-methionyl-leucyl-phenylalanine (determined by microscopic evaluation of polymorphonuclear leukocytes shape change (polarization)). The patients showing no polymorphonuclear leukocyte response or a defective one to formyl-methionyl-leucyl-phenylalanine were all affected by "Systemic Inflammatory Response Syndrome (SIRS)". Therefore the predictive value of the positive test for SIRS is 100%. On the other hand 8.8% of SIRS patients had polymorphonuclear leukocytes < 37.00 arbitrary units of ybar basal value and a "normal" response to formyl-methionyl-leucyl-phenylalanine; the predictive value of the negative test being 90%. Since we demonstrated in vitro a dose-dependent deactivation of endotoxin or lipopolysaccharide-pretreated polymorphonuclear leukocytes, the "normal" response to formyl-methionyl-leucyl-phenylalanine of the "false negative" cases may occur because the endotoxaemia in these patients is too low to prevent it.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple assessment of human neutrophil adhesiveness.

The adhesiveness of human polymorphonuclear leukocytes was assessed in serum-coated polystyrene spectrophotometric cuvettes. Capped cuvettes, containing no more than 2 x 10(6) resting or concanavalin A-treated (100 micrograms/ml) polymorphonuclear leukocytes, were laid horizontally and subjected to three 90 degrees rotations on their major axis at fixed times. After incubation at room temperature, non-adherent cells remaining in suspension were counted on the Coulter counter STKS hematological analyzer. After a 16-min incubation (4 min each side of the cuvette) the adhesion of concanavalin A-activated neutrophils ranged from 98% to 100% and the adhesion of resting neutrophils from 30% to 35% (mean 32.4 +/- 2.2%, n = 10). An 8-min incubation (2 min each side) led to approximately 50% adhesion of concanavalin A-activated neutrophils (mean 49.9 +/- 2.2%, range 46%-54%, n = 16), whereas the adhesion of resting cells was about 21% (mean 21.4 +/- 1.6%, range 19%-24%, n = 16). The variation in percentage adhesion in repeated assays did not exceed 4% using concanavalin A-activated cells and 7.5% with resting neutrophils. The procedure is very rapid, easy to perform and precise, and no special apparatus or glassware is necessary. The method also allows microscopic evaluation of shape changes of adherent neutrophils through the clear sides of the cuvettes.

Instrumental reports and effect of anticoagulants in a case of neutrophil agglutination in vitro.

BACKGROUND: The aim of this study was to investigate a case of EDTA-induced polymorphonuclear leukocyte (PMN) agglutination in vitro on Bayer Technicon H*1. Coulter Counter STKS and STKR analyzers. METHODS: Venous whole blood was anticoagulated with K3.EDTA, sodium citrate, lithius heparinate, acid citrate dextrose (ACD) and with two other anticoagulant mixtures containing citric acid-theophylline-adenosine-dipyridamole (CTAD) or citrate-pyridoxal 5'-phosphate-tris (CPT). RESULTS: PMN agglutination, EDTA--but not temperature--dependent, was found by mere chance in an asymptomatic 48-year-old Caucasian male who did not show detectable PMN antibodies. Pseudoneutropenia without pseudoleukopenia was registered on H*1 exclusively in EDTA anticoagulated blood with a characteristic higher density PMN population on the BASO cytogram. Spuriously low white blood cell (WBC) counts and pseudoneutropenia appeared on STKR and STKS in EDTA anticoagulated blood, but signals on PMN agglutination were unsatisfactory. Accurate total and differential WBC counts were obtained in CTAD or CPT anticoagulated samples on the three analyzers. Heparin was the worst choice because it induced pseudothrombocytopenia and pseudoleukocytosis on STKR and STKS. CONCLUSIONS: Since PMN agglutination was not observed on peripheral smears and was undetected on EDTA anticoagulated samples processed immediately by H*1, the presence in vivo of PMN clumps should be excluded. Further hematological investigations will demonstrate in the long run whether the observed PMN agglutination in vitro is a transient occurrence in an apparently healthy subject not taking drugs at the time of observation.

Evaluation of the liver function of cirrhotic patients based on the formation of monoethylglycine xylidide (MEGX) from lidocaine.

Determination of the functional hepatic reserve is still controversial. Many tests have been proposed, but the assay based on formation of the lidocaine metabolite, monoethylglycine xylidide, seems to offer a promising approach to this problem. In this study we evaluated the effectiveness of the monoethylglycine xylidide test in the clinical evaluation of 31 cirrhotic patients submitted to three different therapeutic options (sclerotherapy, transjugular intrahepatic protosystemic shunt and surgical procedures) and in 1 patient submitted to right hepatectomy for giant hepatic angioma. We found a statistically significant difference between Child A and C patients and between Child B and C patients. The test did not differentiate Child A from Child B patients. We found no correlation between the Child-Pugh score, serum bilirubin, albumin and prothrombin time. There were no differences among the three groups of patients that could be statistically related to their therapy. The monoethylglycine xylidide test seems to be an attractive alternative to previous methods for the evaluation of the functional hepatic reserve, but further studies are necessary to assess the prognostic value of the test in cirrhotics, to separate the independent contribution of portosystemic shunting and hepatocyte dysfunction to monoethylglycine xylidide formation, and to evaluate the test as a prognostic index in cirrhotic patients submitted to general surgery.

Volumetric changes in phorbol myristate acetate activated neutrophils: a rapid and simple assay using Coulter counter STKR and STKS hematological analyzers.

BACKGROUND: The aim of this study was to investigate some physical changes (volume, conductivity and scatter) in human polymorphonuclear leukocytes (PMN) activated with phorbol myristate acetate (PMA). METHODS: Volume changes in PMA-activated neutrophils were assayed by both STKR and STKS, two Coulter hematological analyzers. Scatter changes in PMN activated by PMA in suspensions containing nitro-blue tetrazolium (NBT) were investigated on STKS scattergrams. RESULTS: PMA activation induced PMN volumetric increases that could be assayed with STKR and displayed with STKS. The activation of PMN in suspension containing NBT induced cellular scatter changes on STKS scattergrams. The differences in scatter between resting and PMA-activated neutrophils may thus provide a semiquantitative assay of NBT reduction and superoxide production. Volume changes in PMA-activated neutrophils were due to cellular swelling through water uptake, induced by Na+/H+ antiport activation and Na+ influx. Since volume changes in PMA-activated neutrophils might occur without O2- production and vice versa, in the cases in which PMA activation of protein kinase C cannot be demonstrated by O2- production, the effect of PMA on protein kinase C- mediated Na+/H+ antiport activation, and in turn on PMA volume changes, reflects protein kinase C activation. CONCLUSIONS: A screening of neutrophils unresponsive to volumetric changes from PMA activation may be easily performed using both Coulter STKR and STKS analyzers, whereas expected or defective PMA-induced production of O2- may be semi-quantitatively evaluated by STKS.

Volume, conductivity, and scatter changes of activated polymorphonuclear leukocytes: an estimation by Coulter Counter STKS analyzer.

Suspensions of phorbol myristate acetate-activated polymorphonuclear leukocytes were analyzed with a Coulter Counter STKS hematological analyzer. Phorbol myristate acetate activation induced an increase in polymorphonuclear leukocyte volume and conductivity, while scatter was unchanged. Phorbol myristate acetate-activated neutrophils in a suspension containing nitroblue tetrazolium showed increased scatter. The rise in scatter was phorbol myristate acetate dose dependent, completely inhibited by diphenylene iodonium and partially by dimethyl sulfoxide, two inhibitors of NADPH oxidase. Zymosan-activated polymorphonuclear leukocytes were notably larger with a characteristic position on discriminant function 1 display (volume versus scatter) of the analyzer. Volume and conductivity changes were seemingly inexplicable features of phorbol myristate acetate activation. The rise in scatter was produced by cytoplasmic precipitation of reduced nitroblue tetrazolium and thus by O2-generation in phorbol myristate acetate-activated neutrophils. Zymosan phagocytosis was responsible for the notable rise in polymorphonuclear leukocyte volume. The analysis of activated polymorphonuclear leukocytes by Coulter Counter STKS may provide useful information on their activation and a pragmatic approach for studying function.

Improved neutrophil separation from anti-D-treated Rh(D)-positive whole blood by a discontinuous gradient method.

Neutrophil separation in Rh(D)-positive whole blood with a discontinuous gradient method (Ficoll 1077-1119 and sodium diatrizoate) was improved by adding anti-D reagent to ethylene diaminetetraacetic acid anticoagulated blood (1 drop/mL). The mean yield was raised more than two times (x2.6) without red blood cell contamination. Anti-D reagent did not impair neutrophil viability, purity, nitroblue tetrazolium-formazan production, or shape change response to N-formyl-methionyl-leucinyl-phenyl-alanine.

[Maintenance therapy with beta-methyldigoxin in the elderly. A study of 40 patients]. / Terapia di mantenimento con beta metildigossina nell'anziano. Studio su 40 pazienti.

The efficacy of beta methyldigoxin was examined in a group of 40 elderly patients who had been hospitalised due to congested heart decompensation. A good clinical response was obtained in 95.5% of cases with the presence of slight toxic phenomena in 2 cases only (4.5%). The paper underlines the excellent pharmacokinetic pattern of the substance used in the steady state. Steady state digitalemic values were within the acceptable range in 83.76% of cases, whereas underdosage and overdosage phenomena were observed in 6.87% and 9.37% of patients respectively.

Advantages of a new anticoagulant in routine hematology on the Coulter Counter S-Plus STKR analyzer.

The authors report the advantages of a new anticoagulant-antiaggregant mixture that avoids the deleterious effects of ethylenediaminotetraacetic acid (EDTA) on mean platelet volume and also prevents EDTA-induced platelet clumping. It is suitable for routine cell counting and sizing with the Coulter Counter S-Plus STKR. The values of the common hematologic parameters agree well with those from EDTA-treated samples and are stable for at least eight hours after sampling.

EDTA-induced platelet aggregation can be avoided by a new anticoagulant also suitable for automated complete blood count.

In vitro EDTA-induced platelet aggregation is a fairly rare event but can have serious clinical consequences producing pseudothrombocytopenia and pseudoleukocytosis. Sixteen specimens with previously recognized EDTA-induced platelet aggregation were collected in a new anticoagulant-antiaggregant mixture containing trisodium citrate 17 mmol/l, pyridoxal 5'-phosphate 11.3 mmol/l and Tris 24.76 mmol/l (CPT mixture) and analyzed at various times after venepuncture with four hematological instruments: Coulter Counter S-Plus STKR, Technicon H6000, Technicon H1 and Ortho ELT-8. In CPT-anticoagulated specimens the signals and instrumental flags of platelet clumping were absent, and the platelet number correlated very well with a microscopic count from a finger stick drawn into Unopette. The complete blood count was very similar in "normal" hematological specimens either collected in K3. EDTA or in CPT, although Technicon H1 and Ortho3 ELT-8 required a suitable calibration for MCV and hematocrit in the latter mixture. Mean platelet volume was stable for up to 24 h only in CPT-collected specimens, if it was measured on a Coulter Counter S-Plus STKR. In routine hematological practice CPT can be an alternative anticoagulant to K3. EDTA, most suitable for automated complete blood count and useful in avoiding EDTA-induced platelet clumping.

Determination of high density lipoprotein cholesterol in venous and capillary whole blood.

A procedure is presented and evaluated for separation of plasma high density lipoprotein from either capillary or venous whole blood. The lipoprotein is separated by adding 50 microliter of sample to 250 microliter of 0.15 M NaCl solution containing 99.9 g/l polyethyleneglycol 6000, 0.0374 g/l dextran sulfate (Mr 15,000) and 2.6 mM Mg2+. After gentle mixing for a few minutes and standing 10 min at room temperature, mixtures are centrifuged (1,500 g) for 10 min and cholesterol is measured on 200 microliter of supernatant by an enzymatic-colorimetric method. Comparison studies demonstrate a good correlation between high density lipoprotein cholesterol in plasma and capillary or venous whole blood. The procedure is simple, has the advantage of using either K3-EDTA-anticoagulated whole blood, without the need of centrifugation, or capillary whole blood which can also be collected away from the laboratory.