Monitoring dendritic cells in clinical practice using a new whole blood single-platform TruCOUNT assay.

Dendritic cells (DC) from distinct DC subsets are essential contributors to normal human immune responses. Despite this, reliable assays that enable DC to be counted precisely have been slow to evolve. We have now developed a new single-platform flow cytometric assay based on TruCOUNT beads and the whole blood "Lyse/No-Wash" protocol that allows precise counting of the CD14(-) blood DC subsets: CD11c(+)CD16(-) DC, CD11c(+)CD16(+) DC, CD123(hi) DC, CD1c(+) DC and BDCA-3(+) DC. This assay requires 50 microl of whole blood; does not rely on a hematology blood analyser for the absolute DC counts; allows DC counting in EDTA samples 24 h after collection; and is suitable for cord blood and peripheral blood. The data is highly reproducible with intra-assay and inter-assay coefficients of variation less than 3% and 11%, respectively. This assay does not produce the DC-T lymphocyte conjugates that result in DC counting abnormalities in conventional gradient-density separation procedures. Using the TruCOUNT assay, we established that absolute blood DC counts reduce with age in healthy individuals. In preliminary studies, we found a significantly lower absolute blood CD11c(+)CD16(+) DC count in stage III/IV versus stage I/II breast carcinoma patients and a lower absolute blood CD123(hi) DC count in multiple myeloma patients, compared to age-matched controls. These data indicate that scientific progress in DC counting technology will lead to the global standardization of DC counting and allow clinically meaningful data to be obtained.

Instrumentation for flow cytometry.

Advances in fluidics, optics, electronics, lasers, computers and software have made flow cytometers considerably more complex, but also easier to use. Production of new fluorescent dyes has seen the development of more flexible instruments so that eight or more parameters of correlated data can be collected at once. Flow cytometers can be subdivided into two groups, analysers and sorters. A description of the commercially available analysers and sorters together with their specifications (as at October 1999) will be given.

Antibody penetration of viable human cells. I. Increased penetration of human lymphocytes by anti-RNP IgG.

Antibody penetration of viable cells and interaction with intracellular antigens may have major consequences for immunopathological processes in connective tissue diseases. We have reported previously that antibody can penetrate viable human lymphocytes. To assess further the role of antinuclear antibodies in this process, peripheral blood lymphocytes (PBMC) were incubated with FITC-conjugated IgG fractions from sera containing anti-RNP (anti-RNP IgG), Ro(SS-A), La(SS-B) and dsDNA antibodies and control sera for 24 h. Using crystal violet to quench cell surface staining, intracellular fluorescence of viable lymphocytes was quantified on the flow cytometer. It was noted that anti-RNP IgG entered 46.4 +/- 7.2% of lymphocytes which was significantly higher than anti-Ro(SS-A) (29.9 +/- 4.1%, P less than 0.05), La(SS-B) (22.0 +/- 7.5%, P less than 0.01) IgG and control IgG (28.8 +/- 2.1%, P less than 0.05) and not statistically different from anti-dsDNA IgG (32.6 +/- 14.3%). Inhibition experiments showed that the increased number of cells penetrated by anti-RNP IgG was a specific process. Time-course studies showed that anti-RNP IgG entry into cells was different from pooled control IgG. With anti-RNP IgG, positive-staining lymphocytes gradually increased in number from 12 to 24 h incubation, whilst with pooled control IgG, the peak was reached within 5 min. Dual staining experiments suggested that whereas both anti-RNP IgG and pooled control IgG entered B and NK cells, anti-RNP IgG also entered T cells. Using IgG F(ab')2 and Fc fragments from either anti-RNP IgG or pooled control IgG to compete with their FITC-conjugated counterparts indicated that the entry of anti-RNP IgG into-viable cells appeared to involve both F(ab')2 and Fc fragments, and pooled control IgG depended exclusively on the Fc portion of IgG. Further investigation by incubating anti-RNP IgG with 35S-methionine-labelled monocyte-depleted PBMC (MD-PBMC) suggested that anti-RNP IgG might react with the corresponding antigens either on the cell surface or within the cytoplasm.

Flow cytometry with crystal violet to detect intracytoplasmic fluorescence in viable human lymphocytes. Demonstration of antibody entering living cells.

A new method is described for the detection of intracytoplasmic fluorescence and its differentiation from surface staining of viable human lymphocytes using flow cytometry after addition of crystal violet which quenches surface but not internal fluorescence. This has then been used to study antibody penetration of viable human lymphocytes, using FITC-conjugated IgG from normal serum or serum containing anti-RNP antibody. The results showed that 54 +/- 1% normal lymphocytes were penetrated by anti-RNP antibody and 23 +/- 3% by normal IgG respectively. The lymphocyte population analysed by flow cytometry has been directly demonstrated to be viable by FDA staining. These results provide unequivocal evidence that antibody can penetrate viable human lymphocytes.

Release of galactosyltransferase from human platelets and a subset of monocytes in culture.

We show that human monocytes and platelets release considerable amounts of galactosyltransferase (GT) in serum-free culture as measured by the amount of incorporation of 3H-galactose into ovalbumin. Enzyme production was the greatest among medium-sized mononuclear cells separated by counter-current elutriation. The cells were adherent and positive for the monocyte-specific monoclonal antibody FMC-32. The activity in the monocyte fractions was not due to platelet contamination as shown from experiments in which platelets or platelet antigens were eliminated. Cell viability decreased by less than 3% during the overnight culture, and results from cell disruption experiments showed that the enzyme was not released from dead or dying cells. Cycloheximide inhibited release during 20 hours culture. Approximately 50% of the enzyme in the cell culture supernatant was pelletable at 105,000 g. Platelets released the enzyme more rapidly than did monocytes and were readily stimulated by thrombin to release more GT. Thrombin also increased monocyte GT activity after overnight incubation, but other stimulants, zymosan and lipopolysaccharide (LPS), decreased release. We conclude that GT is released into culture supernatants by platelets and by a subset of peripheral blood monocytes. These sources may account for a significant proportion of the serum enzyme and may be important in modification of extracellular carbohydrates during inflammation and coagulation.

Uremic middle molecules: separation and quantitation.

A chromatographic method for separating uremic middle molecules has been developed, based on a modification of the method of Fürst et al. to permit accurate and rapid (less than 3.5 hours) determination of UMM levels. Semiautomation of the equipment has also resulted in 3 analyses per 8-hr working day. Comparison of UMM results in patients on a carefully controlled study of long (mean 7.5 hr) versus short (mean 3.6 hr) hours of hemodialysis indicate that UMM levels are higher on short dialysis but only peaks 7ao, 7a, 7b, and 7c1 show significant but relatively unimpressive differences between the two regimens. UMM levels have also been obtained in long-term CAPD patients. Data show that UMM levels remain stable, over at least 12 months of CAPD and all UMM levels are lower than in hemodialysis patients. From analysis of generation rate data there is also an indication that UMMs may be dietary related.

Separation and quantification of the "middle molecules" in uremia.

A modification of a two-stage chromatographic procedure (molecular sieve followed by ion-exchange) to separate potentially toxic uremic "middle molecules" from body fluids has been established. The procedure has an analysis time less than half that previously reported with improved resolution. Elution volumes have been found to be reproducible to within 2% and concentrations (as measured by peak height) to within 10%. Careful attention, however, must be paid to artifacts that may arise from sample preparation, drug therapy, and dialysis conditions. Up to ten identifiable subpeaks were observed in sera, urine, and red cell hemolysate samples following ion-exchange separation of a molecular sieve peak in the middle molecule range (300 to 2000 daltons). Red cell concentrations of five of these moieties were significantly higher than those in serum. Four peaks were not detected at all in red cells, and one peak was not detected in sera or urine samples. In addition, although serum concentrations are elevated in uremia, red cell levels in patients with uremia are comparable with those obtained in normal subjects. If any of these species are subsequently shown to be uremic toxins, then their two-compartment distribution within the body has important ramifications in the choice of an appropriate mathematic model to program optimal dialysis therapy.