Effects of resolution reduction on data analysis.

BACKGROUND: There is often a need in flow cytometry to display and analyze histograms at resolutions lower than those native to the data. It is common, for example, to analyze DNA histograms at 256-channel resolution, even though the data were acquired at 1,024 channels or more. The most common method for reducing resolution, referred to as the consecutive summation (CS) method, can introduce distortions into the shape of histograms. Peaks that were symmetric in the original data can become skewed in the reduced-resolution histogram. Data analysis can be negatively affected by the distortions produced by reducing the histogram resolution. An alternative technique for reducing histogram resolution, the unbiased summation (US) method, minimizes shape distortion. This paper describes the US method and examines the benefits it provides in the analysis of DNA histograms. METHODS: Reduced chi-square (RCS) was used to measure the response to three experimental variables in the least-squares analysis of simulated DNA histograms. For each variable (the percentage of coefficient of variation [%CV], number of events, and mean position of the G1 distribution), a test data set of 1,000 histograms was generated at 1,024-channel resolution. Histogram resolutions were reduced with each method and then analyzed with ModFit LT cell-cycle analysis software (Verity Software House, Topsham, ME). S-phase error and processor computation time of each method also were evaluated. A Monte Carlo experiment was performed to compare CS and US methods to theoretically correct reductions. RESULTS: CS method analysis results were negatively affected by changes in %CV, number of events, and G1 peak position. The US method produced consistently lower RCS values (more accurate results) within the tested ranges. The US method eliminated bias in S-phase error and had negligible impact on analysis processing speed. It improved RCS values 44.50% on average (P < 0.0002) with actual DNA histograms. Whereas the CS method became less accurate (chi-square test) as the amount of reduction increased, the US method was unaffected, producing consistently better results. CONCLUSIONS: The US method is recommended for reducing histogram resolution in modeling applications such as DNA cell-cycle analysis. It may have implications in other areas of flow cytometric data analysis.

Storage of platelets in additive solutions: a pilot in vitro study of the effects of potassium and magnesium.

BACKGROUND AND OBJECTIVES: Platelet additive solutions (PAS) have been shown to be suitable for extended platelet storage but have required the carryover of substantial (30%) amounts of plasma for success. Improving platelet quality by optimizing the composition of PAS may allow a reduction to be made in the amount of plasma carried over. Reducing the proportion of plasma carried over would facilitate some methods of viral inactivation and make available greater amounts of plasma for other needs. MATERIALS AND METHODS: Platelets from six pools of 25 buffy coat platelet units and five apheresis platelet units were aliquoted for storage in plasma, or converted to PAS units in either a specific additive solution (PAS-III), with 30% or 20% plasma, or a modification of PAS-III containing 5.0 mm potassium and 1.5 mm magnesium (PAS-IIIM), with 30% or 20% plasma. Units were stored at room temperature with agitation for 7 days with in vitro testing for biochemical, haematological and functional parameters. RESULTS: Storage of platelets in PAS-IIIM resulted in a reduced rate of glycolysis and better retention of pH, morphology score and ATP levels. Platelets initially showed less evidence of activation when stored in PAS-IIIM, with reduced P-selectin expression. Storage in PAS-IIIM with 20% (rather than the standard 30%) plasma appeared to result in the retention of in vitro properties, similarly to storage in standard PAS-III with 30% plasma. CONCLUSIONS: Storing platelets in an additive solution containing magnesium and potassium improves the functionality of the platelets, as measured by in vitro testing, and may allow a reduction of the amount of plasma required to be carried over to the final unit.

Proposal for standardization of flow cytometric reticulocyte maturity index (RMI) measurements.

Flow cytometric (FCM) reticulocyte analysis is more accurate, sensitive, and reproducible relative to previously employed manual microscopic methods in clinical laboratory hematology. FCM reticulocyte analysis using RNA binding fluorochromes additionally allows for the quantification of fluorescence intensity or population distribution of the reticulocyte RNA content. Viewed from the perspective of erythroid maturation, quantification of the fluorescence intensity distribution provides a reticulocyte maturation index (RMI). We performed a systematic study of 18 different methods to express thiazole orange stained reticulocyte fluorescence intensity, compared to standard mean fluorescence intensity quantification, using 185 anemic and non-anemic human blood samples. The method best correlating with the mean fluorescence intensity RMI on 2 different FCM instruments (R2 = 0.93 and 0.86) was a ratio of the highly fluorescent reticulocytes, defined using a normal adult population, and the total number of reticulocytes (HFR%). In contrast to mean fluorescence intensity measurements, a HFR% RMI parameter can provide similar units of expression (0.01-1.00) with good correlation between different FCM instruments (R2 = 0.76). We conclude the HFR% method of RMI expression provides a superior means of interlaboratory standardization and clinical comprehension of this useful diagnostic parameter in clinical laboratory hematology.

Tumor-induced altered gastrointestinal steady-states: absence of MHC restriction in the paraneoplastic gastrointestine.

The growth of a number of experimental rodent tumours including the Lewis lung tumour (LLca) progressively compromises the integrity of the host's gastroinestine by inducing cytokinetic alterations in the small bowel resembling those generally defining the intestinal phase of a graft-versus-host reaction (GVHR). To determine whether the induction of this paraneoplastic gastrointestine (PGI) involves, similar to a GVHR, a disparity between the MHC of the donor (LLca tumour) and the recipient (host), PGI development was evaluated in various LLca tumour-bearing murine strains that were either 'syngeneic' [C57BL/6 and BL/10 (H-2b)], 'semisyngeneic' [B6D2F1 (H-2bd) and B6C3F1 (H-2bk)] or 'allogeneic' [C3H/HeJ (H-2k) and DBA/2 (H-2d)] to the H-2b LLca tumour. The temporal appearance and magnitude of a PGI developing in either LLca-syngeneic or semi-syngeneic hosts, but not the allogeneic strains, suggested that the mechanism(s) involved in PGI development like the GVHR, was restricted by the MHC. Subsequent studies using congenic strains [B10.A (H-2k) and B10.D2/nSn (H-2d)], however, demonstrated that the mechanism(s) responsible for the PGI was restricted by the non-MHC loci of the C57BL mouse. These observations were supported by the appearance of a LLca-induced PGI in various B10.A congenic strains carrying mutations at the I-A or I-E/I-J loci of the MHC. Not unlike the intestinal phase of a GVHR, development of the PGI required the participation of enhanced mucosal mast cells which were limited in the WCB6F1 (S1/S1d) but not the (+/+) murine strains. These observations are discussed in light of the postulated premature migration of immature thymocytes that accompany tumour growth and their ability to non-specifically enhance (or suppress) cell mediated immune reactions in the host.

Comparative study of assays for stimulated fluid pinocytosis in canine and human neutrophils.

We compared a flow cytometric assay of fluid pinocytosis in stimulated neutrophils with previously used methods, including spectrophotofluorometric assay of fluoresceinated dextran (FD) uptake and beta scintillation spectroscopic assay of radiolabeled sucrose uptake. Neutrophils from human and canine blood were stimulated with formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate, respectively. Flow cytometry was used to quantitate uptake of FD and lucifer yellow, spectrophotometry was used to quantitate uptake of FD, and spectroscopy was used to quantitate uptake of 14C-labeled sucrose. The flow cytometric assay, using either FD or lucifer yellow, was found to be superior on the basis of maximal incremental increase observed, minimal variability within replicate experiments, and maximal sensitivity to detect early stimulation of fluid pinocytosis in neutrophils.

Characterization of f-Met-Leu-Phe-stimulated fluid pinocytosis in human polymorphonuclear leukocytes by flow cytometry.

N-formylated chemotactic peptide stimulation of human neutrophils initiates a number of cellular processes, such as lysosomal enzyme release and superoxide anion production, that are indicative of the events of neutrophil activation during the acute inflammatory response in disease. This study characterizes a newly recognized neutrophil activation event, N-formylated chemotactic peptide-stimulated fluid pinocytosis in human neutrophils, using a novel flow cytometric assay for this activity. Fluid pinocytosis was found to be inhibited by acidic pH and low temperature but could be enhanced by cytochalasin B treatment or surface adherence by neutrophils. The activity measured by this new assay of fluid pinocytosis appears to be separate and distinct from lysosomal enzyme release and receptor-mediated adsorptive endocytosis in neutrophils. The physiologic significance of N-formylated chemotactic peptide-stimulated fluid pinocytosis is not known, but a possible relationship to neutrophil locomotion is discussed.

Murine in vitro antigenic modification of tumor cells: effect on susceptibility to natural cell-mediated cytotoxicity.

A 3-methylcholanthrene [(MCA) CAS: 56-49-5]-induced fibrosarcoma cell line and its Friend murine leukemia virus-infected counterpart were assessed for their susceptibility to lysis by so-called "natural" effector cells in a series of 51Cr release assays. Detailed functional and phenotypic analysis of lytic effector cell populations from normal C57BL/6 mouse spleens revealed an identity most closely associated with natural cytotoxic cells. Neither tumor cell line was found to be sensitive to natural killer-mediated lysis. Additionally, virus infection of the MCA-induced fibrosarcoma cell line did not affect susceptibility to natural cell-mediated cytotoxicity.

Role of suppressor cells in feline leukemia virus-associated immunosuppression.

Protein from detergent-disrupted feline leukemia virus was found to suppress lymphocyte blastogenic responsiveness of peripheral blood mononuclear cells from normal cats. When suppressive doses of this protein were substituted for mitogen in the preactivation step of the concanavalin A-induced suppressor cell assay, significant suppression was observed in five of eleven cats tested. These results suggest that feline leukemia virus-associated immunosuppression may be mediated by a virus-induced suppressor cell.

Effect of antioxidants on the proliferative response of canine lymphocytes in serum from dogs with vitamin E deficiency.

The in vitro effect of vitamin E and 3 other antioxidants--ethoxyquin, 2-mercaptoethanol, and ascorbic acid--on proliferation of canine lymphocytes was examined. Lymphocytes from 2 groups of dogs given a vitamin E-deficient diet or whelped from a bitch fed such a diet were cultured with pooled samples of serum from dogs fed a vitamin E-deficient diet or whelped from a bitch fed such a diet, or normal canine serum, and stimulated with phytohemagglutinin. Added vitamin E enhanced the responsiveness in serum from the dogs with vitamin E deficiency, but not in normal canine serum. A similar effect was noted with added ethoxyquin and 2-mercaptoethanol. Ascorbic acid had no effect on proliferation in either serum pool. These results indicated that depressed lymphocyte responsiveness seen with serum from vitamin E-deficient dogs may, at least in part, be due to a loss of antioxidant activity in this serum.

Generation of concanavalin A-induced suppressor cells in the cat.

Preactivation of feline peripheral blood mononuclear cells with mitogenic doses of concanavalin A resulted in suppression of proliferation when these cells were cocultured with untreated autologous cells. The degree of suppression was directly proportional to the preactivation time and ratio of preactivated to untreated cells. The generation, but not the activity, of the suppressor cells was dependent on DNA and protein synthesis. Pokeweed mitogen and phytohemagglutinin were also shown to be capable of inducing suppressor cell activity. Suppression was also observed in allogeneic cocultures. Supernatants from concanavalin A-preactivated cell cultures did not suppress proliferation of untreated cells.

Effect of vitamin E deficiency on the proliferative response of canine lymphocytes.

The effect of vitamin E deficiency on the proliferative response of canine lymphocytes was examined, using the lymphocyte blastogenesis assay, with phytohemagglutinin, concanavalin A, and pokeweed as mitogenic stimulants. A litter (4 pups; group 3) from a bitch fed a complete diet was given a complete diet from the time of weaning (6 weeks) until termination of the experiment, and served as a control group. A litter (4 pups; group 1) from a bitch fed a vitamin E-deficient diet was given a vitamin E-deficient diet from the time of weaning (6 weeks) until termination of the experiment. A litter (6 pups; group 2) from a bitch fed a complete diet was weaned onto a complete diet and then changed to a deficient diet until termination of the experiment. Lymphocytes from group 1 and 2 pups (fed deficient diets) were poorly responsive to mitogen-induced blastogenesis, compared with the lymphocytic response seen in the controls. Washing the lymphocytes from the pups fed vitamin E-deficient diet resulted in enhanced responsiveness to mitogen stimulation. Lymphocytes from controls which had good responses when cultured in bovine fetal serum or normal canine serum were poorly responsive when cultured in serum from groups 1 and 2 pups (fed vitamin E-deficient diet). Reducing the concentration of group 1 and 2 pup sera in the culture medium restored the responsiveness of lymphocytes from control pups. Supplementation of group 1 and 2 pup sera with bovine fetal serum did not result in enhancement of the responses of lymphocytes from controls. The capability of sera from vitamin E-deficient pups (groups 1 and 2) to support blastogenesis of lymphocytes from control dogs was restored following dietary supplementation with vitamin E. It was concluded that the depressed lymphocyte responsiveness in pups fed vitamin E-deficient diet was due to the presense of a suppressive factor in the serum.