A comparison of the cytology of endomyocardial biopsy washings from heart transplants with biopsy histologic study and peripheral blood lymphocyte counts.

Cytospin preparations of endomyocardial biopsy washings were examined on 117 occasions from 13 heart transplant recipients and categorized according to the pattern of cell types observed. Twenty-nine percent of samples were acellular, a further 10% too bloodstained for analysis, and 61% were cellular. Eight lymphocytic samples were found and in all cases there was at least grade 1B rejection (four grade 1B, three grade 2, and one grade 3A) on histologic study. However, histologic study showed at least 1B rejection in 48% of cases when cytospins showed mixed inflammatory cells, 33% of cases when cytospins were histiocytic and in 35% when cytospins were bloodstained or acellular. Furthermore 16 of these rejection episodes with nonlymphocytic cytospins were grade 2. Although the recovery of a lymphocytic cytospin was specific for rejection, the sensitivity of the test was poor. Even when the sample is adequate, this method of biopsy washings will predict only one third of cases of significant acute rejection (grade 2 or worse). The large proportion of unsuitable samples also severely limits the utility of endomyocardial biopsy washings for the diagnosis of rejection. Histiocytic cytospins were seen in 63% of samples when previous biopsy sites were reported on histologic study and also in all three samples when histologic study showed ischemic injury. A mixed inflammatory cell pattern was seen to a lesser extent (31% of samples) in relation to previous biopsy sites. High peripheral blood lymphocyte counts were found when endomyocardial biopsy washings were lymphocytic or mixed inflammatory and also when histologic study showed endocardial lymphocytic infiltration (Quilty effect).

Evaluation of a novel stable whole blood quality control material for lymphocyte subset analysis: results from the UK NEQAS immune monitoring scheme.

The UK NEQAS immune Monitoring Scheme (UK NEQAS) evaluates the performance of laboratories routinely performing T-lymphocyte subset analysis on HIV-infected individuals. The scheme originally issued fresh whole blood, but a significant problem was that of analyte stability, especially 36 h postphlebotomy. To circumvent this problem, we have developed a novel stabilisation procedure that ensures retention of leucocyte light scatter and immunological staining characteristics for up to 300 days. In addition, the stabilised whole blood preparation is fully compatible with flow cytometer technology, incorporating either whole blood lysis or "no wash, no lyse" techniques. The ranges of interlaboratory coefficient of variation for the stabilised material are now tighter than those previously obtained with fresh whole blood. Development of this novel material has enabled overseas laboratories to participate in the UK NEQAS immune Monitoring Scheme and could in the future lead to the production of reference and/or calibration reagents for leucocyte immunophenotyping.

Circulating CD20dim T-lymphocytes increase with age: evidence for a memory cytotoxic phenotype.

The CD20 antigen has been regarded as a B lineage specific, 35 kDa, non-glycosylated membrane phosphoprotein, which functions as either a Ca2+ ion channel or as a regulatory protein of such a channel. Weak expression of CD20 (CD20dim), however, has recently been reported on a sub-population of T lymphocytes. We present results which confirm the existence of a CD20dim T lymphocyte population and show that such cells have a reduced antibody-binding capacity, when compared to CD20bright B-cells (10337 +/- 642 and 346311 +/- 24264 respectively). In addition, CD20dim cell counts vary with age, with the highest levels occurring in octogenarians: cord blood 0.3 +/- 0.1% (n = 13), 20-60 year-old group 2.1 +/- 1.1% (n = 18) and individuals > or = 61 years of age 6.9 +/- 3.2% (n = 10) (P < 0.001). Further characterization of CD20dim T cells, using three colour flow cytometry, demonstrated a predominantly memory cytotoxic phenotype, in that the cells were CD8+CD28+CD45RO+T-CR alpha beta +CD38-HLA-DR-.

Determination of leucocyte antibody binding capacity (ABC): the need for standardization.

The flow cytometric determination of antigen density, or cellular antibody binding capacity, is now an accepted technique for the characterization of cells in health and disease. In HIV infection, for example, antigen density changes in CD38 expression may be an important indicator of disease progression. Our experience of using one such method, Quantum Simply Cellular, which measures antibody binding capacity (ABC), has highlighted several technical factors which can affect the results. We report the influence of pH, incubation temperature and time, antibody fluorochrome and titre, as well as lysing reagent (FACS Lysing Solution v. Ortho-mune Lysing Reagent) on the ABC of anti-CD3, CD4 and CD8 of normal lymphocytes. In addition, the effect of single, double or triple-staining was assessed. The results indicate that the ABC values are influenced by all the variables studied. The pH range tested (6.0-9.0) demonstrated that pH 7.4 gave maximal binding. Furthermore, temperature also influenced the pH of the two lysing solutions, and thus potentially the ABC. Antibody concentration, fluorochrome and staining technique are also important factors with an observed difference of up to 458,855 ABC between the various fluorochromes. In addition a maximal difference of 130,119 ABC was observed between single and triple staining techniques. In conclusion, if antigen quantification is to be used in the clinical setting, an internationally standardized method is required to ensure the reproducibility of results from centre to centre. Our data suggests that single staining, using fluorescein isothiocynate (FITC) conjugated antibodies with all reagents at pH 7.4 + 0.1, with incubation and lysing carried out at 20 + 1 degrees C, could be used as a 'benchmark' method for ABC determination using the QSC system.

Absolute CD4+ T-lymphocyte and CD34+ stem cell counts by single-platform flow cytometry: the way forward.

To determine the potential advantage of single-platform technology in the enumeration of CD4+ T lymphocyte and CD34+ stem cells, data has been analysed from the UK NEQAS for Leucocyte Immunophenotyping schemes. The inter-laboratory CVs for CD4+ T lymphocyte counts were consistently lower for single-platform (mean 13.7%, range 10-18.3%) compared to dual-platform methodology (mean 23.4%, range 14.5-43.7%). Subgroup analysis of single-platform users demonstrated mean overall inter-laboratory CVs of 17.2%, 13% and 7.1% for the FlowCount, TruCount and volumetric approach respectively. The lowest inter-laboratory CVs obtained for a single sample by each single platform approach were 4% (TruCount), 4.4% (volumetric), 4.6% (FACSCount) and 12.7% (FlowCount). Similarly, the mean inter-laboratory CV for CD34+ stem cell enumeration using non-standardized single-platform approaches was 18.6% (range 3.1-36.9%) compared to 28.6% (range 19-44.2%) for the dual-platform technology. Our results suggest absolute cell subset enumeration should be performed by single-platform technology and that such an approach should improve the quality control of multi-centre clinical trial data for CD4+ T lymphocyte and CD34+ stem cells.

Quality assessment of CD34+ stem cell enumeration: experience of the United Kingdom National External Quality Assessment Scheme (UK NEQAS) using a unique stable whole blood preparation.

CD34+ peripheral blood stem cell (PBSC) mobilization and harvesting has rapidly replaced autologous bone marrow as a source of stem cells for transplantation. Timing and adequacy of harvests rely upon the accurate enumeration of circulating CD34+ cells. However, previous EQA programmes have reported interlaboratory CVs as high as 284%, suggesting the need for greater standardization. In addition the routine use of fresh and/or frozen cells as analytes also introduces antigen instability as a variable factor. To circumvent this problem and achieve a true reflection of interlaboratory variation, we have used a novel whole blood preparation in which the antigenic profiles of PBSCs, as determined by flow cytometry, are retained for > 200 d. This international scheme, currently the largest in the world, distributes aliquots of stabilized whole blood bi-monthly to 91 laboratories in 20 countries (44 U.K., 47 overseas). Participants are required to determine the percentage and absolute values for CD34+ PBSCs using in-house techniques. Adopting such a preparation, a more accurate determination of interlaboratory variation has been possible when compared to previous EQA studies, with CVs as low as 22% and 24% for percentage and absolute counts. In addition the programme has established that a wide range of methods are in routine use, emphasizing the urgent requirement for national/international consensus guidelines.

Standardization of lymphocyte antibody binding capacity - a multi-centre study.

As quantitative flow cytometry is being increasingly used to characterize non-malignant and malignant disorders, interlaboratory standardization becomes an important issue. However, the lack of standardized methods and process controls with predefined antibody binding capacity values, limits direct interlaboratory comparison. The present study has addressed these issues using a stable whole blood product and a standardized antigen quantification protocol. It was demonstrated that: (i) a standard technical protocol can result in a high degree of interlaboratory concordance; (ii) interlaboratory variation of less than 12% can be achieved for CD4 antibody binding capacity values; and (iii) stable whole blood can be used as a process control with predefined antibody binding capacity values. Furthermore, using such an approach, a normal range was established for CD3, CD4 CD8 and CD19. These antigens appear to be expressed in a hierarchical manner, a factor that could be used as a procedural quality control measure.

Swallowing food without chewing; a simple way to reduce postprandial glycaemia.

1. The degree to which disruption by mastication affects the glycaemic response to four different carbohydrate foods was investigated in healthy human volunteers; each food was eaten by six subjects. 2. Subjects ate meals of sweetcorn, white rice, diced apple or potato on two occasions; on one occasion they chewed the food thoroughly, on the other occasion they swallowed each mouthful without chewing it. 3. When the foods were chewed the postprandial blood glucose levels rose to levels which varied according to the food ingested. 4. Swallowing without chewing reduced the glycaemic response to each food, achieving a similar effect as administration of viscous polysaccharides or 'slow-release' carbohydrates.