Blast counts in blood progenitor cell (BPC) correlate with CD34+ cells and CFU-GM and are useful predictor of haemopoietic recovery after autologous BPC transplantation collections.

Assessment of the quality of blood progenitor cell (BPC) collections is based mainly on CD34+ cell enumeration by flow cytometry, or scoring of granulocyte-macrophage colony-forming cells (CFU-GM). A minimum cell dose for haemopoietic recovery can be defined by both assays; however, the CFU-GM assay can not be used for 'real-time' decisions, whereas CD34+ cell scoring requires facilities and expertise which are not universally available. We have investigated the possibility of using morphologically defined blast cells within BPC harvests as a surrogate marker of harvest haemopoietic stem/progenitor cell content, as well as their correlation with CD34+ cells and CFU-GM within the harvests. We have found that blast counts correlate strongly with both CD34+ cell counts and CFU-GM within BPC harvests, as well as with time to granulocyte and platelet recovery after autologous BPC transplantation (ABPCT). Furthermore, we have defined a threshold value of 1.3 x 10(6)/kg blasts, above which there is a high probability of rapid haemopoietic recovery after ABPCT. We conclude that blast count is a simple, rapid and reliable method of assessing BPC harvest quality.

Blood polymorphonuclear leukocytes from the majority of sickle cell patients in the crisis phase of the disease show enhanced adhesion to vascular endothelium and increased expression of CD64.

There is increasing interest in the role of blood polymorphonuclear leukocytes (PMNs) in the pathogenesis of sickle cell crisis. We studied the adherence of PMNs from 18 sickle cell patients in crisis, 25 out of crisis, and 43 healthy subjects (controls) to monolayers of human umbilical cord endothelium that were either untreated or pretreated with tumor necrosis factor alpha (TNFalpha). Overall, the PMNs from patients in crisis were more adherent than control PMNs to untreated endothelial monolayers (mean 53% increase; P < .001) and TNFalpha-treated monolayers (mean 41% increase; P < .002). Increased adhesiveness was not associated with an abnormal expression of CD11a, CD11b, CD11c, CD18, CD62L, or CD15. There was an increase in the number of PMNs expressing CD64 in patients in crisis (median value, 44%) compared with patients out of crisis (median, 21%; P = .025) and controls (median, 6.5%; P < .001). Sera from patients in crisis had normal levels of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interferon-gamma, TNFalpha, interleukin-1 (IL-1), IL-6, or IL-8 and did not modify the adherence of PMNs or their expression of CD64. Only IFN-gamma induced CD64 expression on PMNs, but this effect was not associated with enhanced binding to endothelium. Because PMNs bound to endothelial monolayers were CD64(+) and CD64-enriched PMNs were 7 times more adherent to endothelial monolayers than CD64-depleted PMNs, it is likely that CD64 is a marker of adherent PMNs. Two of the three anti-CD64 antibodies used in our antibody blocking studies (clones 32.2 and 197) partially inhibited the binding of sickle cell PMNs to untreated endothelium (mean inhibitions of 33% [P = .01] and 21% [P = .03], respectively), whereas only one (clone 197) inhibited binding to TNFalpha-treated endothelium (mean inhibition, 29%; P = . 004). In some patients with sickle cell disease, an enhanced PMN adhesion to vascular endothelium could contribute to the vascular occlusion that characterizes the acute crisis of the disease.

'Low-risk' myelodysplastic syndrome is associated with excessive apoptosis and an increased ratio of pro- versus anti-apoptotic bcl-2-related proteins.

We performed flow cytometric analysis of CD34+ cell apoptosis in 59 patients with myelodysplastic syndrome (MDS) or acute myeloid leukaemia (AML) secondary to MDS (MDS-AML) using annexin V-FITC, which binds to exposed phosphatidylserine on apoptotic cells. Apoptosis was significantly increased in FAB subtypes RA, RARS and RAEB (<10% blasts) (56.5% (15.1-86.5%)) compared to normal controls (18.5% (3.4-33.4%), P<0.0001) and RAEB-t/MDS-AML (16% (2.1-43.2%), P<0.0001). There was no correlation between % apoptosis, Full blood count or cytogenetics in any disease category. Two-colour cytometric analysis of permeabilized CD34+ cells stained with antibodies to Bcl-2, Bcl-X (anti-apoptotic), Bax and Bad (pro-apoptotic), demonstrated significantly higher ratios of pro- v anti-apoptotic proteins in early MDS (2.47 (1.19-9.42) compared to advanced disease (1.14 (0.06-3.32), P=0.0001). Moreover, using repeated measures of variants (ANOVA), we found that variations between individual Bcl-2-related proteins differed significantly according to disease subtype (P<0.0005). Our results confirm that CD34+ cell apoptosis was significantly increased in MDS subtypes RA and RARS and fell with disease progression. Early MDS was also associated with a significantly higher CD34+ cell pro- v anti-apoptotic Bcl-2-family-protein ratio than advanced disease. Furthermore, patterns of expression of individual Bcl-2 related proteins differed significantly between different disease categories. However, no correlation between pro- v anti-apoptotic Bcl-2-family-protein ratios and the degree of apoptosis was observed.

Persistent polyclonal B-cell lymphocytosis in identical twins.

This is the first report of the unusual syndrome of persistent polyclonal B-cell lymphocytosis occurring in monozygotic twins. The syndrome is characterized by a lymphocytosis, with circulating atypical, binucleated lymphocytes, mild splenomegaly and raised serum IgM. It occurs predominantly in females, with serological evidence of previous EBV infection, and is associated with cigarette smoking and HLA-DR7 phenotype. The association with .DR7 suggests a genetic predisposition. Its occurrence in identical twins, documented here, provides stronger support for a hereditary/genetic basis for the syndrome.

Neonatal screening for haemoglobin variants using filter paper-dried blood specimens.

Neonatal screening for haemoglobinopathies utilizing cord blood samples is well established, although it has a high miss rate and has the inherent problem of possible misdiagnosis from maternal contamination of the sample. The use of dried Guthrie card samples which are taken at six days of age avoids these problems and has the advantage of using an established system of sample collection. Controversy exists as to the method of choice for analysis of dried samples, this study of 2406 samples found that Iso-electric focusing (IEF) analysis of dried specimens gives excellent correlation when compared with cellulose acetate/citrate agar electrophoresis of liquid cord blood samples. The IEF results were clear and relatively simple to interpret even when the samples had been stored at room temperature for 4 weeks. The commercial enzyme linked immunosorbent assay (ELISA) screening test JOSHUA reliably determines the presence or absence of haemoglobin S in dried specimens. It could therefore be used as a relatively cheap and simple method for the confirmation of sickle cell trait in neonatal screening programmes based on dried specimens.