The total circulating red cell volume (RCV) is a better guide to the oxygen-carrying capacity of the blood in the whole circulation than is the haemoglobin concentration (Hb) or haematocrit in a blood sample. Pre- and post-transfusion RCV (and blood volume (BV)) may be determined by flow cytometry by exploiting antigen differences between transfused donor red cells and the recipient's red cells. This paper describes the use of red cell antigen differences of Duffy, Kidd, MN and RhD between donor and recipient. In 20 infants, transfused on 21 occasions, pretransfusion RCV ranged from 12 to 39 mL kg(-1) body weight. Only at one transfusion could no usable donor-recipient antigen differences be exploited. Measurement of RCV, used routinely, may determine the transfusion requirements of sick infants more accurately, with the aim of normalizing RCV and BV--securing euvolaemia--at the end of the transfusion. This may allow a complete correction of the RCV deficiency at the first occasion of transfusion. This approach may reduce donor exposures and also optimize oxygen transport and organ perfusion of the infant undergoing intensive management, perhaps leading ultimately to improved survival rates and fewer long-term complications of neonatal intensive care.

RAS mutations arise at high frequency (20–40%) in both acute myeloid leukemia and myelodysplastic syndrome (which is considered to be a manifestation of preleukemic disease). In each case, mutations arise predominantly at the N-RAS locus. These observations suggest a fundamental role for this oncogene in leukemogenesis. However, despite its obvious significance, little is known of how this key oncogene may subvert the process of hematopoiesis in human cells. Using CD34+ progenitor cells, we have modeled the preleukemic state by infecting these cells with amphotropic retrovirus expressing mutant N-RAS together with the selectable marker gene lacZ. Expression of the lacZ gene product, β-galactosidase, allows direct identification and study of N-RAS–expressing cells by incubating infected cultures with a fluorogenic substrate for β-galactosidase, which gives rise to a fluorescent signal within the infected cells. By using multiparameter flow cytometry, we have studied the ability of CD34+ cells expressing mutant N-RAS to undergo erythroid differentiation induced by erythropoietin. By this means, we have found that erythroid progenitor cells expressing mutant N-RAS exhibit a proliferative defect resulting in an increased cell doubling time and a decrease in the proportion of cells in S + G2M phase of the cell cycle. This is linked to a slowing in the rate of differentiation as determined by comparative cell-surface marker analysis and ultimate failure of the differentiation program at the late-erythroblast stage of development. The dyserythropoiesis was also linked to an increased tendency of the RAS-expressing cells to undergo programmed cell death during their differentiation program. This erythroid lineage dysplasia recapitulates one of the most common features of myelodysplastic syndrome, and for the first time provides a causative link between mutational activation of N-RAS and the pathogenesis of preleukemia.

The role of p21 RAS in the proliferation and differentiation of myeloid cells has been studied by analysing the changes in the level of expression of p21 RAS proteins by flow cytometry upon differentiation down the granulocytic and monocytic pathways. Differentiation resulted in upregulated p21 RAS expression despite a marked decline in the number of dividing cells. On the other hand, growth inhibition, without differentiation, resulted in a decline in expression. Cell cycle analysis showed that the increase in p21 RAS occurred throughout the cell cycle. These results suggest that p21 RAS has a role in the process of myeloid differentiation.

198 patients with MDS were followed cytogenetically for up to 90 months. There were significant differences in survival between patients with a normal karyotype, single abnormalities (p < 0.05) and multiple abnormalities (p < 0.0001). Survival differences were also seen in each of the FAB sub-types but were only significant in RAEB/RAEB-t and CMML (p = 0.001) where a normal karyotype was associated with prolonged survival. Single and multiple abnormalities of chromosomes 7 and 8 but only multiple abnormalities of chromosome 5 were also associated with reduced survival. 126 patients were successfully investigated on more than one occasion. Karyotype evolution occurred in 15 and was associated with reduced overall survival in those patients who had previously been karyotypically normal (p < 0.05). Median survival following evolution was only 10 months. 29 patients developed leukaemia. The incidence of transformation was significantly higher in patients with multiple abnormalities than in those with a normal karyotype (p < 0.05) or single abnormalities (p < 0.05).

The prognostic significance of clonal karyotype status in myelodysplastic syndrome (MDS) is assessed after an extended follow‐up period of 5 years. There are three prognostic sub‐groups according to the presence of a normal karyotype, single abnormalities or multiple abnormalities at the time of referral. However, there is no correlation between the size of the abnormal clone and prognosis. Karyotype status has independent prognostic significance in ‘high risk’MDS so that patients with a refractory anaemia with excess of blasts (RAEB)/RAEB in transformation (RAEB‐ t ) and a normal karyotype survive significantly longer than those with an abnormal karyotype ( P <0·001) and do not differ significantly from patients with refractory anaemia (RA). Significant differences in survival according to karyotype status are also seen in patients with chronic myelomonocytic leukaemia ( P <0·001) but not in those with primary acquired sideroblastic anaemia and RA. Among patients studied sequentially, those who retained a normal karyotype survived significantly longer than those who developed an abnormality on follow‐up ( P <0·001). The risk of leukaemic transformation was also increased in patients who presented with or subsequently developed a clonal karyotype abnormality compared with those who remained normal ( P <0·05).

Measurements of peripheral blood lymphocyte surface transferrin receptor using flow cytometry show that phytohaemagglutinin (PHA) stimulation causes a marked increase in both the number of cells bearing receptors and the absolute number of receptors. This increase is accompanied by the interleukin-2 receptor and there is a progressive increase of cells in the S phase of their cycle. Furthermore, ferritin synthesis in proliferating cells, as determined by immunoprecipitation techniques, increases significantly compared to non-stimulated cells. Intracellular concentrations of both spleen-type and heart-type ferritin are also increased with a preferentially high proportion of heart-type ferritin. These results suggest that expression of transferrin receptor and synthesis of ferritin is regulated by a complex mechanism. Cellular proliferative activity increases the expression of transferrin receptor and could modulate the biosynthesis of intracellular ferritin that is normally controlled by iron. These two processes do not appear to be linked.

ABSTRACT: Determination of circulating red cell volume (RCV) in anemic preterm infants is, in theory, a better indicator of transfusion needs than Hb concentration. Our study reports the results of RCV measurement using biotin labeling of red cells on 40 occasions in preterm infants of 25–34 wk gestation. In 20 infants, who had estimations made within 24 h of birth, the RCV varied between 17.7 and 66 mL/kg. Twenty measurements were made at a later age at the time of a blood transfusion. RCV values were between 13.1 and 41.5 mL/kg before transfusion. In 13 infants, RCV was determined simultaneously using two methods, biotin and dilution of autologous HbF with donor HbA at transfusion. There was no significant difference between the results of RCV estimations using these two methods. Our study demonstrates that biotin labeling is an effective method for determining RCV in preterm infants.

The time interval between the development of a new technique or methodology and its acceptance, if successful, as a recognized clinical application can be many years. The application of flow cytometry to reticulocyte counting, for example, has taken 8 years from the appearance of the first publication, and in 1990 it is still in its infancy as a clinical method. It is therefore a challenging task to anticipate which of the methodologies currently under development will achieve acceptance. It would be impossible to deal with all the candidates in the space available, and so a review is provided to those methods that may have potential applications in clinical haematology, together with some of the more practical details of methods that have recently been demonstrated to be viable in the clinical laboratory. The first category consists of leukocyte enumeration and studies on bone marrow, neutrophils, platelets and cellular DNA content, whilst the second covers reticulocyte counting and total red cell volume measurement. The contribution of flow cytometry to the field of immunophenotyping haematological disorders is probably unique in already being clinically acceptable. Finally, the question of quality control is addressed, as this is an essential prerequisite to the adoption of any new method in the clinical laboratory.

DNA index (DI) determined by flow cytometry and karyotype determined by conventional methods were obtained on bone marrow samples from 43 haematologically normal subjects and 54 patients with myelodysplastic syndrome (MDS). Twenty one patients had a clonal karyotype abnormality but an additional five had a DI outside the normal range, showing evidence of aneuploidy that was not available from chromsome preparations. When patients were grouped into those with excess chromosomal material, those with diploid karyotypes, and those with a loss of chromosomal material, there was a significant difference among the mean DIs of each group, normal subjects being different from all patient groups. In these patients DI measurements were of value when carried out together with conventional chromsomal analysis in gaining the maximum amount of genetic information when a satisfactory karyotype might not be available or where failure of an abnormal cell population to proliferate might give an incomplete cytogenetic picture. The contribution of non-clonal chromsome loss to the DI is probably significant but has not been quantitated.

We have used flow cytometry to quantitate nuclear c-myc protein, at each phase of the cell cycle, during in-vitro differentiation of CD34-positive stem cells isolated from normal human bone marrow by the monoclonal antibody, MY10. Mean c-myc protein levels in CD34-positive cells, consisting of >70% blasts, are lower than a marrow fraction containing myeloid cells of intermediate maturation, but have an invariant proportional relationship, with regard to nuclear mass, over the cell cycle. The majority of these primitive cells are non-cycling, as revealed by DNA content. Under our assay conditions, nuclear c-myc protein distribution over the cell cycle did not change as these progenitors entered a proliferative phase in culture. In cultures containing factors supporting myeloid maturation, mean G0G1 p62c-myc levels initially decline, then rise above starting values as promyelocytes and myelocytes differentiate from CD34-positive cells, and as proliferation begins. With further myeloid maturation, and while cell numbers are increasing, c-myc protein continues to increase. C-myc protein kinetics differ in cultures in which macrophages, rather than myeloid cells, predominate. These data indicate that a complex relationship exists between c-myc gene expression and proliferation, maturation and lineage in haemopoietic cells, and lend support to the notion that early down regulation may be causally associated with the differentiation process.

c-myc and c-myb mRNAs have been found to be tightly regulated during hemopoietic differentiation. We have studied nuclear c-myc and c-myb oncoproteins through the cell cycle, during macrophage, granulocyte, erythroid, and megakaryocytic differentiation of KG1, HL60, and HEL cells. p62c-myc and p75c-myb content of propidium iodide-stained nuclei was quantitated by flow cytometry using fluoresceinated antibodies CT14-G4 and MB4.3, respectively. In uninduced cells p62c-myc content is highest in HL60, followed by HEL, then KG1, while p75c-myb is highest in HEL, followed by HL60 and KG1. All lines showed a less than 2-fold increment in both oncoproteins over the cell cycle. Macrophage induction of KG1 and HL60 resulted in early increase in both oncoproteins, followed by a decline to less than starting values by 48 h, concurrent with a reduction of S phase cells and the appearance of adherent alpha-naphthyl acetate esterase-positive cells. p62c-myc changes were more pronounced in HL60 and p75c-myb changes in KG1. Different patterns of oncoprotein expression were found when different inducing agents were used for granulocyte differentiation of HL60. Under all conditions, however, both oncoproteins declined to basal levels before granulocyte maturity. Hemin-induced erythroid differentiation of HEL to hemoglobin-containing cells resulted in biphasic p62c-myc and p75c-myb kinetics. In contrast, dimethyl sulfoxide-induced megakaryocytic differentiation of HEL was accompanied by an early and steady decline in both oncoproteins. Despite considerable reduction in oncoprotein levels, HEL cells were still actively cycling at 120 h. It appears that c-myc and c-myb proteins decline with differentiation, well before proliferation ceases in some lineages. The kinetics of the decline differ between the two oncogenes and vary with the lineage induced and the nature of the inducing agent used. The cell cycle distribution of the oncoproteins does not change during maturation. These data suggest disparate roles for c-myc versus c-myb during hemopoietic differentiation and the existence of multiple signal transduction pathways for down-regulation of these genes.

Stimulation of lymphocytes with phytohaemagglutinin (PHA) causes an increase in the rate of ferritin and total protein synthesis which is not enhanced by supplementing the medium with iron. There is an increase in intracellular ferritin content which is amplified by the presence of iron but this is also observed in the absence of PHA stimulation. The increased cellular ferritin is greater for heart type ferritin than for spleen type ferritin. Ferritin secreted into the medium from the cells shows a relative preponderance of spleen type ferritin compared to the intracellular protein. The data shows a dissociation between ferritin synthesis, cellular accumulation and secretion for which the mechanisms have still to be elucidated.

A method is described for the measurement of DNA index and cell cycle distribution in purified erythroid and myeloid populations from human bone marrow. Erythroid cells were prepared after complement mediated lysis of non-erythroid marrow cells. Myeloid cells were obtained by fluorescence activated cell sorting by forward and wide angle light scatter. Mononuclear marrow cells were prepared with a density gradient. Nuclei prepared from the separated populations were stained with propidium iodide. Myeloid cells had a higher DNA index than erythroid cells, and the mononuclear preparation had an intermediate value. There were more erythroid than myeloid cells in the S and G2M phases of the cell cycle. These lineage differences are particularly relevant when considering data derived from unseparated bone marrow cells, and further experiments are needed to determine the origin of these anomalies.

Flow cytometric studies of T-lymphocytes in breast cancer patients show that the number of cells bearing ferritin on their surface is significantly greater than normal. The number of ferritin-bearing T-cells does not appear to be related to the clinical stage of the disease nor to the serum ferritin concentration, though this is higher in cancer patients than in normal women. There is no difference in the number of T-cells positive for interleukin 2 or transferrin receptors nor in the absolute number of T-cells, T-helper cells and B-cells between normal women and those with breast cancer or benign breast disease. However, there is a significant increase in the level of HLA DR-positive T-cells and T-suppressor cells in breast cancer patients. While the significance of ferritin-bearing T-cells is not known an increase in their number appears to be associated with cancer.

SummaryHuman c‐myc protein, p62c‐myc, has been quantitated by flow cytometry in the nuclei of normal marrow and peripheral blood cells, and the HL60 cell line. Marrow and peripheral blood cells exhibit nuclear c‐myc protein throughout the cell‐cycle, at an average level 2–3–fold lower than HL60 cells. In no cells did p62c‐myc vary more than 2‐fold throughout the cell cycle. A small subset of marrow Go/G1 cells, enriched in early myeloid and blast cell fractions, contained p62c‐myc at levels equal to or even exceeding those of HL60.Overall c‐myc protein content was higher in myeloid, compared to erythroid and lymphoid marrow fractions. Within the myeloid lineage, the highest average p62c‐myc level was present in cells of intermediate maturation, i.e. myelocytes and metamyelocytes. In the erythroid lineage, c‐myc protein level was highest in the most immature cells and declined with maturation. Significant amounts of p62c‐myc were present in post‐mitotic, end‐stage neutrophils, but were barely detectable in cycling late erythroblasts or in quiescent lymphocytes and monocytes. HL60 cells, despite c‐myc gene amplification and increased gene expression, contain c‐myc protein at a level corresponding to promyelocytes in normal human marrow. The virtual absence of p62c‐myc in cycling late erythroblasts, and its presence in post‐mitotic end‐stage granulocytes, suggests that c‐myc protein may have functions unrelated to cell proliferation.