Multiple platelet defects identified by flow cytometry at diagnosis in acute myeloid leukaemia.

Summary Previous findings of megakaryocytic hypogranulation and dysmegakaryocytopoietic features in acute myeloid leukaemia (AML) strongly indicate defects in platelet production. The bleeding tendency of these patients may result from dysregulated platelet production, resulting in thrombocytopenia as well as qualitative platelet defects. The present study examined platelet function at diagnosis in 50 AML patients by whole blood flow cytometry. Following in vitro platelet agonist stimulation, platelet activation markers were analysed and compared with 20 healthy individuals. To detect recent in vivo platelet activation, plasma soluble P-selectin (sP-selectin) was measured. Flow cytometric analysis of platelet activation markers demonstrated reduced CD62P [35.6 vs. 118.5 x 10(3) molecules of equivalent soluble fluorochrome (MESF); P < 0.0001], CD63 (11.3 vs. 50.7 x 10(3) MESF; P < 0.0001), and PAC-1 (41.5 vs. 90.5%; P = 0.0001) while reductions in CD42b were abnormal (45.6 vs. 70%; P < 0.0001). sP-selectin levels were similar in patients and healthy controls (0.04 vs. 0.27 fg/platelet; P = 0.84). The presented data indicate that AML pathogenesis may result in multiple platelet defects, involving adhesion, aggregation, and secretion and demonstrate that flow cytometry is a feasible method for platelet function analysis in patients with thrombocytopenia.

Impaired circulating myeloid DCs from myeloma patients.

BACKGROUND: In clinical trials, cancer patients have received immunotherapy based on DCs generated from leukapheresed blood. It would therefore be an advantage to be able to measure blood levels and estimate the phenotype of DC before leukapheresis, to estimate the yield required for preparation of vaccines, or ex vivo stimulation of T cells for adoptive immunotherapy. METHODS: Recently, circulating lineage negative (Lin-) myeloid DC cells and their precursors have been identified by flow cytometry. We apply this strategy to the screening of blood samples from patients with multiple myeloma, in an attempt to characterize and quantitate the subset. By a direct flow cytometry approach, the blood levels of circulating lineage (CD3, CD19, CD14) negative, CD33++, HLA-DR+ cells were estimated before and following ex vivo cell differentiation, and phenotyped by MAbs with specificity against HLA-DR, HLA-ABC, CD1a, CD11c, CD33, CD40, CD49d, CD49e, CD54, CD80, CD83, and CD86. RESULTS: This study demonstrated that multiple myeloma patients have a 50% reduced blood level of Lin-, CD33++, HLA-DR+ myeloid DC, but a DC-precursor level within normal range. Furthermore, GM-CSF and IL-4 ex vivo stimulated DCs demonstrated an impaired up-regulation of the co-stimulatory molecule CD80 and the adhesion molecule CD54. DISCUSSION: These results may have clinical implications as a predictor for yield and functionality of the harvested DCs to be used in vaccination of myeloma patients.

Flow cytometric detection of growth factor receptors in autografts and analysis of growth factor concentrations in autologous stem cell transplantation: possible significance for platelet recovery.

In order to improve prediction of hematopoietic recovery, we conducted a pilot study, analyzing the significance of growth factor receptor expression in autografts as well as endogenous growth factor levels in blood before, during and after stem cell transplantation. Three early acting (stem cell factor (SCF), Flt3 ligand (Flt3) and fetal antigen 1 (FA1)) and three lineage-specific growth factors (EPO, G-CSF and thrombopoietin (Tpo)) were analyzed by ELISA in 16 patients with multiple myeloma (MM) and 16 patients with non-Hodgkin's lymphoma (NHL). The relative number of SCF, Flt3, Tpo and G-CSF receptor positive, CD34+ progenitor cells were measured by flow cytometry in the leukapheresis product used for transplantation in a subgroup of 15 patients (NHL, n = 8, MM, n = 7). Three factors were identified as having a significant impact on platelet recovery. First, the level of Tpo in blood at the time of the nadir (day +7). Second, the percentage of re-infused thrombopoietin receptor positive progenitors and finally, the percentage of Flt3 receptor positive progenitors. On the other hand, none of the analyzed factors significantly predicted myeloid or erythroid recovery. These findings need to be confirmed in prospectively designed studies.

Characterisation of non-P-glycoprotein multidrug-resistant Ehrlich ascites tumour cells selected for resistance to mitoxantrone.

An Ehrlich ascites tumour cell line (EHR2) was selected in vivo for resistance to mitoxantrone (MITOX). The resistant cell line (EHR2/MITOX) was 6123-, 33-, and 30-fold-resistant to mitoxantrone, daunorubicin, and etoposide, respectively, but retained sensitivity to vincristine. The resistant cells showed moderate sensitisation to mitoxantrone on treatment with verapamil or cyclosporin A. Compared with EHR2, the multidrug resistance-associated protein mRNA was increased 13-fold in EHR2/MITOX. Western blot analysis showed an unchanged, weak expression of P-glycoprotein. Topoisomerase IIalpha was reduced to one-third in EHR2/MITOX relative to EHR2 cells, whereas topoisomerase IIbeta was present in EHR2 but could not be detected in EHR2/MITOX. In the resistant subline, net accumulation of MITOX (120 min) and daunorubicin (60 min) was reduced by 43% and 27%, respectively, as compared with EHR2. The efflux of daunorubicin from preloaded EHR2/MITOX cells was significantly increased. EHR2/MITOX microsomes had a significant basal unstimulated ATPase activity. The apparent K(i) value for vanadate inhibition of the ATPase activity in EHR2/MITOX microsomes was not significantly different from the K(i) value for P-glycoprotein-positive cells. However, whereas verapamil (50 microM) inhibited the ATPase activity of EHR2/MITOX microsomes, it stimulated the ATPase activity of microsomes derived from P-glycoprotein-positive cells. In conclusion, the resistance in EHR2/MITOX was multifactorial and appeared to be associated with: 1) a quantitative reduction in topoisomerase IIalpha and beta protein; 2) reduced drug accumulation, probably as a result of increased expression of a novel transport protein with ATPase activity; and 3) increased expression of MRP mRNA.

CD34+ megakaryoblastic leukaemic cells are CD38-, but CD61+ and glycophorin A+: improved criteria for diagnosis of AML-M7?

The aim of this flow cytometry study in acute megakaryoblastic leukaemia (AML-M7) was to describe the membrane phenotype of CD34+ progenitor subsets and compare these with the phenotypes expressed by other AML FAB types. Following conventional histopathological diagnosis mononuclear cells from bone marrow and blood were examined in seven patients with AML-M7 and compared with results from 26 sequential patients with AML-M0 to AML-M6. The CD34+ subsets in AML-M7 patients differed from that of patients with AML-M0 to AML-M6 as the CD34+ CD61+ and the CD34+ Glycophorin A+ subsets were median 31% and 20%, respectively, compared to 4% and 2% in the AML-M0 to AML-M6 (P = 0.0005). Only 1% of the CD34+ progenitors were CD34+ CD38+ in AML-M7 compared to 72% in other AML subtypes (P < 0.000). These findings suggest that the CD34+ cell compartment in AML-M7 consists of early lineage-specific progenitors. In conclusion, flow cytometry analysis of CD34+ subsets may improve the diagnostic safety in AML-M7 and consequently the prognostic significance of immunophenotyping in acute leukaemia.

Lymphocyte subset composition in peripheral blood from normal subjects may be influenced by both spleen size and plasma norepinephrine.

The aim of the present study was twofold: (a) to confirm a previously observed negative relationship between plasma catecholamines and the percentage of natural killer (NK) cells in peripheral blood from resting human subjects, and (b) to examine the relationship between the size of the spleen and the composition of circulating lymphocyte subsets in resting subjects. A total of 14 young healthy male subjects were investigated in a supine resting position. Lymphocyte subset composition was determined with two-colour flow cytometry, and lymphocyte subsets were expressed as percentages of mononuclear cells. Spleen size was evaluated with ultrasonography. Plasma catecholamines were determined. Plasma norepinephrine and the percentage of NK-cells (CD3-CD56+) were negatively correlated (rs = -0.62, p = 0.019). The CD4/CD8 ratio and plasma norepinephrine were positively correlated (rs = 0.57, p = 0.037) and the major part of this correlation was due to a correlation between plasma norepinephrine and the percentage of CD4+ cells. The percentage of NK cells (CD3-CD56+) was predicted by a multiple regression model including the percentage of CD8+ cells and the spleen index, a measure of spleen size (r = 0.93, p < 0.001). The correlation in the resting state between plasma norepinephrine and the percentage of NK cells (negative correlation) on the one hand and the CD4/CD8 ratio (positive correlation) on the other contrasts with the acute mobilizing effects of epinephrine, isoproterenol and exercise on lymphocyte subsets. These relationships remain unexplained, but results accord with the hypothesis that catecholamines may have dual effects on lymphocyte subsets. The results support the view that the spleen may have a depot function for NK cells.

[Occupational accidents in the fishing industry. A survey of fishing-related accidents treated at the Bornholm Central Hospital in Rønne as well as in general practice 1987-1990]. / Arbejdsulykker i fiskeriet. Opgørelse af behandlede fiskeriskader på Bornholms Centralsygehus i Rønne og i loegepraksis 1987-1989.

Over a two year period from 1987-1989 all fishing-related injuries treated in the Emergency Room at Bornholm's central hospital in Rønne and at some general practices were registered prospectively. One hundred injuries were registered altogether. Accidents occurring in connection with putting out and recovering apparatus constituted respectively 14% and 36%. Working with the trawler scoop was responsible for 18% of the accidents and was the most common direct cause, followed by work with winches which constituted 11%. Falls made up almost a quarter of the accidents. Finger injuries constituted 33%, and hand and wrist injuries 17%. Trawler scoop injuries most commonly involved the fingers and hands and fish-cleaning injuries the hands, wrists and fingers. Prophylactic efforts concerning safety in work with trawler scoops, winches and steering systems should be made.

Interleukin-2 receptor alpha and beta chain expression by circulating alpha beta and gamma delta T cells in inflammatory bowel disease.

The pathogenetic role of activated alpha beta and gamma delta T cells in inflammatory bowel disease (IBD) is not well defined. To elucidate this, interleukin-2 receptor (IL-2R) alpha and IL-2R beta single chain expression and coexpression by peripheral blood TCR alpha beta + cells and TCR gamma delta + cells was studied in 21 patients with ulcerative colitis (UC), 25 with Crohn's disease (CD), and 15 controls. The percentages of IL-2R alpha + beta-, IL-2R alpha-beta +, and IL-2R alpha + beta + TCR alpha beta + cells were increased in IBD patients with moderate and severe disease activity, as compared to controls (P < 0.01). In contrast, the percentages of IL-2R alpha-beta + and IL-2R alpha + beta + TCR gamma delta + cells were increased in patients with inactive UC (P < 0.01), but not in CD. The results suggest that activated alpha beta T cells are involved in the development of IBD. The differences in gamma delta T cell IL-2R expression between inactive UC and CD may correspond to a yet undefined etiopathogenetic difference between these two diseases.

Individual lymphocyte subset composition determines cAMP response to isoproterenol in mononuclear cell preparations from peripheral blood.

The aim of the present study was to examine to what extent isoproterenol-stimulated cAMP production in mononuclear cells, isolated from resting human subjects, was correlated with lymphocyte subset composition. Peripheral blood was collected from 14 healthy male subjects, who rested in the sitting position. Mononuclear cells were prepared by density centrifugation and subset composition was determined by two-colour flow cytometry. Cyclic AMP production was determined by a radioimmunoassay after inhibition of phosphodiesterase and stimulation with isoproterenol. NK-cells (CD3-CD56+) were positively correlated to total cAMP concentrations (basal cAMP plus increase in cAMP). Tcytotoxic cells (CD8+) were positively correlated to the relative increase in cAMP. Thelper cells (CD4+) were negatively correlated to total cAMP concentrations and B-lymphocytes (CD(19+20)+) were negatively correlated to the relative increase in cAMP. The results support the view that NK-cells and Tcytotoxic cells have higher beta 2-adrenoceptor densities and more receptors with high affinity than Thelper and B-cells. In conclusion, our study demonstrates that cAMP levels in blood mononuclear cells obtained from normal subjects show substantial variation. This variation is at least in part due to differences in lymphocyte subset composition in the same subjects. Therefore, it is important to know the lymphocyte subset composition when evaluating cAMP accumulation in mononuclear cell preparations from peripheral blood.

[Renaissance of the reticulocyte]. / En renaessance for retikulocytten.

Flowcytometric enumeration of reticulocytes using RNA-binding fluorochromes, a method both sensitive and reproducible, is rapidly gaining ground. It performs conspicuously better than the traditional manual method of reticulocyte counting, which is unacceptably inaccurate. The reticulocyte count is a valuable indicator of erythropoietic activity, of essential importance in discerning between the aregeneratory anaemias and anaemias accompanied by an adequate, partial or complete compensatory erythropoietic response. The reticulocyte count can be applied to the monitoring of the response to treatment including both allogeneic and autologous bone marrow transplantation, as well as to the evaluation of the response to recombinant human growth factors. The mean fluorescence intensity of reticulocytes enumerated by the flowcytometric analysis, is a variable possessing an independent clinical value for calculation of a reticulocyte production index and an erythropoiesis index.

Percentage of NK-cells in peripheral blood in resting normal subjects is negatively correlated to plasma adrenaline.

The purpose of the present study was to examine, if there was a correlation between natural killer cells in blood and plasma catecholamines in a resting situation. Lymphocyte subsets, especially, the NK-cells (CD3-CD56+), plasma adrenaline and noradrenaline were determined in peripheral blood from healthy male subjects resting in the supine position. Median age was 32 years. A negative correlation was observed between resting plasma adrenaline and the percentage of (CD3-CD56+) mononuclear cells (p = 0.048, r = -0.61). Previous studies have shown however, that adrenaline may increase the number of natural killer cells in blood within minutes. We suggest that adrenaline may have a dual effect on NK-cells: an acute effect by which NK-cells are mobilized from depots and a chronic effect, which decreases the number of lymphocytes and especially NK-cells in peripheral blood.

Different membrane expression of CD11b and CD14 on blood neutrophils following in vivo administration of myeloid growth factors.

During the administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) or granulocyte-macrophage CSF (rhGM-CSF) we studied the early and late changes of membrane antigen density on neutrophils. RhG-CSF and rhGM-CSF both caused an early transient reduction in blood neutrophilic granulocyte-concentration within the first 30 min after treatment followed by a marked later increase during the subsequent 24 h. During the early neutropenia quantitative flow cytometry showed an associated marked increase in the density of membrane CD11b from 169 x 10(3) before to 568 x 10(3) A.U. per cell induced by rhGM-CSF but a non-significant change by rhG-CSF, suggesting that different mechanisms may be responsible for the transient neutropenia. The subsequent neutrophil granulocytosis was followed by a significantly (P < 0.05) increased density of the CD14 antigen from 6.1 x 10(3) before to 15.9 x 10(3) A.U. per cell during treatment with rhG-CSF, but not by rhGM-CSF administration. These results demonstrate that the two cytokines may affect the function of neutrophilic granulocytes in different ways. The increased expression of CD11b could explain some of the side-effects during treatment with rhGM-CSF. The upregulation of CD14 induced by rhG-CSF may be clinically relevant, as CD14 is an opsonic receptor for lipopolysaccharide binding proteins, acting in the defence against Gram-negative bacterial infections.

Increased yield of myeloid progenitor cells in bone marrow harvested for autologous transplantation by pretreatment with recombinant human granulocyte-colony stimulating factor.

Pretreatment with haemopoietic cytokines prior to marrow harvest may result in improved quality of bone marrow harvested for autologous bone marrow transplantation (BMT). Such improvements may reduce the risk for graft failure and decrease time to engraftment. Patients undergoing autologous BMT received recombinant human G-CSF (rhG-CSF) immediately prior to marrow harvest. rhG-CSF was administered as daily subcutaneous injections for 5 days at 5 micrograms/kg body weight. Comparison of bone marrow samples before and after rhG-CSF treatment showed an increased bone marrow cellularity and a ninefold increase in the number of marrow leucocytes per volume aspirated. The mean marrow myeloid:erythroid ratio increased from 2.6 to 4.0. The mean numbers of immature (CD38 positive) and proliferating (CD71 positive) myeloid cells increased significantly from 41.6 to 50.8% and from 17.0 to 34.8%, respectively. Other subsets studied, including CD34 positive stem cells, were unchanged. The relative numbers of day 7 and 14 granulocyte-macrophage colony-forming units (day 7/14 GM-CFU) were unchanged. Long-term marrow cultures revealed that the numbers of 'long-term culture initiating cells' were unchanged after rhG-CSF treatment in spite of the ninefold increase in cellularity. To date, five of the patients have been transplanted with autologous marrow harvested after rhG-CSF treatment. Time to trilineage engraftment was unchanged compared with historical controls. We conclude that pretreatment with rhG-CSF prior to marrow harvest may improve the graft by increasing the total number of myeloid lineage restricted progenitor cells, resulting in stable but not accelerated myeloid engraftment of autologous marrow.

Quantitative buffy coat analysis in haematological patients compared to standard laboratory methods.

We have tested a Quantitative Buffy Coat (QBC) instrument (Becton Dickinson, USA), and compared results obtained with this instrument to results obtained with standard methods in a haematology clinic. The basic principle of the method is a classical haematocrit centrifuge. The analysis provides a haematocrit value, platelet count, a total white blood count, and separates the white blood cells in granulocytes and mononuclear cells (lymphocytes plus monocytes). The instrument is easy to use but requires a trained observer. All results are available in 15 min. We have found the accuracy of the method good for all parameters. The precision of the instrument is good but for estimation of granulocytes and lymphocytes plus monocytes we did not find the high sensitivity claimed by the manufacturer (manufacturer's lower limit 0.02 x 10(9)/l; standard deviation for low levels of granulocytes: 0.300 x 10(9)/l and lymphocytes plus monocytes: 0.235 x 10(9)/l). A large fraction of samples (leukocytes 27%, platelets 40%) from a haematological clinic falls beyond the limits for reliable results set by the manufacturer, which reduces the utility of the instrument in such patients. Furthermore, in 21% and 10% of samples within the recommended range for leukocytes and platelets, respectively, QBC results could not be read owing to ill-defined boundaries. For granulocytes and lymphocytes plus monocytes 25% and 34% of the samples, respectively, could not be read owing to ill-defined boundaries. The instrument is not constructed to protect against blood contamination of the centrifuge and, therefore, the safety of the instrument is not satisfactory.

The DANOP-DATA system: a low-cost personal computer based program for monitoring of wound infections in surgical ward.

A low-cost personal computer program to monitor surgical wound infections was developed in parallel to the Danish national guidelines for recording postoperative wound infections. Internationally accepted definitions were used. The program offers three fixed-data entry screens and produces user-specified variations of four standard tables, comprising most of the epidemiological data needed for surveillance and infection control. The program was tested in Danish hospitals and was found to serve well as a simple local tool for the operating staff, offering fast information on infection rates. Results from two hospitals consisting of 3904 operations are presented. Infections occurring after discharge were included. Overall infection rates for clean wounds were 2.3%, clean-contaminated wounds 4.7%, contaminated wounds 4.3% and dirty operations 8.3%. None of the hospitals had used infection surveillance systems before.

[Registration of postoperative wound infections by ADB. A trial of the Danop program]. / Registrering af postoperative sårinfektioner på edb. En afprøvning af programmet Danop.

The EDB programme, Danop, which was developed in cooperation with Statens Seruminstitut which is the central department for hospital hygeine in Denmark, was employed during the period 1.7.1987-30.6.1988 for consecutive registration of operative interventions and postoperative wound infection. During the period, 1,660 interventions were registered with a total frequency of infection of 3.5%; 2.6% of these in clean cases and 13.8% in infected cases. About one third of the infections were demonstrated by the general practitioners after discharge. In connection with introduction of registration, a considerable decrease in the number of postoperative wound infections occurred. It is decisive for the employability of the system that a reliable procedure for registration and return information is organized. The system is easy to use and functions well and does not require prior knowledge of edb and it provides excellent current control of and review of postoperative wound infections in one or more departments.

Blood monocyte elastolytic activity enhanced by immune complex stimulation in patients with rheumatoid arthritis and normal controls. Effect of aurothiomalate or D-penicillamine.

Blood monocyte elastolytic activity in patients with rheumatoid arthritis and in normal controls was studied by a new in vitro technique. The enzyme activity of live cells was measured by soluble [3H]elastin hydrolysis first under basic conditions and then after immune complex stimulation. The cells from patients had a higher elastolytic potential than cells from controls (p less than 0.02) and responded to smaller amounts of immune complex (p less than 0.01), even in patients treated with D-penicillamine or aurothiomalate. Treatment of normal monocytes in vitro with aurothiomalate did not influence the elastolytic response. These findings indicate that monocytes from patients with rheumatoid arthritis have an enhanced elastolytic activity compared with cells from normal controls and may invoke greater tissue damage on immune complex stimulation.

Degradation of solubilized [3H]elastin by intact human monocytes stimulated by immune complexes in vitro.

A simple assay is described that permits determination of elastolytic activity released from human monocytes. The substrate, solubilized [3H]elastin, is coated onto cell culture dishes. Blood monocytes are allowed to adhere, and during subsequent serum-free culture, radioactive degradation products of elastolysis are released into the culture medium. Using this assay we have demonstrated an elastolytic activity by normal human monocytes of 20 +/- 2 ng elastin/hour/3 X 10(6) mononuclear cells. Contaminating granulocytes and lymphocytes do not cause any significant elastolysis. On stimulation with immune complexes in vitro, the monocytes respond with an enzymatic burst that is 3.6 times the basic enzyme activity. This assay could be a useful tool in the evaluation of elastolytic activity of cells from other sources as well as from human monocytes.

Maternal-serum-alphafetoprotein screening for fetal malformations in 28 062 pregnancies. A four-year experience from a low-risk area.

From 1st March, 1980 and up to 29th February 1984 a multicenter serum alpha-fetoprotein (S-AFP) screening project was carried out for the detection of severe fetal malformations. S-AFP was determined by a radio-immunoassay in 28 062 pregnant women between the 16th and 20th week of gestation. Patients with elevated S-AFP values, e.g. above 95 percentile, were examined further with a second S-AFP and by ultrasound scan. 244 amniocenteses (0.9%) were carried out to detect 62 malformations (21 anencephalies, 14 spina bifidas, 2 encephaloceles, 7 omphaloceles, 5 gastroschises, 4 chromosome abnormalities and 9 other malformations). Fifteen of the 16 cases of spina bifida could not be verified by ultrasound scan, whereas all other malformations except chromosome abnormalities were confirmed by ultrasonography. Two cases of spina bifida and one case of skin-covered encephalocele had normal S-AFP concentrations and were therefore not detected. There were no definitive false-positives, e.g. therapeutic abortion of a normal fetus. Our conclusion is that a nationwide S-AFP screening should be recommended.