A multicentre study of reference intervals for haemoglobin, basic blood cell counts and erythrocyte indices in the adult population of the Nordic countries.

Eight haematological quantities were measured in EDTA anticoagulated venous blood specimens collected from 1826 healthy male and female individuals between 18 and 90 years of age in the Nordic countries (Denmark, Finland, Iceland, Norway and Sweden). The samples, collected between November 1999 and November 2001 as part of the Nordic Reference Interval Project (NORIP), were analysed on 12 different types of modern automated haematology instruments currently in use among the 60 laboratories participating in the study. Non-parametric reference intervals (between 2.5 and 97.5 percentiles) have been calculated for B-Haemoglobin (females 117-153 g/L, males 134-170 g/L), B-Erythrocytes (females 3.94-5.16 x 10(12)/L, males 4.25-5.71 x 10(12)/L), B-EVF (females 0.348-0.459, males 0.395-0.500), B-MCV (82-98 fL), Erc-MCH (27.1-33.3 pg), Erc-MCHC (317-357 g/L), B-Trc (females 165-387 x 10(9)/L, males 145 x 348 x 10(9)/L) and B-Lkc (3.5-8.8 x 10(9)/L). Partitioning of data according to age and gender was done according to a standardized procedure. For most variables the calculated reference intervals corresponded well with older and less well-defined reference intervals. The mean concentration of B-Haemoglobin increased by 0.08 g/L per year of age in women, and decreased by 0.1 g/L per year of age in men. B-Haemoglobin increased with body mass index in both men and women. Smoking increased the mean of B-Lkc by 1.1 x 10(9)/L and regular use of alcohol increased the mean of B-MCV by 0.8 fL. The influence of these factors was small overall and did not promote specific reference intervals.

Differential counting of blood leukocytes using automated microscopy and a decision support system based on artificial neural networks--evaluation of DiffMaster Octavia.

The morphological appearance of blood cells has an established association to clinical conditions. A novel system, DiffMaster Octavia for differential counting of blood leukocytes, has been evaluated. The system consisted of a microscope, 3-chip color charge coupled device (CCD) camera, automated motorized stage holder, electronic hardware for motor and light control and software for automatic cell location and image processing for preclassification of blood cells using artificial neural networks. The DiffMaster test method, was evaluated on 322 routine blood samples (400 cells per sample) using manual microscopy as reference method. The results showed a correlation of determination (r(2)) of 0.8-0.98 for the normal cell classes and blast cells. The DiffMaster correctly preclassified 89% of all leukocytes with a good reproducibility. After verification of the cell classes, the agreement between the test and reference method was 91% whether the sample was abnormal or normal. The clinical sensitivity was 98% and specificity 82%. The sensitivity to identify blast cells was slightly higher with the DiffMaster than manual microscopy. Similar levels of short-term imprecision for the two methods were found for all cell classes. In conclusion this study shows that the DiffMaster can provide a decision support system which, together with a qualified morphologist, can generate leukocyte differential count reports of high quality.

Increased serum erythropoietin concentration after allogeneic compared with autologous blood transfusion.

Serum erythropoietin (sEPO) level is known to increase as hemoglobin (Hb) concentration decreases during and after preoperative autologous blood donation (PAD). The endogenous erythropoietin (EPO) production after allogeneic blood transfusion has not to our knowledge, been studied. The aim of the present study was to determine whether there is, after surgery, any change in sEPO concentration after allogeneic blood transfusion, and whether there is any difference in EPO response after autologous or allogeneic blood transfusion. Thirty-one patients approaching total hip-joint replacement surgery, were randomized to receive either allogeneic red blood cells (n = 15) or predeposited autologous whole blood transfusion (n = 16). The relationship between Hb, sEPO, and reticulocytes in the recipients were repeatedly analyzed before, during and after surgery. The Hb followed an expected pattern, with a decreased concentration after PAD in the autologous group, then in both groups after surgery. The sEPO concentration was significantly higher in the allogeneic than in the autologous group on day one and day 4-5 postoperatively. The reticulocyte level, on the contrary, was higher in the autologous patients before, one hour after, and one day after surgery. The study showed a greater increase in sEPO concentration after allogeneic blood transfusion than after autologous blood transfusion. There may be an inverse relationship between sEPO and the reticulocyte level.

No increased frequency of trisomies 8 and 9 by fluorescence in situ hybridization in untreated patients with essential thrombocythemia.

Essential thrombocythemia (ET) is one of the diseases included among the myeloproliferative disorders in which trisomies for chromosomes 8 and 9 commonly occur. In ET, only a few patients are known to show clonal abnormalities. With fluorescence in situ hybridization (FISH), interphase cells can be evaluated and clones can be detected even though not revealed by conventional cytogenetic methods. By using FISH for enumeration of chromosomes 8 and 9 in bone marrow cells, we studied 22 patients with ET; 20 of them were investigated at the time of diagnosis when they were still untreated with myelosuppressive agents. Only two patients had trisomy 8; one of them was also found to have +8 with conventional cytogenetics. None of the patients had trisomy 9; two patients had borderline values in comparison to a control group. Thus, in ET, no increased frequency of patients with trisomy for 8 or 9 at the time of diagnosis could be detected with FISH.

Basis of hematopoietic defects in platelet-derived growth factor (PDGF)-B and PDGF beta-receptor null mice.

Platelet-derived growth factor (PDGF)-B and PDGF beta-receptor (PDGFR beta) deficiency in mice is embryonic lethal and results in cardiovascular, renal, placental, and hematologic disorders. The hematologic disorders are described, and a correlation with hepatic hypocellularity is demonstrated. To explore possible causes, the colony-forming activity of fetal liver cells in vitro was assessed, and hematopoietic chimeras were demonstrated by the transplantation of mutant fetal liver cells into lethally irradiated recipients. It was found that mutant colony formation is equivalent to that of wild-type controls. Hematopoietic chimeras reconstituted with PDGF-B(-/-), PDGFR beta(-/-), or wild-type fetal liver cells show complete engraftment (greater than 98%) with donor granulocytes, monocytes, B cells, and T cells and display none of the cardiovascular or hematologic abnormalities seen in mutants. In mouse embryos, PDGF-B is expressed by vascular endothelial cells and megakaryocytes. After birth, expression is seen in macrophages and neurons. This study demonstrates that hematopoietic PDGF-B or PDGFR beta expression is not required for hematopoiesis or integrity of the cardiovascular system. It is argued that metabolic stress arising from mutant defects in the placenta, heart, or blood vessels may lead to impaired liver growth and decreased production of blood cells. The chimera models in this study will serve as valuable tools to test the role of PDGF in inflammatory and immune responses. (Blood. 2001;97:1990-1998)

Masked monosomy 7 in myelodysplastic syndromes is uncommon and of undetermined clinical significance.

Masked monosomy 7, i.e. detected by FISH but not by conventional cytogenetics, has been reported in varying frequency in MDS. To establish the prevalence and possible clinical significance of the aberration, we studied the 123 previously karyotyped MDS patients using FISH and a DNA probe specific for chromosome 7. Metaphase cytogenetics revealed ten patients (8%) with monosomy 7 (6 RAEB and 4 RAEB-t). FISH confirmed this result and detected four more cases (4%) with masked monosomy 7 (3 RA and 1 RARS). Thus, masked monosomy 7 is less common than has been suggested, and does not seem to carry the same prognostic weight as monosomy 7 diagnosed by metaphase cytogenetics.

Hydroxyurea treatment reduces haematopoietic progenitor growth and CD34 positive cells in polycythaemia vera and essential thrombocythaemia.

The aim of the present work was to investigate the effect of hydroxyurea (HU) treatment on haematopoietic progenitors and CD34 positive (CD34+) cells in patients with polycythaemia vera (PV) and essential thrombocythaemia (ET). Of the PV patients were 10 treated with phlebotomy only and 10 were on HU therapy. Seven ET patients were untreated and 10 received HU. In each subject peripheral blood was obtained for in vitro colony growth, determination of CD34+ cells and plasma erythropoietin (EPO) concentration. The mean number of EPO independent erythroid colonies (EEC) was higher in the group of PV patients on phlebotomy therapy compared to the PV patients treated with HU (74.4 and 8.0 colonies/10(5) cells, respectively) but the difference did not reach statistical significance. The corresponding means for the untreated ET patients and ET patients treated with HU were 13.0 and 1.3 colonies/10(5) cells, respectively, this difference being statistically significant (p = 0.012). The mean EEC for combined groups of PV and ET without myelosuppressive treatment were compared with the results for PV and ET patients on HU therapy; this difference was statistically significant (p = 0.014). The same pattern was observed for total erythroid growth with EPO. The relationship between the concentration of CD34+ cells and total EEC in peripheral blood was statistically significant for both PV (p<0.005) and ET (p<0.01). This finding supports the hypothesis that the level of CD34+ cells in peripheral blood could be used as a proliferation marker in these two myeloproliferative entities. No relationship between plasma EPO and EEC was present. It therefore appears that the reported differences in plasma/serum EPO concentrations between PV patients on phlebotomy treatment compared to patients on myelosuppressive treatment are not likely to be found at the production site for erythrocytes.

Cytogenetic studies in patients with mastocytosis.

Chromosomal aberrations in hematopoietic cells are common in malignant hematological disorders and have also been reported in some patients with mastocytosis. In this study, 34 patients with either urticaria pigmentosa or systemic mastocytosis were investigated by cytogenetic analysis of bone marrow cells. A follow-up investigation was performed in 22 patients. Clones with chromosome abnormalities were found in 32% of the patients at the first examination and in 27% at the second examination; in total, 41% of the patients had an abnormal clone in at least one examination. No clinical correlation was found with regard to cytogenetic results, with the exception of four patients who had an associated hematological disease and poor prognosis. In the second examination, only 6 patients had an unchanged chromosome pattern, and 4 of the patients with an initial normal pattern had appearance of abnormal clones; however, in 7 patients, the initial abnormal cells disappeared. The abnormalities were, among others, deletions of chromosomes 5, 7, 11, and 20. The proportion of cells with structural or numerical chromosome changes was higher in comparison with reported control groups. The frequency and type of chromosome abnormalities in bone marrow cells from patients with mastocytosis was about the same as observed in other chronic myeloproliferative disorders and myelodysplastic syndromes, diseases which also developed in 4 of our patients. An association between malignant hematological disorders and mastocytosis have been suggested by us and others. The chromosome abnormalities maybe reflect a genetic instability of the hematopoietic cells in mastocytosis.

Plasma erythropoietin concentrations in polycythaemia vera with special reference to myelosuppressive therapy.

In 80 patients with polycythaemia vera (PV) a total of 108 venous blood samples were obtained and analysed for EDTA-plasma erythropoietin (EPO) concentration. At the time of study 21 of the PV patients were newly diagnosed and had prior to blood sampling neither received phlebotomy treatment nor therapy with myelosuppressive agents; these subjects had a mean plasma EPO concentration of 0.5+/-0.9 IU/L. Thirty-seven patients treated with phlebotomy only had a mean plasma EPO concentration of 2.5+/-2.9 IU/L. The mean plasma EPO concentrations for 26 patients treated with hydroxyurea, 13 patients treated with radiophosphorous and 11 patients given a combination of myelosuppressive agents were 8.9+/-8.0, 10.9+/-12.6 and 7.2+/-7.4 IU/L, respectively. Untreated patients and patients on phlebotomy only had significantly lower values for plasma EPO than patients on therapy with myelosuppressive drugs. This finding persisted also after a correction for differences in haemoglobin levels had been introduced. Thereby, the present results would suggest a difference in the EPO feedback system in untreated and phlebotomised PV patients compared to PV patients treated with myelosuppressive agents.

Low megakaryocyte ploidy in Ph-positive chronic myelogenous leukemia measured by flow cytometry.

In chronic myeloproliferative disorders, the megakaryocytes differ in size and maturation compared with those of healthy individuals. In the present study, by using a 2-color flow cytometry technique, we determined the frequency of bone marrow megakaryocytes in different ploidy classes in 13 newly diagnosed and untreated patients with Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML) and in 12 healthy volunteers. The results showed a significant difference in megakaryocyte ploidy distributions between these 2 study groups. On the average, patients with CML had 59% of their megakaryocytes in ploidy classes 2N to 8N; in contrast, the healthy volunteers had only 22% of their megakaryocytes in classes 2N to 8N. Two patients with complex Ph translocation and 2 patients with a small clone with a chromosome abnormality in addition to Ph had the same ploidy distribution as those with only Ph translocation. The platelet count did not correlate with the megakaryocyte mean ploidy.

Bone marrow fibrosis and radiological changes of the long bones in children with acute megakaryocytic leukaemia.

The diagnosis of acute megakaryocytic leukaemia (AMkL) may be difficult to establish owing to difficulties in obtaining adequate bone marrow aspirates secondary to bone marrow fibrosis. We describe three children without Down's syndrome under 2 y of age with AMkL. Although none of the patients had the non-random t(1;22) (p13;q13) translocation, bone marrow cells from all patients exhibited chromosome abnormalities with complex karyotypes, including trisomy 21 in two cases. All patients had profound bone marrow fibrosis and characteristic lamellar diaphyseal radiological changes of the long bones.

Serum methylmalonic acid and total homocysteine in patients with suspected cobalamin deficiency: a clinical study based on gastrointestinal histopathological findings.

We compared the sensitivity and specificity of the two metabolite tests, methylmalonic acid (MMA) and total homocysteine (Hcy) in serum, and serum cobalamin (Cbl) in patients referred to our hospital because of suspected cobalamin deficiency and a serum cobalamin value at the referring unit <200 pmol/L. All 111 patients included were investigated using upper gastrointestinal endoscopy with biopsy specimens taken from the gastric and duodenal mucosa to find a morphological basis for cobalamin malabsorption as well as the Schilling test for the validation of the serum tests. All patients were treated with cobalamin and new blood samples were taken after 4 weeks. We found no difference in sensitivity and specificity between serum MMA, Hcy, and Cbl in identifying patients with and without conditions compatible with cobalamin malabsorption. Elevated serum MMA and Hcy were also found in about 15% of the group of patients with normal Schilling tests and without a morphological basis for cobalamin malabsorption. Moreover, most patients in this group responded with decreased values of the metabolite tests following cobalamin treatment, suggesting that neither elevated metabolites nor a decrease in these values following cobalamin treatment are specific for cobalamin deficiency.

Increase of CD34 positive cells in polycythaemia vera.

The purpose of the present work was to evaluate the proliferative character of polycythaemia vera (PV). Therefore, in 15 patients with different stages of PV we assessed the level of CD34 positive (CD34+) cells in peripheral blood and bone marrow, erythroid colony growth of bone marrow cells and plasma erythropoietin (EPO). The mean concentration of CD34+ cells in blood was significantly increased in PV patients (9.0 +/- 11.2 x 10(3)/mL) compared to healthy controls (2.0 +/- 1.7 x 10(3)/mL). In aspirated bone marrow no such difference between PV and control subjects was present. Six patients with splenomegaly and/or requirement for chemotherapy had significantly higher mean blood levels of CD34+ cells compared to the remaining PV patients. All PV patients presented EPO independent erythroid colonies. Three PV patients with anaemia and long disease duration had high EPO levels.

Bullous pyoderma gangrenosum in a patient with myelodysplastic syndrome during granulocyte colony-stimulating factor therapy.

An unusual case of bullous pyoderma gangrenosum in a patient with myelodysplastic syndrome during treatment with granulocyte colony-stimulating factor (G-CSF) is reported. The possible relationship between G-CSF therapy and pyoderma gangrenosum, as well as the beneficial effect of cyclosporin A therapy, is discussed.

Prognostic impact of bone marrow karyotype in childhood acute lymphoblastic leukaemia: Swedish experiences 1986-91.

The prognostic value of cytogenetic classification in acute lymphoblastic leukaemia (ALL) was evaluated in Swedish children below 16 years of age (n = 372) diagnosed between 1986 and 1991. A bone marrow karyotype was obtained in 281 cases, of which 149 (53%) showed clonal abnormalities. Event-free survival (p-EFS) was 0.64-0.69 in patients with diploid and pseudodiploid karyotype. Patients with massive hyperdiploidy (> 50 chromosomes) had the best outcome (p-EFS = 0.76) and those with hypodiploidy (< 46 chromosomes) had the worst (p-EFS = 0.33). White blood cell count and age were the strongest predictors of outcome. The karyotype reached borderline significance. The diagnostic karyotype was also a predictor of outcome after relapse, with hyperdiploid patients doing better than the others. The presence of a structural chromosomal abnormality did not constitute a negative prognostic factor when intensive chemotherapy was given.

Spontaneous remission during two pregnancies in a patient with essential thrombocythaemia.

A woman with untreated essential thrombocythaemia (ET) exhibited spontaneous normalization of her elevated platelet count during two pregnancies. After delivery the platelet count increased rapidly to the same high level as before. Low platelet adhesion as well as adrenalin- and collagen-induced platelet aggregation, present before pregnancy, increased to normal values during pregnancy. Furthermore, no erythropoietin-independent colony growth was observed during pregnancy, while such colonies were clearly demonstrated when the patient was assayed after pregnancy. This is the second case in the literature demonstrating that remission of ET can occur without treatment during pregnancy.

Flow cytometric analysis of megakaryocyte ploidy in chronic myeloproliferative disorders and reactive thrombocytosis.

Megakaryocyte (MK) ploidy patterns were analysed by flow cytometry in 29 newly diagnosed and previously untreated patients with chronic myeloproliferative disorders (MPD) and concomitant thrombocytosis, in 9 patients with reactive thrombocytosis (RT) and in 12 healthy individuals. Unfractionated bone marrow from routine aspirates was used. MKs were identified with a fluorescein labelled monoclonal antibody specific for glycoprotein IIIa (GPIIIa) and DNA was stained with propidium iodide. For the 12 healthy volunteers the mean modal ploidy number was 16 N; the 9 patients with RT displayed an identical MK ploidy pattern. The frequency of MKs with a ploidy > or = 32 N was 45% among the patients with essential thrombocythaemia (ET) compared to 32% among the healthy volunteers (p < 0.001). MKs with ploidy number > or = 64 N, comprising approximately 13% of the total number of MKs, was a characteristic finding in the patients with ET. Similar findings were present in 8 patients with polycythaemia vera (PV). In patients with PV 34% and 6% of the MKs displayed ploidies > or = 32 N and > or = 64 N, respectively. In contrast, a distinct shift towards lower ploidy number, with 63% of MKs < or = 8 N, was found among the 4 patients with chronic myeloid leukaemia (CML). The present results indicate that by using flow cytometric analysis of MK ploidy distribution in patients with thrombocytosis, those with a reactive cause are likely to be discriminated from patients with myeloproliferative thrombocytosis, i.e. PV and ET on one hand and CML on the other hand. The distinction between ET and PV, however, has to be made on other grounds.

Bone marrow progenitor cell growth and karyotype changes in healthy 88-year-old subjects.

Previous studies have indicated a decline in bone marrow progenitor cell function in subjects aged 75-82 years, possibly causing lower Hb concentrations. We studied the bone marrow with in vitro colony assays and cytogenetic analysis in 24 apparently healthy 88-year-olds with Hb concentrations ranging from moderate anaemia to normal levels. Twenty-two healthy younger subjects, aged 21-57 years, were used as a control group. The 88-year-olds showed significantly lower numbers of myeloid bone marrow progenitors than the controls, and the elderly men had lower numbers of both erythroid and myeloid progenitors than the elderly women. There were no in vitro growth differences between elderly subjects with "low" or "normal" Hb concentrations. Ten out of 14 men had bone marrow cells with a missing Y-chromosome, which did not seem to have any relationship to the erythroid function. No morphological or other cytogenetic indications of a clonal progenitor cell disorder were found. A more rapid decline in Hb concentrations in healthy elderly men as compared to elderly women might be explained by differences in bone marrow progenitor cell function. However, progenitor cell abnormalities do not seem to explain differences in Hb concentrations within groups of apparently healthy men and women of advanced age.

Mice deficient for PDGF B show renal, cardiovascular, and hematological abnormalities.

Platelet-derived growth factor (PDGF) affects the growth, migration, and function in vitro of mesenchymal cells, but little is known about its normal physiological functions in vivo. We show here that mice deficient for PDGF B die perinatally and display several anatomical and histological abnormalities. Kidney glomerular tufts do not form, apparently because of absence of mesangial cells. Instead, a single or a few distended capillary loops fill the glomerular space. The heart and some large arteries dilate in late-stage embryos. Most PDGF B mutant embryos develop fatal hemorrhages just prior to birth. Their hematological status includes erythroblastosis, macrocytic anemia, and thrombocytopenia. On the basis of these findings, we conclude that PDGF B has crucial roles in vivo in establishing certain renal and circulatory functions.