Reticulocyte population data in different erythropoietic states.

AIMS: To assess changes in reticulocyte impedance volume, conductivity and light scatter (reticulocyte population data or RPD) obtained with the volume, conductivity and laser light scatter (VCS) technology in healthy subjects and in different erythropoietic states. METHODS: Blood samples were analysed with the Beckman-Coulter LH750 system, using the VCS method, from a group of 40 healthy subjects and three groups of patients with different types of untreated or treated anaemia: 24 cases of iron deficiency at the time of diagnosis, 16 patients with iron deficiency anaemia during intravenous iron administration, and 57 patients with chronic kidney disease undergoing dialysis and administration of rHu-erythropoietin and intravenous iron. RESULTS: RPD data were reproducible. Average mean channels for volume were 50.9 for controls and 49.2, 55.7 and 64.0 for the three patient groups, respectively. Average mean channels for conductivity were 52.0 for controls and 59.8, 55.7 and 56.1 for the three patient groups. Average mean channels for light scatter were 108.4 for controls and 113.3, 117.3 and 128.2 for the three patient groups. SD data indicated increased dispersion in the patient groups. CONCLUSION: When values in the patient groups are compared with reference values obtained in healthy controls, the main differences are found in impedance volume and light scatter, which were both increased in patients with stimulated erythropoiesis. These data indicate the opportunity to further evaluate the clinical usefulness of RPD in haematological diseases.

Immature platelet fraction (IPF) in hospitalized patients with neutrophilia and suspected bacterial infection.

OBJECTIVES: Neutrophilia is frequently in hospitalized patients, so a screening test for infections would be very useful. The immature platelet fraction (IPF) is provided by the automated blood analyzer XE 2100 (Sysmex, Kobe, Japan) as a proportional value of the total optical platelet count [IPF%; an absolute count of immature platelets can also be obtained (AIPC)] to indicate the rate of platelet production. IPF could help to identify the aetiology of thrombocytopenia and to recognize the increased bone marrow activity as occurred during the course of infectious disease. MATERIALS AND METHODS: We selected from daily routine cell blood counts 535 samples from adult patients (age >14 years) with neutrophilia (> 8 x 10(9)/L) and platelets > or =150 x 10(9)/L, to avoid bias due to physiological increase of IPF. In 153 patients blood cultures (BC) were done the same day of IPF determination because of clinically suspected infection (fever > or =38 degrees C). RESULTS: Eighty-nine samples (58.2%) had positive BC (75 Gram-positive agents, 14 Gram-negative). Using our IPF reference normal range [IPF% 2.39% (0.8-5.1); AIPC 5.15 x 10(9)/L (2-12.6)], we found a significantly (p<0.0001) higher level of IPF in samples with positive BC [mean IPF% 4.86+/-2.67%, median 3.8 (range 2.4-15.8); mean AIPC 14.74+/-9.26 x 10(9)/L, median 11.7, (range 4.7-44.9)] than in BC negative samples [mean IPF% 1.79+/-0.63%, median 1.7 (range 0.5-4.7); mean AIPC 6.55+/-3.58 x 10(9)/L, median 5.45 (range 1.6-20.4)]. CONCLUSIONS: The increased IPF shows a statistically significant correlation with BC positivity. This parameter could therefore be used in the daily laboratory routine in patients with neutrophilia as a low cost screening test for bacterial infection.

Reticulocyte hemoglobin equivalent: an indicator of reduced iron availability in chronic kidney diseases during erythropoietin therapy.

Anemia is a common complication of chronic kidney disease (CKD), particularly in dialysis patients. Correction of anemia in CKD patients includes the administration of both recombinant human erythropoietin and intravenous iron. An optimization of iron treatment requires obtaining a target hemoglobin level and avoiding an excessive body-iron overload. The reticulocyte hemoglobin content (CHr) has been shown to be an early indicator of iron-restricted erythropoiesis. The recent European guidelines for anemia treatment in CKD assessed the value for CHr >29 pg/cell as the reticulocyte parameter to evaluate a patient's iron needs. The reticulocyte hemoglobin equivalent (RET-He), recently introduced to determine the forward scatter of fluorescence-labeled reticulocytes, seems to be a sensitive indicator of iron-deficiency anemia. This study evaluates the concordance between the CHr parameter, used as a reference, and the new RET-He in a cohort of 57 dialysis patients referred to the Nephrology Unit of our hospital. All patients received erythropoietin, and iron was administered intravenously to maintain the hemoglobin level between 10 and 12 mg/dL. A total of 285 determinations were performed with both instruments. In the dialysis population, the 95% central range for CHr of 24.8 to 36.3 pg corresponds to a range for RET-He of 23.3 to 40.1 pg, with a mean bias of 1.12 pg between the 2 parameters. In comparison with CHr, the value of 30.5 pg for RET-He appeared to be the best cut-off point with a very good sensitivity and specificity to determine patients needing iron supplementation. Our study showed an excellent diagnostic efficiency of RET-He to evaluate patients needing iron support and demonstrated a strict correspondence between the classic CHr and the new Ret-He. This correspondence was independent of clinical changes, frequently occurring in dialysis patients. Both parameters could be used soon to guide and monitor iron treatment in dialysis patients.

Aberrant methylation of DAP-kinase in therapy-related acute myeloid leukemia and myelodysplastic syndromes.

Death-associated protein kinase (DAP-kinase), a proapoptotic serine/threonine kinase, is a candidate tumor suppressor gene. We studied the methylation status of DAP-kinase of 194 bone marrow samples from 160 patients with acute myeloid leukemia (AML) and 34 with a myelodysplastic syndrome (MDS) at the time of initial diagnosis by polymerase chain reaction (PCR). Hypermethylation of DAP-kinase was present in 27.5% (44 of 160) of AML and in 47% (16 of 34) of MDS specimens and significantly correlated to loss of DAP-kinase expression (P =.008). It was significantly more frequent in AML secondary to therapy for other malignancies (s-AML; 14 of 29, 48.3%), as compared to de novo AML (30 of 131, 22.9%, P =.01). DAP-kinase hypermethylation in AML was associated with myelodysplastic changes in the bone marrow at the time of the initial diagnosis (P =.002) and with the presence of cytogenetic abnormalities (P =.02). Alteration in the apoptotic response due to the loss of DAP-kinase function may be an early event in the transformation pathway to secondary leukemia via myelodysplasia.

Overexpression of the polycythemia rubra vera-1 gene in essential thrombocythemia.

PURPOSE: To examine the utility of polycythemia rubra vera-1 (PRV-1)-specific reverse transcriptase polymerase chain reaction (RT-PCR) to discriminate essential thrombocythemia (ET) and polycythemia vera (PV) from secondary thrombocytosis (ST) or secondary erythrocytosis (SE). PATIENTS AND METHODS: We analyzed the expression of PRV-1 in granulocytes isolated from 37 patients with ET, 37 patients with PV, 25 patients with ST, 10 patients with SE, 25 patients with secondary leukocytosis (SL), five patients with chronic myelogenous leukemia (CML), five patients with chronic idiopathic myelofibrosis (IM), five patients with myelodysplastic syndrome (MDS), and 20 normal individuals by PRV-1-specific RT-PCR. In female patients, PRV-1 expression was correlated with clonality analysis as assessed by the human androgen receptor polymorphism assay. RESULTS: PRV-1 was not expressed in granulocytes isolated from normal individuals or from patients with ST, SE, CML, IM, MDS, and inflammatory/infectious SL. On the contrary, all ET patients, 35 of 37 PV patients, and five patients with acute postsurgery and posttraumatic SL overexpressed PRV-1. All the cases with monoclonal hematopoiesis (17 of 21 with ET and 12 of 12 with PV) expressed PRV-1, yet PRV-1 overexpression extended also over the cases of ET showing polyclonal hematopoiesis (four of 20). CONCLUSION: The overexpression of PRV-1 seems to be a useful tool for discriminating ET and PV from ST and SE, thus offering an innovative diagnostic approach on the basis of the detection of positive diagnostic criteria instead of exclusion criteria.