Administration of granulocyte-colony-stimulating factor for allogeneic hematopoietic cell collection may induce the tissue factor-dependent pathway in healthy donors.

The hypercoagulable state caused by the use of recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been cited in anecdotal reports. Since tissue factor (TF) is the main initiator of the coagulation cascade, we examined if rhG-CSF had an inductive effect on the TF-dependent pathway in 18 healthy donors receiving rhG-CSF (10 microg/kg/day x 5 days) for peripheral blood progenitor cell mobilization. After rhG-CSF, there were increases both in TF antigen (TF:Ag) (P=0.01) and TF procoagulant activity (TF:PCA) (P=0.06) plasma levels and in TF:Ag cytofluorimetric expression on CD33 (+) cells (P=0.04). Mean activities of FVIII and vWF also increased significantly. Thrombin time was slightly prolonged (P=0.06) due to significant increases in plasma D-dimer levels. In addition, while FIX activity remained stable, there were marked reductions in mean plasma FX and FII activities and a slight decrease in FVII activity that resulted in a significant prolongation of prothrombin time within normal ranges. In conclusion, the administration of rhG-CSF led to a "prothrombotic state" via stimulation of TF and increased endothelial markers, such as F VIII and vWF. In the light of these findings, the use of rhG-CSF for stem cell mobilization should be undertaken cautiously in healthy donors with underlying thrombotic risk factors.

In vivo use of all-trans retinoic acid prior to induction chemotherapy improves complete remission rate and increases rhodamine 123 uptake in patients with de novo acute myeloid leukemia.

All-trans retinoic acid (ATRA) is used in the treatment of acute promyelocytic leukemia. Because ATRA has effects (increase in apoptosis, suppression of bcl-2), it has also been used for the treatment of other French-American-British (FAB) subtypes of acute myelogenous leukemia (AML). To find out the in vivo and in vitro effects of ATRA in AML, we analyzed 37 patients with de novo AML. Twenty-seven patients received ATRA before remission-induction (RI) treatment (ATRA group). Results were compared to a control group (10 patients) that received induction without ATRA during the same time period. Bone marrow or peripheral blood samples were collected from all patients on d 0 and 4. The immunphenotype, myeloperoxidase (MPO), reac tion and the efflux uptake of rhodamine 123 (Rh123) were analyzed on myeloblasts in these samples. In the myeloblasts from patients treated with ATRA, the uptake of Rh123 was increased significantly (p = 0.026) from d 0 to d 4, and all other parameters remained unaltered. ATRA administration increased the complete remission (CR) rate (88%, 22/25 vs 55%, 5/9) significantly (p = 0.042). Logistic regression analysis revealed that ATRA administration was the important factor in CR, among other potential factors including age, white blood count, bcl-2 expression, and the uptake and efflux of Rh123 (p = 0.05). Estimated disease-free survival and overall survival were similar between these two groups (43% vs 37.5% and 51.2% vs 37.5%, respectively). In conclusion, ATRA treatment prior to RI treatment may improve the CR rate in patients with de novo AML, which seems to be related to its beneficial effect on multidrug resistance.

CD41+ and CD42+ hematopoietic progenitor cells may predict platelet engraftment after allogeneic peripheral blood stem cell transplantation.

The objective of this study was to quantify subpopulations of CD34+ cells such as CD41+ and CD42+ cells that might represent megakaryocyte (MK) precursors in peripheral blood stem cell (PBSC) collections of normal, recombinant human granulocyte-colony stimulating factor (rhG-CSF) primed donors and to determine whether there is a statistical association between the dose infused megakaryocytic precursors and the time course of the platelet recovery following an allogeneic PBSC transplantation. Twenty-six patients with various hematologic malignancies transplanted from their HLA identical siblings between July 1997 and December 1999 were used. All patients except one with severe aplastic anemia who had cyclophosphamide (CY) alone received busulfan-CY as preparative regimen and cyclosporine-methotrexate for GVHD prophylaxis. Normal healthy donors were given rhG-CSF 10 microg/kg/day subcutaneously twice daily and PBSCs were collected on days 5 and 6. The median number of infused CD34+, CD41+ and CD42+ cells were 6.61 x 10(6)/kg (range 1.47-21.41), 54.85 x 10(4)/kg (5.38-204.19), and 49.86 x 10(4)/kg (6.82-430.10), respectively. Median days of ANC 0.5 x 10(9)/L and platelet 20 x 10(9)/L were 11.5 (range 9-15) and 13 (8-33), respectively. In this study, the number of CD41+ and CD42+ cells infused much better correlated than the number of CD34+ cells infused with the time to platelet recovery of 20 x 10(9)/L in 26 patients receiving an allogeneic match sibling PBSC transplantation (r = -0.727 and P < 0.001 for CD41+ cells, r = -0.806 and P < 0.001 for CD42+ cells, r = -0.336 and P > 0.05 for CD34+ cells). There was an inverse correlation between the number of infused CD41+ and CD42+ cells and duration of platelet engraftment. Therefore, as the number of CD41+ and CD42+ cells increased, duration of platelet engraftment (time to reach platelet count of > or = 20 x 10(9)/L) shortened significantly. Based on this data we may conclude that flow cytometric measurement of CD41+ and CD42+ progenitor cells may provide an accurate indication of platelet reconstitutive capacity of the allogeneic PBSC transplant.

The effect of G-CSF on lymphocyte subsets and CD34+ cells in allogeneic stem cell transplantation.

The effect of granulocyte colony-stimulating factor (G-CSF) on peripheral blood lymphocytes (PBL) and CD34+ cell frequency in the apheresis product has been determined in 25 healthy stem cell donors. Peripheral blood mononuclear cells (PBMNC) were collected after five days of G-CSF 10 microg/kg/day s.c., which was well tolerated. The median number of leukocytes increased eight-fold over that of pretreatment levels. Collection of PBMNC lasted a median of two (range, 1-3) days. The mean mononuclear cell (MNC) count and total lymphocyte percentage were 6.69 x 10(8)/kg and 59.08%, respectively, and the frequency of CD34+ cell expression was 2.1% in the apheresis product. The frequency of CD3+, CD4+, CD25+, NK and CD122+ cell expressions in mobilized PBMNC and PBL showed no significant difference. However, the frequency of CD8+, CD8+28+, CD3+DR+, CD19+, CD20+ and CD22+ B cells expression in the apheresis product increased significantly compared to steady-state PBL. In contrast, the frequency of the CD11 a+ and CD8+38+ cell expressions in the apheresis product was decreased compared to the steady-state PBL. The mean yield of CD34+ and CD3+ cells were 13.6 x 10(6) and 2.69 x 10(8)/kg of recipient body weight (RBW), respectively. Following allograft all patients engrafted with >0.5 x 10(9)/l neutrophil and < or = 20 x 10(9)/l platelets on a median of day 13 and 12, respectively. Nine patients had grade II-IV acute GVHD and chronic GVHD occurred in eight patients. Four patients died due to transplant-related complications. There was one late engraftment failure which occurred on the fifth month. Thirteen patients are still alive. In conclusion, these results indicate that administration of G-CSF at 10 microg/kg/day in normal donors alters the lymphocyte subsets and there are significant differences in the lymphocyte contents of the recipients before apheresis and in apheresis product.

Effects of combined conventional and modified ultrafiltration in adult patients.

BACKGROUND: Modified ultrafiltration (MUF) improves hemodynamics and postoperative recovery in children. Ultrafiltration (UF) may have similar benefits in adults. The purpose of this study was to investigate the effects of UF in adult patients. METHODS: A total of 40 adult patients undergoing cardiac surgery were randomized into a study group of conventional UF during bypass + venovenous MUF after bypass and a control group with no UF. Perioperative clinical variables, cytokines, and endothelin-1 levels were compared between groups. RESULTS: There was no mortality in either group. The patients in the study group had a greater rise in hematocrit (5.7% +/- 2.4% vs 1.2% +/- 1.9%, p < 0.001), hemoglobin (1.7 +/- 0.8 mg/mL vs 0.5 +/- 0.6 mg/mL, p < 0.0005), and platelet levels (27,800 +/- 29,200 vs -9,000 +/- 30,970, p < 0.001). Mean arterial blood pressure and CI increased after MUF (from 64.2 +/- 16.9 mm Hg to 72.3 +/- 14.1 mm Hg, p = 0.05, and from 2.4 +/- 0.7 to 2.8 +/- 0.6, p < 0.03, respectively). Postoperative oxygenation was better in the study group (alveolo-arterial PO2 tension gradient 74.6 +/- 43.9 mm Hg vs 107.2 +/- 27.8 mm Hg, p = 0.03). Ultrafiltration reduced postoperative bleeding (522.2 +/- 233.4 mL vs 740 +/- 198.4 mL, p < 0.003). CONCLUSIONS: A combination of conventional and modified UF is effective and safe in adult patients undergoing cardiac surgery. Ultrafiltration improved hemodynamics, hemostatic, and pulmonary functions. We recommend the use of combined UF in high-risk adult patients.

Beneficial effects of iloprost during experimentally induced hemorrhagic shock.

BACKGROUND: The aim of the study was to evaluate the efficacy of iloprost on myocardial insufficiency associated with hypovolemic shock in dogs. We designed the study as a controlled randomized study. METHODS: Sixteen mixed-breed dogs were included into the study and divided into two equal groups as the control and iloprost groups. Mean arterial pressure was reduced to 45 mmHg by withdrawing the arterial blood into citrated bags. The control group did not receive any drug but the other group received iloprost at a rate of 20 ng/kg/min by an infusion pump. Iloprost infusion was started 30 min after the blood pressure was reduced to 45 mmHg. All measurements were made before removal of blood, 45 min after exsanguination and at 1 hour intervals for 3 hours. Left ventricular stroke work index was measured 72 hours after the study. The hemodynamic and biochemical parameters and blood gas analysis were obtained. RESULTS: After hemorrhage, cardiac index (CI) decreased significantly from 132 +/- 14 to 51 +/- 8 ml/kg/min in the control group and from 128 +/- 11 ml/kg/min to 47 +/- 13 ml/kg/min in the iloprost group, respectively but at the end of the third hour it was 81 +/- 8 ml/kg/min in the control group and 105 +/- 6 ml/kg/min in the iloprost group (p < 0.05). Tumor necrosis factor-alpha (TNF alpha) was 41 +/- 8 pg/ml in the control group and 18 +/- 6 in the iloprost group 3 hours after bleeding (p < 0.05). Tumor necrosis factor-alpha concentration was significantly higher in the control group than in the iloprost group. There was no significant difference in pH between the groups but actual bicarbonate concentrations were different between the groups (p < 0.05). At the end of the third hour total body oxygen consumption was 105 +/- 11 ml/min in the control group and 132 +/- 12 ml/min in the iloprost group (p < 0.05). Oxygen delivery 3 hours after hemorrhage was 201 +/- 19 ml/min in the control group and 252 +/- 24 ml/min in the iloprost group (p > 0.05). Left ventricular stroke work index was higher in the iloprost group (p < 0.05). CONCLUSIONS: Hemorrhagic shock causes tumor necrosis factor-alpha release which may lead to multiple organ failure. Organ dysfunction still persists even after the appropriate treatment. Iloprost attenuates the release of tumor necrosis factor-alpha which may improve the adverse effects of hemorrhagic shock.

Thrombopoietic cytokines in patients with iron deficiency anemia with or without thrombocytosis.

Iron deficiency anemia is a cause of reactive thrombocytosis. A moderate increase in platelet numbers is common but sometimes counts may exceed 1,000 x 10(9)/l. The mechanisms causing reactive thrombocytosis are unclear. In this study, we evaluated 15 women with iron deficiency anemia and thrombocytosis (platelets >450 x 10(9)/l) and 16 women with iron deficiency anemia with normal platelet counts. Serum samples were taken before oral iron replacement therapy, after 1 and 3 months and at the end of replacement therapy. Thrombopoietin, erythropoietin (EPO), leukemia inhibitory factor, interleukin-6 and interleukin-11 levels were assayed. There was no change in the levels of thrombopoietic cytokines except for EPO. The correlation between high EPO levels and high platelet counts may suggest that EPO increases platelet counts, but the same EPO level changes can also be demonstrated in women with iron deficiency anemia but normal initial platelet counts. The fact that the levels of other cytokines remained unchanged during treatment suggests that either these cytokines have no effect on reactive thrombocytosis or the change in platelet counts in our patients is in a narrow range and is thus not affected by the cytokine levels.

Soluble adhesion molecules (sICAM-1, sL-Selectin, sE-Selectin, sCD44) in healthy allogenic peripheral stem-cell donors primed with recombinant G-CSF.

BACKGROUND: We analysed the effects of rhG-CSF (Amgen-Roche, USA) on serum changes of four soluble adhesion molecules (SAM) (sICAM-1, sL-Selectin, sE-Selectin and sCD44) in healthy peripheral allogeneic stem-cell transplantation donors and their correlation with acute GvHD and effect on engraftment kinetics. METHODS: Serum SAM of 15 consecutive healthy HLA identical-sibling donors (median age 30 years, male:female ratio, 7:8) were monitored using a commercial ELISA Kit (Bender Med, Austria) prior to, on the day of first apheresis and 24 h after the cessation of rhG-CSF (10 microg/kg/day s.c. on 5 days) administration. Leukapheresis was started on the fifth day of rhG-CSF administration, using a continuous-flow blood separator (Cobe Spectra, COBE BCT, Inc, Lakewood, CO). Apheresis cycles were continued daily until a target of 4.0 x 10(6) CD34(+) cells/kg was reached. RESULTS: The results indicate a steady rise of sL-Selectin, sE-Selectin, and sCD44, but not of sICAM-1. Median number of mononuclear cells (MNC) and CD34(+) cells transfused were 7.7x 10(8)/kg and 6.0 x 10(6)/kg, respectively. There was a near-significant correlation between the sL-Selectin levels and CD34(+) cell yield (r = 0.49, 0.06). Median granulocyte and platelet engraftment days were 11 (10-18) and 12 (9-33), respectively. There was a significant inverse correlation between the CD34(+) cell dose and granulocyte levels (r = -0.68, p = 0.022), but not for platelet engraftment. The only correlation between SAM levels and engraftment was for sICAM-1 levels. Increasing sICAM-1 levels were a sign of prolonged neutropenia (r = 0.72, p = 0.011). No correlation between the apheresis day serum levels of adhesion molecules and acute GvHD was documented. DISCUSSION: Analysis of sICAM-1, sL-Selectin, sE-Selectin and sCD44 levels during allogeneic PBSC apheresis did not reveal any significant effect on engraftment and GvHD, except the correlation of sL-Selectin levels and collected CD34(+) cells. More research and data about the role of not only SAM levels, but also antigenic expression of SAM are required to enlighten leukocyte-endothelial cell interactions and egress of stem cells during G-CSF administration.

The beneficial effects of aminophylline administration on heparin reversal with protamine.

The aim of this study was to demonstrate the beneficial effects of aminophylline on protamine cardiotoxicity. Thirty-four patients were examined, 17 of whom received aminophylline 3 mg/kg before protamine administration, being the study group, while the other 17, being the control group, did not. All cardiac output and biochemical measurements were evaluated 5 min following protamine administration. The cAMP level was 43.4 +/- 3.51 pmol/ml in the study group and 18.7 +/- 2.98 in the control group (P < 0.0001) before protamine administration, while the oxygen extraction rate decreased from 49% to 44 +/- 2% in the control group, and from 51.2% to 47 +/- 3% in the study group (P < 0.03). The N-acetyl glucosaminidase value was 16.9 +/- 13.9 pmol/ml in the study group and 27.8 +/- 1.47 pmol/ml in the control group (P < 0.01), and myocardial lactate extraction was -0.20 +/- 0.03 in the control group and -0.07 +/- 0.07 in the study group (P < 0.001). The left ventricular stroke work index was 28.6 +/- 3.14 gm/m2 in the control group and 37 +/- 6.77 gm/m2 in the study group (P < 0.002). The findings of this study led us to conclude that the adverse effects of heparin neutralization using protamine can be relieved by aminophylline.

Thiamine-responsive megaloblastic anemia with diabetes mellitus and sensorineural deafness.

This study introduces a patient who has thiamine and thiamine pyrophosphokinase (TPKase) enzyme deficiency associated with diabetes mellitus, sensorineural deafness and thiamine-responsive megaloblastic anemia. Diabetes mellitus was diagnosed when she was 20 months old. After 1 year, macrocytic anemia developed and the thiamine therapy was started at 75 mg/day. During the follow-up, the insulin requirement decreased and even ceased, and macrocytic anemia improved with thiamine treatment. After thiamine therapy was ceased an increase in insulin requirement was observed and macrocytic anemia developed again.

Effect of in vitro hemolysis on 25 common biochemical tests.

Clinical chemists frequently encounter hemolyzed samples. Our study examines the effects of hemolysis on the results of 25 common biochemical tests. We collected 60 15-mL blood samples from inpatients and outpatients and mechanically hemolyzed 10 mL of the samples in a two-step procedure. We classified serum from these samples as being nonhemolyzed, moderately hemolyzed, or severely hemolyzed and then performed 25 common biochemical tests. Statistical analysis of the results showed that hemolysis had the greatest effect on the lactate dehydrogenase, acid phosphatase, and potassium tests.