Evaluation of a new protocol for peripheral blood stem cell collection with the Fresenius AS 104 cell separator.

In this report we analyzed sixty leukapheresis procedures on 35 patients with a new protocol for the Fresenius AS 104. Yields and efficiencies for MNC, CD 34+ cells, and CFU-GM indicate that the new protocol is able to collect large quantities of hemopoietic progenitors. Procedures were performed processing 8.69 +/- 2.8 liters of whole blood per apheresis and modifying 3 parameters: spillover-volume 7 ml, buffy-coat volume 11.5 ml, centrifuge speed 1,500 rpm; blood flow rate was 50 ml/min and the anticoagulant ratio was 1:12. No side effects were observed during apheresis procedures except for transient paresthesia episodes promptly resolved with the administration of calcium gluconate. Yields show a high capacity of the new program to collect on average MNC 17.28 +/- 10.85 x 10(9), CD 34+ 471 +/- 553.5 x 10(6) and CFU-GM 1278.7 +/- 1346.3 x 10(4) per procedure. Separator collection efficiency on average was 49.91 +/- 23.28% for MNC, 55.1 +/- 35.66% for CFU-GM, and 62.97 +/- 23.09% for CD 34+ cells. Particularly interesting are results for MNC yields and CD 34+ efficiency; these results make the new program advantageous or similar to the most progressive blood cell separators and capable to collect a sufficient number of progenitor cells for a graft with a mean of 1.80 +/- 0.98 procedures per patient.

Evaluation of two different protocols for peripheral blood stem cell collection with the Fresenius AS 104 blood cell separator.

OBJECTIVES: Reconstitution of hematopoiesis by means of peripheral blood stem cells is a valid alternative to autologous bone marrow transplantation. The aim of this investigation was to increase the efficiency of collection of circulating blood progenitor cells and to obtain a purer product for transplant. METHODS: We carried out leukapheresis procedures with the Fresenius AS 104 blood cell separator, using two different protocols, the previously used PBSC-LYM and a new mononuclear cell collection program. RESULTS: Both programs were highly effective in collecting mononuclear cells (MNC) and CD34+ cells. Some differences were found, especially regarding MNC yield and efficiencies. There are remarkable differences in the efficiency of collection of CD34+ cells (62.38% with the new program as opposed to 31.69% with the older one). Linear regression analysis showed a negative correlation between blood volume processed and MNC efficiency only for the PBSC-LYM program. Differences were also observed in the degree of inverse correlation existing in both programs between patients' white blood cell precount and MNC collection efficiency. The inverse correlation was stronger for the PBSC-LYM program. Seven patients with solid tumors and hematologic malignancies received high dose chemotherapy and were subsequently transplanted with peripheral blood stem cells collected using the new protocol. All patients obtained a complete and stable engraftment with the reinfusion product collected with one or two leukapheresis procedures. CONCLUSIONS: High efficiencies and yields were observed in the new protocol for MNC and CD34+ cells. These were able to effect rapid and complete bone marrow recovery after myeloablative chemotherapy.

Prognostic factors of patients with acute myeloid leukemia (AML) allografted in first complete remission: an analysis of the EORTC-GIMEMA AML 8A trial. The European Organization for Research and Treatment of Cancer (EORTC) and the Gruppo Italiano Malattie Ematologiche Maligne dell' Adulto (GIMEMA) Leukemia Cooperative Groups.

The Leukemia Cooperative Groups of the EORTC and the GIMEMA conducted a prospective randomized phase III trial, in order to assess the value of autologous BMT (ABMT) vs a second intensive consolidation course (IC2), following a common intensive consolidation course (IC1) for patients with AML. Patients with an HLA-identical sibling donor were not randomized, but were included in an allogeneic BMT (alloBMT) program. This is an analysis of prognostic factors which influence the outcome of treatment after alloBMT in first complete remission (CR). The study included 730 patients < 46 years of age in CR, 270 having a histocompatible sibling donor. In 169 of these patients alloBMT was performed in first CR. Early remitters (122 patients achieving CR with one course of treatment) had a DFS at 3 years of 67%, significantly longer than that of 44% for late remitters (47 patients achieving CR after more than one course of treatment) (P = 0.006). The relapse risk for early vs late remitters was 16 and 40% at 3 years (P = 0.001) and the treatment-related mortality (TRM) at 2 years was 21 vs 27%. Age appeared to be a prognostic factor for TRM, WBC for DFS, whereas the FAB classification was not of prognostic importance. Patients with poor risk cytogenetic abnormalities showed a trend towards a higher relapse risk. Patients transplanted shortly after achieving CR appeared to have a worse prognosis than those transplanted further into remission. Overall, the number of courses of induction therapy needed to achieve CR was the most important prognostic factor for outcome after allogeneic BMT.

Instrument effect on the activated protein C resistance plasma assay performed by a commercial kit.

In order to evaluate the influence of the coagulation instrument on the activated protein C (APC)-resistance plasma assay performed by a commercial kit, we tested 70 plasma samples on 4 different instruments during a simultaneous session run using a same lot of Coatest APC-resistance (Chromogenix). The results were analyzed employing three different modes of expression (aPTT prolongation in the presence of APC, APC-sensitivity ratio, normalized APC-sensitivity ratio) and three different diagnostic threshold values (below the control mean--2 standard deviations or the lowest control value or the 5th percentile of the control values). The inter-instruments variability in the mean values of the control individuals can be limited expressing the results as normalized-APC-sensitivity ratio (range 0.99-1.05). The overall diagnostic yield in thrombotic patients and their relatives depended mainly on the instrument employed and only in some cases on the mode of expression of the results and on the diagnostic threshold value. The sensitivity of the commercial assay on heterozygotes for factor V Leiden diagnosed by gene analysis was overall satisfactory (75-100%) but in some cases a lower diagnostic yield was noticed, depending on the type of instrument employed and/or the type of expression of the results and/or the diagnostic threshold values. Thus the instrument system adopted should be carefully considered in the interpretation of the results using the commercial kit.

The combination of erythropoietin and granulocyte colony-stimulating factor increases the rate of haemopoietic recovery with clinical benefit after peripheral blood progenitor cell transplantation.

In order to investigate the effects of erythropoietin (EPO) plus granulocyte colony-stimulating factor (G-CSF) administration after peripheral blood progenitor cell transplantation (PBPCT) we performed a phase I/II study in patients with high-risk cancer. 15 consecutive patients were treated wit recombinant human G-CSF (rhG-CSF) at the dose of 5 micrograms/kg subcutaneously (s.c.) every 24 h until day + 12 and with recombinant human EPO (rhEPO) at the dose of 150 IU/kg s.c. every 48 h until day + 11 following PBPCT. Their haemopoietic recovery was compared to that obtained in eight historic and control patients who did not receive any cytokines after PBPCT. No side-effects were observed during EPO plus G-CSF treatment and the treatment was not discontinued in any of the patients before completion of the treatment plan. The administration of EPO plus G-CSF after PBPCT produced a significant increase in the rate of white blood cell (WBC) (P = 0.0005), polymorphonuclear leucocyte (PMN) (P = 0.0005) and platelet (PLT) (P = 0.0105) recovery compared to the control group. The acceleration in haemopoietic recovery observed in the EPO plus G-CSF-treated patients produced a significant reduction of the days with WBC < 1 x 10(9)/l (P = 0.0009), PMN < 0.2 x 10(9)/l (P = 0.0030) and PMN < 0.5 x 10(9)/l (P = 0.0006). EPO plus G-CSF-treated patients required a significantly lower number of single donor PLT transfusions (P = 0.0142) and did not experience neutropenic fever, but historic control patients experienced fever > 38 degrees C for a median period of 4 d (0-12) with a medial period of parenteral antibiotic administration of 7.5 d (0-17). The length of the hospital stay was significantly shorter in the study group than in the historic control group (P = 0.0264). In conclusion, we can confirm that EPO plus G-CSF treatment is feasible and potentiates the haemopoietic recovery after PBPCT, thus simplifying the clinical management of cancer patients who undergo high-dose chemotherapy.

High-dose chemotherapy with autologous peripheral stem cell support in advanced ovarian cancer.

Twenty patients with advanced (stage III-IV), previously untreated ovarian carcinoma were treated by: (a) induction chemotherapy (40 mg/m2 cisplatin, days 1-4; 1.5 g/m2 cyclophosphamide, day 4; every 4 weeks for two cycles) followed by (b) intensification chemotherapy (100 mg/m2 cisplatin, day 1; 650 mg/m2 etoposide, day 2; 1.8 g/m2 carboplatin, day 3). Eligibility criteria further included: age less than 55 years, moderately good to poor tumour grade, macroscopic (> 0.5 cm) residual tumour. Autologous peripheral stem cells were recruited after the induction cycles and, to ensure haematological support, autologous bone marrow harvesting was routinely performed in the first 14 cases. Haematological support consisted of autologous peripheral stem cells and autologous bone marrow transplant in 16 and four patients, respectively. All patients are evaluable for toxicity and 19 for pathological response, one being dead of systemic mycosis 35 days after the autologous bone marrow transplant. Severe extra-haematological toxicities were the following: gastrointestinal (100%), neurological (10%), hepatic (10%). Pathological response was detected in 84% of cases (CR 37%, microscopic PR 26%, macroscopic PR 21%). Median follow-up times of 48 and 41 months have been reached respectively from enrolment and second-look. Four-year 62% overall and 57% progression-free survivals have been reached. Ten patients are still alive with NED (six of seven with CR, three of five with microscopic PR, and one of four with macroscopic PR). Autologous peripheral stem cell transplant significantly reduced the duration of aplasia compared with autologous bone marrow transplant, and toxicity was proved to be manageable in those patients undergoing autologous peripheral stem cell transplant. The prolonged disease-free survival in patients showing CR and microscopic PR suggests that further investigation on this new approach is worthwhile.

Kinetics of Free Platelet Decrease After ADP: Effect of Fibrinogen Binding Inhibitors.

Evaluation of platelet aggregation in vitro has generally been performed by the turbidometric method. This technique is largely insensitive to early events of aggregation which, are best evaluated by following the decrease in number of free-platelets after agonist addition. In this study the kinetics of free-platelet decrease after adenosine-5-diphosphate (ADP) addition was analyzed in platelet rich plasma and in whole blood samples obtained from 29 normal donors and 5 thrombocytopenic subjects. Analysis of experimental data using a single exponential decay equation allowed us to compute the maximal velocity and the rate constant of the reaction as well as the number of un-reactive platelets. The velocity of aggregation was positively correlated with platelet number and was markedly increased by the presence of red blood cells. The process was unaffected by platelet cyclooxygenase inhibition and was also independent of thrombospondin or von Willebrand factor binding to GpIIb-IIIa. Fibrinogen receptor blockade by either anti-GpIIb-IIIa monoclonal antibody or by the RGDS analogue, MK-852, reduced, in a dose dependent manner, the aggregation velocity. Simple decay estimations based on the difference between counts made before and 15 s after ADP addition, were used to evaluate the anti-aggregating effect of MK-852. Compared to turbidometry, the method provided a more accurate dose-response curve and gave, in addition, a significantly higher IC, value (0.21±0.05 vs 0.11±0.04 pmol/l p<0.01, means±SD). We conclude that the kinetics of free-platelet decay allows characterization of early events of the platelet response to ADP by quantitative parameters. In addition, this technique may represent a significant improvement over turbidometry in the laboratory monitoring of the anti-aggregating effect of fibrinogen binding inhibitors.

Clinical manifestations and management of inherited thrombophilia: retrospective analysis and follow-up after diagnosis of 238 patients with congenital deficiency of antithrombin III, protein C, protein S.

The clinical history of 238 patients with inherited thrombophilia (AT III = 94, PC = 103, PS = 41) was analyzed retrospectively at diagnosis and in the follow-up period after diagnosis. At diagnosis 129 patients (54%) had suffered from thrombosis, with a recurrence rate of 48%. The most frequent onset manifestation was deep vein thrombosis of lower limbs (58%). Thrombotic history started before 40 in 80% of the cases. Forty-nine percent of the venous thromboses were preceded by a triggering event, in most cases pregnancy (17%) and surgery (12%). After diagnosis, follow-up lasted a total of 1,113 pt-years. A policy of short-term prophylaxis during risk situations for all patients and long-term prophylaxis in symptomatic patients failed to prevent venous thrombotic episodes (diagnosed by objective methods) in 4 previously asymptomatic subjects and recurrence in 7 previously symptomatic subjects. After knowledge of the patients' diagnosis the incidence of venous thrombosis/100 pt-years was reduced as compared before diagnosis as total episodes (onset+recurrencies) (1.0 vs 1.9), onset episodes (0.7 vs 1.3) and recurrent episodes (1.3 vs 4.8), even though the differences were not statistically significant. However most of the venous thromboses occurred at a more advanced age (67% after 40 years) and without any apparent cause (83%), at significant variance with the period preceding the diagnosis; in particular the incidence of venous thrombotic onset in patients younger than 40 passed from 1.3/100 pt-years to 0.2/100 pt-years. In 6 recurrences after diagnosis a poor compliance for antithrombotic treatment was recognized.(ABSTRACT TRUNCATED AT 250 WORDS)

Biocompatibility in hemapheresis: blood coagulation.

Hemapheresis may influence the coagulation system with effects of activation and dilution. Dilution can lead to reduced levels of platelets, fibrinogen and antithrombin III. Activation initially causes increased clotting activity, but the consumption of the activated factors generally induces a subsequent phase of hypocoagulability. In the donor, apheresis diminishes platelet count and function, as well as the levels of many other clotting factors. Depletion of fibrinogen and antithrombin III are less transient than others because their rates of synthesis are lower. In spite of the wide variety in hemapheretic procedures, all of them (or at least, those that are the most commonly used) are associated with similar activation phenomena, that appear to be mediated by the formation of a fibrinogen layer on the artificial surfaces of the circuitry.

Replacement therapy with a purified protein C concentrate during initiation of oral anticoagulation in severe protein C congenital deficiency.

The case of an adult patient with moderately severe protein C deficiency (antigen 16%, activity 12%) is reported. Both parents had protein C levels compatible with heterozygous deficiency. Unlike other reported cases of severe protein C deficiency in adults, the onset of thrombotic symptoms occurred at 1 month of age; however, a symptom-free period until age 17 followed. Replacement therapy with a monoclonal antibody purified protein C concentrate was carried out during the initiation of oral anticoagulation after a course of i.v. heparin for deep vein thrombosis. The administration of the concentrate allowed maintenance of protein C above 50% until a stable therapeutic anticoagulation level could be obtained. This was reached within a short time, thus allowing safe administration of a loading dose of warfarin. We conclude that this approach to the prevention of skin necrosis seems more rapid and safer than previous schedules of oral anticoagulation in protein C-deficient patients.

Tamoxifen-induced immune-mediated platelet destruction. A case report.

Drug-induced immunologic thrombocytopenia, a fairly common disorder, is characterized by drug-dependent antiplatelet antibodies that destroy circulating platelets in the presence of the provoking drug or its metabolites. The development of reliable methods for the detection of platelet-bound immunoglobulins causing in vivo platelet destruction, such as the use of monoclonal antibodies tagged with fluorescein and flow cytofluorimetric analysis, has ushered in a new era to differentiate between immune and non-immune thrombocytopenias. A severe thrombocytopenia developed in an elderly female patient treated with tamoxifen, a non-steroidal anti-estrogen drug, after surgery for breast cancer. A tamoxifen-dependent platelet antibody was detected in the patient's serum and linked on the platelet membranes. This antibody reacted only in the presence of the offending drug and showed platelet specificity. Withdrawal of drug restored platelet count to normal levels.

Further investigations on the expression of HLA-DR, CD33 and CD13 surface antigens in purified bone marrow and peripheral blood CD34+ haematopoietic progenitor cells.

We evaluated the HLA-DR, CD33 and CD13 antigen expression on CD34+ haematopoietic progenitor cells (HPC) isolated from the bone marrow (BM) and peripheral blood (PB) of normal donors. The majority of both BM and PB CD34+ HPC expressed CD13 and HLA-DR. The coexpression of CD34 and CD33 was found in a minor CD34+ subset. After 7 d of culture in the presence of interleukin-3 and granulocyte-macrophage colony-stimulating factor, CD33 expression was detected in about 50% of HPC. At this point CD34 antigen expression was lost and CD13 and HLA-DR expression was partially lost. After 14 d of culture, the majority of HPC were CD33+. HPC maintained the capacity to generate colony forming unit granulocyte-macrophage but they lost the capacity to generate burst forming unit-erythroid. A correlation was found between the percentage of CD34+/HLA-DR+ cells and the total number of colony forming cells in unfractionated samples from BM and PB of patients with malignancies. These studies demonstrate that, in normal conditions, only a minor subset of CD34+ cells coexpress CD33 antigen either in BM or in PB and CD33 antigen is a lineage marker which is coexpressed with HLA-DR and CD13 on a progenitor committed to the granulocytic-macrophagic lineage.

Autologous stem cell transplantation: sequential production of hematopoietic cytokines underlying granulocyte recovery.

We investigated the serum concentrations of a variety of cytokines [granulocyte-macrophage-colony-stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), interleukin (IL) 1 alpha, IL-3, IL-6, IL-8, erythropoietin, tumor necrosis factor alpha, gamma-interferon in 10 patients with advanced ovarian cancer undergoing autologous peripheral blood stem cell (PBSC) harvesting followed by treatment with high-dose cisplatin, etoposide, and carboplatin and PBSC transplantation (chemotherapy was administered on days 1 through 3, PBSCT on day 6). Preliminary observations on cytokine serum levels were performed for 4 patients; on this basis, the kinetics of cytokines was then investigated in greater detail at closely sequential times in 6 further patients. We observed a consistent pattern of sequential GM-CSF, G-CSF, and IL-8 release after chemotherapy/PBSCT in all 10 cases, including the 6 patients monitored in detail: (a) at days 5-10 a GM-CSF peak; (b) at days 12-14 a pronounced release of both G-CSF and IL-8, which always preceded granulocyte recovery by approximately 7 days. At days 17-23, a second GM-CSF peak was monitored in 5 of the 6 patients analyzed in detail, as well as in the other 4 cases. Particularly relevant are the observations that: (a) the peak of G-CSF serum concentration and neutrophil number in the recovery phase are strikingly and directly correlated, thus indicating a key role for G-CSF in granulocyte rescue; (b) the time courses of G-CSF and IL-8 levels are strictly parallel, thereby suggesting a coordinate stimulus for production of granulocytes, mediated by G-CSF, and their activation/migration capacity, mediated by IL-8. Results were essentially negative for IL-3, tumor necrosis factor alpha, and gamma-interferon concentrations (except in one case for each cytokine). An early peak of IL-1 alpha was observed in all 3 analyzed patients, while an IL-6 peak was monitored at days 13-15 in all 4 patients analyzed in detail. The present results indicate a sequential coordinate pattern of cytokine release after ablative therapy and PBSCT and shed light on the mechanisms mediating the recovery of granulocytes, and more generally of hematopoiesis, after stem cell transplantation. Furthermore, these studies may contribute to the design of improved protocols for cytokine administration following myelosuppressive anticancer therapy, as well as to the prediction of granulocytic response.

The linkage between binding of the C-terminal domain of hirudin and amidase activity in human alpha-thrombin.

A method derived from the analysis of viscosity effects on the hydrolysis of the amide substrates D-phenylalanylpipecolyl-arginine-p-nitroaniline, tosylglycylprolylarginine-p-nitroanaline and cyclohexylglycylalanylarginine-p-nitroalanine by human alpha-thrombin was developed to dissect the Michaelis-Menten parameters Km and kcat into the individual rate constants of the binding, acylation and deacylation reactions. This method was used to analyse the effect of the C-terminal hirudin (residues 54-65) [hir-(54-65)] domain on the binding and hydrolysis of the three substrates. The results showed that the C-terminal hir-(54-65) fragment affects only the acylation rate, which is increased approx. 1.2-fold for all the substrates. Analysis of the dependence of acylation rate constants on hirudin-fragment concentration, allowed the determination of the equilibrium binding constant of C-terminal hir-(54-65) (Kd approximately 0.7 microM). In addition this peptide was found to competitively inhibit thrombin-fibrinogen interaction with a Ki which is in excellent agreement with the equilibrium constant derived from viscosity experiments. These results demonstrate that binding of hir-(54-65) to the fibrinogen recognition site of thrombin does not affect the equilibrium binding of amide substrates, but induces only a small increase in the acylation rate of the hydrolysis reaction.

Evaluation of a new whole blood cytometric lymphocyte transformation test for immunological screening.

Lymphocyte transformation test (LTT) is usually performed by 3H-thymidine (3H-TdR) incorporation of phytohemagglutinin stimulated lymphocytes. This test presents a lack of standardization as well as a great variability of results making it difficult to compare the results carried out by different laboratories. Flow cytometry (FCM) can precisely evaluate cell kinetics by measuring the DNA content of stained nuclei. We tested a new ready-to-use kit for the blastogenic response of mitogen stimulated lymphocytes in FCM for clinical immunological screening (Blastest, Ylem). Separated peripheral blood lymphocytes from the same donors were also tested with the 3H-TdR method at 72h and results compared to FCM. Our results show a significant correlation between data obtained from FCM and 3H-TdR method. We show that there are no differences in results between different cytometers if CVs are low. Flow cytometry is more precise and reproducible and makes a better standardization feasible, it also offers a simpler and faster way for routine LTT enabling a wider clinical use.

Increased thromboxane biosynthesis in patients with polycythemia vera: evidence for aspirin-suppressible platelet activation in vivo.

Increased thromboxane (TX) production and modified aspirin sensitivity has been detected in vitro in platelets isolated from patients with polycythemia vera. To verify the relevance of these capacity-related measurements to the actual rate of TXA2 biosynthesis in vivo and its suppression by oral aspirin, we have investigated the urinary excretion of major enzymatic metabolites of TXB2 in 17 patients with polycythemia vera and 23 gender- and age-matched controls. Urinary 11-dehydro-TXB2 and 2,3-dinor-TXB2 were measured by previously validated radioimmunoassays. In addition, urinary immunoreactive leukotriene (LT) E4 was measured to explore the 5-lipoxygenase pathway of arachidonate metabolism. Polycythemic patients had significantly (P < .001) higher excretion rates of both 11-dehydro-TXB2 (1,033 +/- 1,050 v 117 +/- 45 pmol/mmol creatinine; mean +/- SD) and 2,3-dinor-TXB2 (725 +/- 676 v 82 +/- 43 pmol/mmol creatinine) than controls. In contrast, urinary LTE4 was not significantly different. Enhanced metabolite excretion did not correlate with the platelet count or with the hematocrit value, and was not related to the current treatment or to a clinical history of thrombotic complications. Platelet TX receptor studies did not show any significant changes in the binding characteristics of two different ligands. A platelet-selective regimen of aspirin therapy (50 mg/d for 7 to 14 days) was associated with greater than 80% suppression in metabolite excretion in nine patients. These results are consistent with abnormal stimuli operating in polycythemia vera to induce a selective enhancement in the platelet biosynthesis of TXA2 without changes in receptor binding. This in vivo abnormality in platelet biochemistry can be largely suppressed by low doses of aspirin.