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Biomedical subjects

J Riggert

Publications and source records attributed to J Riggert.

At least 19 recordsLinked to original sources
PubMed2005

Leukapheresis for the extraction of monocytes and various lymphocyte subpopulations from peripheral blood: product quality and prediction of the yield using different harvest procedures.

BACKGROUND AND OBJECTIVES: Leukapheresis of non-mobilized healthy donors is performed to harvest monocytes and lymphocyte subpopulations for use in various therapeutic regimens. In this methodological study, we compared two different leukapheresis programs, using equivalent volumes of processed blood over similar processing periods, to determine the influence of the procedures on the donor peripheral blood count and to establish the procedure that yields the highest quality product. MATERIALS AND METHODS: The target variables obtained in 41 healthy blood donors who underwent short-term leukapheresis (80-105 min) were retrospectively compared. Twenty-one volunteers were processed on a COBE Spectra machine at the MNC setting and 20 volunteers were processed at the AutoPBSC setting. Data were collected on pre- and postleukapheresis samples and on the product. RESULTS: AutoPBSC and MNC procedures resulted in a decrease of haemoglobin (5-7%), platelets (17-20%), monocytes (22%) and lymphocytes (23-27%), but not of granulocytes in peripheral blood. Both procedures produced nearly identical leucocyte and lymphocyte yields. AutoPBSC products contained a greater number of granulocytes, monocytes and red cells, but fewer platelets. The preleukapheresis values correlated with the yields for monocytes, T-helper and T-suppressor cells, B-lymphocytes and natural killer cells, but not for granulocytes or platelets. CONCLUSIONS: Leukapheresis is a safe and efficient procedure for collecting large numbers of peripheral blood monocytes and different lymphocyte populations from non-mobilized donors. The two programs yield comparable leucocyte harvests. Based on our results, yields can be predicted from the peripheral cell counts.

Blood Cell Count↗
PubMed2001

Prospective, randomized, sequential, crossover trial of large-volume vs. normal-volume leukapheresis procedures: effects on subpopulations of CD34(+) cells.

Some data exist on the influence of leukapheresis volume on the number of harvested peripheral blood hematopoietic progenitor cells (HPC), but less is known about the influence on the composition of HPC. We therefore performed a prospective, randomized crossover trial to evaluate the effect of large-volume (LVL) vs. normal-volume leukapheresis (NVL) on subpopulations of CD34(+) cells in the harvest product of 15 patients with breast cancer and 8 patients with non-Hodgkin's lymphoma. Patients were randomly assigned to start either with an LVL on day 1 followed by an NVL on day 2 or vice versa. The number of HPC, the extraction efficiency defined as difference between yield in the harvest and decrease in peripheral blood, and the relative proportion as well as the absolute numbers of CD34(+) cells coexpressing CD38, CD90, HLA-DR, CD117, CD7, CD19, CD41, or CD33 were evaluated. There was no significant difference with regard to the percentages of the subsets on comparison of LVL to NVL procedures. Only the absolute median number of CD34(+)HLA-DR(-) cells was significantly (P=0.02) higher in LVL harvests compared with the corresponding NVL components, which can be explained on the basis of the higher yield and the higher extraction efficiency in LVL compared with NVL. LVL results in a higher yield of CD34(+) cells and leads to an intra-apheresis recruitment of HPC but the relative composition of the harvested CD34(+) cells is not changed significantly. In addition, the amount of early, HLA-DR(-), hematopoietic HPC seems to be increased by an LVL.

Adult↗
PubMed2001

Ex vivo generation of human anti-melanoma autologous cytolytic T cells by dentritic cell/melanoma cell hybridomas.

Due to their central role in controlling immunity, dendritic cells are logical targets for priming naive cytotoxic T lymphocytes against tumour cells. In a strictly autologous system, we fused dendritic cells with melanoma cells, both of which were derived from patients with metastatic malignant melanoma. Hybridomas were positive for major histocompatibility complex (MHC) class II, CD40, CD54, CD83, CD86, and the pro-inflammatory cytokine interleukin-12. Autologous T lymphocytes were co-incubated with hybridomas. After 6 days, in-vitro-primed T lymphocytes revealed a strong proliferation activity and released Th-1-associated, but not Th-2-associated, cytokines. Furthermore they showed effective anti-melanoma activity, resulting in death of 70 +/- 9% of autologous melanoma cells. After depletion of CD4+ cells from the mixed population of primed T lymphocytes, the remaining CD8+ cells were able to kill 63+/-8% of autologous melanoma cells. Following depletion of CD8+ cells, however, the cytotoxic capacity of the remaining T lymphocytes caused death in only 32+/-6% of autologous melanoma cells. Blocking of MHC class I, but not class II, molecules on hybridomas impaired T cell proliferation, secretion of Th-1-associated cytokines, as well as the cytotoxic activity of primed T cells. These findings strongly suggest that hybridomas deliver melanoma-associated antigens via MHC class I molecules to T lymphocytes, resulting in the generation of CD8+ cytotoxic T lymphocytes with effective anti-melanoma activity in vitro. The data may serve as a basis for the use of hybridomas in the immunotherapy of malignant melanoma in vivo.

Antigen Presentation↗
PubMed2001

Successful transplantation and engraftment of peripheral blood stem cells after cryopreservation, positive and negative purging procedures, and a second cryopreservation cycle.

Transplantation of peripheral blood stem cells (PBSC), positively and/or negatively selected immediately after harvest, has become a widely applied therapeutic option in hematological or oncological patients. The following case of peripheral blood stem cell transplantation represents the first case of successful transplantation of PBSC, cryopreserved twice and purged after cryopreservation. PBSC were harvested in a 44-year-old female patient with a low-grade non-Hodgkin's lymphoma stage IV after mobilization with chemotherapy and G-CSF. A total number of 15.2 x 10(6) CD34+ cells/kg bodyweight was harvested with a 36.9% contamination of tumor cells coexpressing CD5 and CD20. After subsequent chemotherapy cycles and cyclophosphamide mobilization, only 0.77 x 10(6) CD34+ cells/kg bodyweight, not sufficient for transplantation, were achieved after positive selection. Therefore, 10.8 x 10(6) cryopreserved CD34+ cells/kg bodyweight were thawed and a positive selection was carried out with the BAXTER Isolex 300i machine. Before additional negative selection, the 0.77 x 10(6) positively selected CD34+ cells/kg bodyweight from the second mobilization were added. A total quantity of 4.4 x 10(6) CD34+ cells/kg bodyweight with a purity of 93.1% representing a recovery of 38% was obtained. Cells were again cryopreserved, stored and retransfused after conditioning the patient with TBI and high-dose cyclophosphamide. The patient engrafted with a WBC count > 1000/microliter on day eight and a platelet count > 20,000/microliter without transfusion support on day 12 post-transplantation. This case indicates that purging procedures can successfully be carried out with cryopreserved cell material and that purified CD34+ cells can be cryopreserved a second time before transplantation, without affecting their hematopoietic capacity.

Adult↗
PubMed2001

A multicentre evaluation of a new filtration protocol for leucocyte depletion of high-haematocrit red blood cells collected by an automated blood collection system.

BACKGROUND AND OBJECTIVES: A multicentre trial was set up to evaluate the performance of a new leucodepletion protocol. MATERIALS AND METHODS: Filtration at high haematocrit was started during collection of red blood cell (RBC) products by apheresis with Trima. SAG-M was added after filtration through the filter. Haematocrits and haemoglobin of the filtered RBCs were measured. Residual leucocytes were determined by Nageotte counting. RESULTS: One-hundred and forty seven procedures were carried out. The haematocrit and haemoglobin contents were 57.3 +/- 3.0% and 55.1 +/- 4.3 g/unit, respectively. All products showed low residual leucocyte levels (< or = 0.75 x 106/unit; 99.31% < 1 x 106). CONCLUSION: Immediate, on-line, high-haematocrit filtration of red cells collected on Trima resulted in leucoreduced RBCs, which met the AABB and Council of Europe criteria.

Blood Cell Count↗
PubMed2001

Filtration of methylene blue-photooxidized plasma: influence on coagulation and cellular contamination.

BACKGROUND: Virus inactivation of plasma can be achieved by photodynamic methods in the presence of phenothiazine dyes such as methylene blue (MB). Subsequent filtration may increase the efficacy of virus inactivation and reduce adverse effects of WBC contamination and MB. STUDY DESIGN AND METHODS: This study examined the effect of filtration with three different filters (MBF1, MBF2, and MBF3) on MB concentration, residual cells, coagulation factors, and activation measures of coagulation, fibrinolysis, and complement in MB-treated (1 microM/L) plasma units. RESULTS: Filtration reduced the concentration of MB by > or = 89 percent. WBCs were depleted by 92 percent (MBF1) and >99.9 percent (MBF2 and MBF3). Treatment with MB significantly decreased the coagulation potency from levels in untreated plasma, as measured by thromboplastin time ratio (112 +/- 18% vs. 95 +/- 11%), activated partial thromboplastin time (40 +/- 3 sec vs. 44 +/- 3 sec), thrombin time (16.9 +/- 1.1 sec vs. 18.6 +/- 1.5 sec), factor VIII (1.09 +/- 0.21 U/mL vs. 0.85 +/- 0.13 U/mL), and vWF (0.94 +/- 0.65 U/mL vs. 0.65 +/- 0.24 U/mL). Filtration did not further decrease these values, while factor XI (0.75 +/- 0.22 U/mL vs. 0.37 +/- 0.20 U/mL) and prekallikrein values decreased in MB plasma units filtered with the MBF3. In addition, activated factor XII (0.7 +/- 0.5 microg/L vs. 4.5 +/- 1.0 microg/L) increased. CONCLUSION: WBCs and MB can be eliminated from MB-treated plasma units by filtration. Differences in biocompatibility of the different filters, especially the influence on the contact phase of coagulation, must be taken into consideration.

Blood Coagulation↗
PubMed2000

A prospective, randomized, sequential crossover trial of large-volume versus normal-volume leukapheresis procedures: effects on serum electrolytes, platelet counts, and other coagulation measures.

BACKGROUND: LVL procedures with the administration of heparin as an additional anticoagulant are increasingly performed because of the potentially higher yield of autologous peripheral blood HPCs. A prospective, randomized crossover trial was performed to evaluate the influence of leukapheresis volume-that is, large versus normal-on serum electrolytes, platelet count, and other coagulation measures in 25 patients with breast cancer and 14 patients with non-Hodgkin's lymphoma. STUDY DESIGN AND METHODS: Patients were randomly assigned to start either with an LVL on Day 1 followed by a normal-volume leukapheresis (NVL) on Day 2 or vice versa. In LVL, heparin was administered in addition to ACD-A. Bleeding complications, transfusion support, whole-blood counts, and several coagulation measures as well as plasma heparin levels were evaluated. RESULTS: Although the duration, the infused amount of ACD-A, the flow rate, the drop in platelet count, and the drop in potassium were significantly greater in LVL, and although LVL patients also received heparin, there was no significant difference in clinical tolerance or bleeding complications. After LVL, patients exhibited a significantly longer activated partial thromboplastin time (APTT), with a median of 70 seconds (range, 44-100 sec), and a median anti-factor Xa activity of 0.69 IU per mL (range, 0.10-1.29 IU/mL). The value of the APTT after LVL correlated with anti-factor Xa activity (r = 0.37, p<0.05), but not with platelet count or heparin infusion rate. Markers for coagulation activation did not increase during NVL or LVL. CONCLUSION: LVL with heparin as an additional anticoagulant seems to be a safe procedure in patients with low preleukapheresis platelet counts. No activation of coagulation occurred after NVL or LVL procedures.

Adult↗
PubMed2000

Testing of individual blood donations for HCV RNA reduces the residual risk of transfusion-transmitted HCV infection.

BACKGROUND: To allow cost-effective RNA testing with NAT techniques, the national authorities of several countries have planned or already introduced tests of mixed specimens, that is, plasma pools. STUDY DESIGN AND METHODS: High-throughput extraction, amplification, and detection of HCV RNA from individual blood donations were optimized and validated. The feasibility of the method and the frequency of anti-HCV-negative, HCV RNA-positive donations were determined in a prospective study of 27,745 allogeneic and 792 autologous individual donations. RESULTS: The 50- and 95-percent detection limits of the method were determined at 44 IU per mL and 162 IU per mL, respectively (World Health Organization HCV reference material). When 201 HCV RNA-positive sera were taken as a reference, the sensitivity was 97.5 percent. The assay specificity was determined at 99.77 percent. During a 20-month period, two seronegative blood donors tested positive in HCV PCR. The viral load of these donations was 6 x 10(6) and 3 x 10(7) copies per mL, respectively. Thus, the yield of HCV RNA testing in this study was 7. 63 per 100,000 screened donations (95% CI, 1.25-22.07). In both PCR-positive donors, seroconversion was found in subsequent blood samples. CONCLUSION: This study compares the feasibility of single-donation HCV RNA screening, with the detection of a relatively high percentage of window-phase donations, to data reported from groups using HCV RNA testing of plasma pools. The relative yield of NAT of individual donations versus minipools should be directly investigated in the near future.

Blood Donors↗
PubMed2000

A cell-kinetic model of CD34+ cell mobilization and harvest: development of a predictive algorithm for CD34+ cell yield in PBPC collections.

BACKGROUND: Mobilization and homing of PBPCs are still poorly understood. Thus, a sufficient algorithm for the prediction of PBPC yield in apheresis procedures does not yet exist. STUDY DESIGN AND METHODS: The decline of CD34+ cells in the peripheral blood during apheresis and their simultaneous increase in the collection bag were determined in a prospective study of 18 consecutive apheresis procedures. A cell-kinetic, four-compartment model describing these changes was developed. Retrospective data from 136 apheresis procedures served to further improve this model. A predictive algorithm for the yield was developed that considered the sex, weight, and height of the patient, the number of CD34+ cells in peripheral blood before apheresis, the inlet flow, and the duration of the apheresis. The accuracy of this algorithm was evaluated by comparison of the predicted and the observed yields of CD34+ cells in 105 prospective autologous and 148 retrospective allogeneic apheresis procedures. RESULTS: The correlation between predicted and observed yields was good for the autologous and allogeneic groups with a correlation coefficient (r) of 0.8979 and 0.8311 (p<0.0001), respectively. The regression is described by the equations log (measured value [m]) = 1.0118 + 0.8595 x log (predicted value [p]) for the autologous and log (m) = 2.226 + 0.7559 x log (p) for the allogeneic group. The respective equations for the zero-point regression are log (m) = 1.014 x log (p) and log (m) = 1.026 x log (p). The probability that the measured value was 90 percent or more of the predicted value was 83.8 percent for the autologous and 90.5 percent for the allogeneic apheresis procedures. CONCLUSION: The predictive accuracy of the algorithm and the slope of the zero-point regression curve were higher for allogeneic than autologous PBPC collections. The predictive algorithm may be a useful tool in PBPC harvest, enabling the adaptation of the size of the apheresis to the needs of each patient.

Algorithms↗
PubMed2000

Hepatitis C virus transmission through quarantine fresh-frozen plasma.

In 1994, quarantine fresh-frozen plasma (Q-FFP) was introduced in Germany in order to reduce the risk of HIV and HCV transmission. In 1998, an acute HCV infection of a patient was reported to us. The look-back revealed that this patient had received two Q-FFP from a donor who had seroconverted for HCV in the meantime. Recipients of further plasma donations from this donor were identified. Back-up specimens of these donations were investigated in several laboratories. A total of 25 additional HCV-PCR positive plasma units had been transfused to 12 further patients. HCV infections were diagnosed in seven of these recipients, three patients had already been deceased. One of the remaining two recipients was already HCV positive prior to transfusion, in the other patient, no HCV infection was detectable. This patient had received three units of an "early" plasma donation , which was tested negative by PCR in one laboratory, but positive in the other. The subsequent, clinically infectious donation had the same discrepant PCR results. Thus, eight cases of HCV transmission were revealed and classified as "certain" with regard to causality, also due to an identical HCV genotype, i.e. 3e. Some of these infections would have been prevented by application of a different anti-HCV assay. The assay used in the respective plasmapheresis station was in-sensitive in this individual case for more than 400 days after the first PCR positive donation. This caused the release of the above mentioned infectious units. Upon re-testing the backups, three of four other anti-HCV assays revealed a positive result already 104 days after the first PCR-positive donation. The donor had increased ALAT levels (> 23 IU/L) at nine of 28 donations, two of these were higher than 2.5 times the upper normal limit, and two were higher than 68 IU/L, which is the cut-off value for male blood donors in Germany. The results of these (look-back) studies arouse several queries, i.e. differences in the diagnostic sensitivity between current anti-HCV and PCR tests, the accuracy of risk-estimates (especially when based on hemovigilance studies for Q-FFP), the value of ALAT testing, and currently practised release algorithms for Q-FFP.

Adult↗
PubMed1999

Quantitative PCR for counting residual white blood cells in blood products.

Leukocyte depleted blood components are frequently used to reduce alloimmunization and the risk of transfusion transmitted infection. Counting residual white blood cells in filtered blood products requires sensitive and reliable techniques. After separation of white blood cells from 500 microliters of 20 non-filtered and 54 filtered blood products we used polymerase chain reaction (PCR) and fluorimetric detection for the quantification of genomic DNA. The results were compared with results from Nageotte chamber counting. The accurate limit of detection of PCR was determined at 1 WBC/microliter (intra-assay coefficient of variation: 16.3%). PCR correlated well with Nageotte chamber counts (r = 0.77, p < 0.001, n = 74). Concordant results were obtained in 51 filtered and 20 non-filtered blood products. Discrepant results were obtained in 3 filtered whole blood units: In these blood products > 12 WBC/microliters were counted in Nageotte chamber and PCR gave a negative result. After component preparation fresh-frozen plasma and red cell concentrates of these units contained < 1 WBC/microliter using both methods. In conclusion we describe a quantitative PCR method which had about the same sensitivity and specificity as Nageotte chamber testing. However, PCR is more laborious than the standard method. As well, as reliable PCR testing requires expensive instruments and staff experienced in molecular biology, the standard method is more cost effective.

Fluorometry↗
PubMed1999

A prospective, randomized, sequential, crossover trial of large-volume versus normal-volume leukapheresis procedures: effect on progenitor cells and engraftment.

BACKGROUND: The influence of leukapheresis size on the number of harvested peripheral blood progenitor cells is still unclear. A prospective randomized crossover trial was thus performed, to evaluate the effect of large-volume leukapheresis (LVL) versus normal-volume leukapheresis (NVL) on progenitor cells and engraftment in 26 patients with breast cancer and 15 patients with non-Hodgkin's lymphoma who were eligible for peripheral blood progenitor cell transplantation. STUDY DESIGN AND METHODS: Patients were randomly assigned to undergo either LVL on Day 1 and on Day 2 or vice versa. The number of progenitor cells was evaluated in the harvest and before and after leukapheresis in the peripheral blood. RESULTS: The number of harvested CD34+ cells (4.8 x 10(6) vs. 3.4 x 10(6)/kg body weight, p < 0.001) and colony-forming units-granulocyte-macrophage (3.1 x 10(5) vs. 2.4 x 10(5)/kg body weight, p = 0.0026) was significantly higher for LVL procedures than for NVL procedures. The median extraction efficacy, defined as the difference between the yield in the harvest and the decrease in the total number of CD34+ cells in peripheral blood during leukapheresis, was significantly (p < 0.0001) higher for LVL than for NVL (2.6 x 10(8) and 8 x 10(7), respectively). In patients with breast cancer, the median amount of CD34+ cells in the harvest and the median extraction efficacy were higher for LVL than for NVL (p < 0.0001). This was not found for patients with non-Hodgkin's lymphoma. CONCLUSION: LVL results in a higher yield of CD34+ cells and colony-forming units-granulocyte-macrophage than NVL, but only in patients with breast cancer and with high numbers of CD34+ cells in the peripheral blood before leukapheresis.

Adult↗
PubMed1999

Leukaemic blasts differ from normal bone marrow mononuclear cells and CD34+ haemopoietic stem cells in their metabolism of cytosine arabinoside.

Different metabolites of cytosine arabinoside (AraC) contribute to its cytotoxicity including incorporation of AraCTP into DNA, the incorporation of AraUMP into RNA, inhibition of polymerase alpha and beta (AraCMP/CTP), an impairment of repair mechanisms (AraCTP), alterations of phospholipid metabolism (AraCDP-choline), a direct membrane interaction (AraC), the alteration of signal transduction pathways (AraCDP-choline, AraCTP) and the induction of apoptosis. Since little is known about the potential differences in AraC metabolism between leukaemic blasts and normal haemopoietic progenitor cells, the formation of all known AraC metabolites was determined in bone marrow samples from patients with acute myeloid leukaemia (AML), healthy volunteers and specimens of cellsorted CD34+ haemopoietic stem cells. Highly significant differences were found for phosphorylated AraC metabolites (AraCMP, -CDP, -CTP, AraUMP) between AML and normal mononuclear bone marrow (ng/107 cells respectively 1.30 v 2.66; 2.65 v 7.50; 33.68 v 99.0; 1.18 v 5.70). The highest differences were found for formation of AraCDP-choline (3.75 v 12.86) which might be relevant for the high efficacy of high-dose AraC regimens. In contrast, no differences were found in the deamination product AraU (2.01 v 2.91). Only minute amounts of phosphorylated AraU derivatives were detected, providing an explanation for the lacking contribution of AraU to cytosine arabinoside cytotoxicity. Results in normal CD34+ haemopoietic stem cells did not differ significantly from normal bone marrow mononuclear cells and therefore justify their use as a surrogate in determining AraC-induced haematotoxicity. These data suggest a metabolic basis for the relative selectivity of AraC cytotoxicity for AML blasts and provide a means to determine the role of different metabolites and their related mechanism of action for overall AraC cytotoxicity.

Acute Disease↗