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P Kühnl

Publications and source records attributed to P Kühnl.

At least 19 recordsLinked to original sources
PubMed2006

Comparison of flow cytometry vs. a haematology cell analyser-based method to guide the optimal time-point for peripheral blood stem cell apheresis.

BACKGROUND AND OBJECTIVES: For timing the onset of apheresis, parameters obtained by flow cytometry and by a haematological cell analyser were compared. MATERIALS AND METHODS: Haematopoietic cell counts (n = 159) were performed by two different methods; CD34 analyses by flow cytometry, immature myeloid information (IMI) and human progenitor cell counts (HPC) by a haematological cell analyser. RESULTS: Comparing the IMI total results with CD34+ analyses (n = 159) revealed a correlation of r = 0.46 (P < 0.05). Similar results were obtained for HPC (r = 0.44; P < 0.05). CONCLUSION: The haematology analyser-based method does not allow the precise determination of absolute haematopoietic stem cell numbers and is thus not able to replace flow cytometry for the monitoring of peripheral blood stem cell counts.

Blood Component Removal↗
PubMed2000

[Flow-cytometric analysis of platelet activation during rotablation].

Acute occlusion and subacute restenosis of the coronary artery are still the limiting factors of the otherwise successful interventional cardiology. Platelets and especially activated platelet populations play a key role concerning these typical and sometimes fatal complications. In this study we used flow-cytometry to determine the influence of the modern interventional technique of rotablation on platelet antigens and their possible alteration. A PTCA control group was included. We analyzed the fluorescence expression of structural antigens CD41a (GPII-IIIa) and CD42b (GPIb-V-IX), and of the activation-dependent antigens CD62p (P-selectin, PADGEM, GMP-140) and CD63 (GP53). Furthermore we analyzed the binding of fibrinogen to the platelet flow-cytometrically. CD41a and CD42b did not show significant alternations in fluorescence before, directly after and thirty minutes after finishing PTCA and rotablation (PTCA: CD41a p=0.8 and 0.9; CD42b p=0.5 and 0.2; rotablation: CD41a p=0.2 and 0.2; CD42b p=0.4 and 0.1). But platelet activation could be detected directly after PTCA and rotablation measuring the mean channel fluorescence intensity (MCFI) of CD62p, CD63 and fibrinogen binding (all p<0.05). Thirty minutes after finishing the procedures there were again significant changes in MCFI in PTCA (CD62p, CD63, fibrinogen binding; all p<0.05), but not in rotablation (CD62p p=0.1; CD63 p=0. 9; fibrinogen binding p=0.5). But MCFI for CD62p and fibrinogen binding in rotablation was higher than in PTCA. The results of our study show that rotablation also induces significant platelet activation that is higher than in PTCA alone. Flow cytometry is a sensitive and specific, multiparametric tool in establishing platelet activation. The individual platelet activation process is part of a complex cascade of events happening in the rotablated coronary segment leading to a vascular-molecular inflammatory process and consecutive clinical problems in some patients.

Angioplasty, Balloon, Coronary↗
PubMed1999

Influence of stent length and heparin coating on platelet activation: a flow cytometric analysis in a pulsed floating model.

Platelets are involved in acute and subacute thrombotic occlusions of coronary stents and also may play a role in the pathophysiology of in-stent restenosis. This study sought to investigate the expression of activation dependent glycoproteins on platelets by flow cytometry and time until stent thrombosis in an in vitro model of stent thrombosis. Coronary stents were placed in parallel silicon tubings with circulating citrated platelet rich plasma to measure 1) influence of stent length on platelet antigens; 2) influence of heparin coating on platelet antigens; and 3) time until stent thrombosis. After recalcification aliquots of platelet-rich plasma were taken over 10 minutes in 2-minute intervals and immediately fixed and stabilized. For flow cytometric analysis monoclonal antibodies to CD41a (glycoprotein IIb/ IIIa), CD42b (glycoprotein Ib-V-IX), CD62p (P-selectin), and CD63 (glycoprotein 53) were used. Within 2 minutes after start of circulation, the expression of CD62p and CD63 increased. Longer stents resulted in more platelet activation than shorter stents (25 mm vs. 15 mm; p<0.001. Time until stent thrombosis was reduced (25 mm vs. 15 mm; p<0.05). Heparin coating did not significantly influence flow cytometry detectable platelet activation but prolonged time until stent thrombosis (coated vs. uncoated; p<0.005). In control tubing systems without stents platelet activation was less pronounced (p<0.0001). Antibodies to CD41a and CD42b did not show significant changes. In this model platelet activation detected by flow cytometry and time until stent thrombosis were dependent on stent length and coating. In vitro testing could be useful to optimize stent design and material.

Adult↗
PubMed1998

The HTLV type I sequence from an asymptomatic German blood donor is related to sequences from South America.

In most parts of Europe only a limited number of sporadic cases of HTLV-I infections have been identified. So far, the few cases found in Germany were individuals from endemic areas or with relations to endemic areas. Here we report an HTLV-I infection from an asymptomatic female German blood donor whose only known potential risk was a former partner from South America, where HTLV-I is known to be endemic. The DNA sequence of the LTR region was determined and a phylogenetic analysis indeed suggested homologies with HTLV-I sequences from South America.

Base Sequence↗
PubMed1997

German consensus on immunogenetic donor search for transplantation of allogeneic bone marrow and peripheral blood stem cells.

In Germany allotransplantation of bone marrow or peripheral blood stem cells is presently performed by 34 different teams operating more or less independently. Thus, strategies of immunogenetic donor search, use of the various tissue typing techniques and policy on acceptable HLA mismatches in related and unrelated settings may vary considerably from one transplant centre to another. This paper summarises the results of the first German consensus meeting on immunogenetic donor search for bone marrow/peripheral blood stem cell grafting. The main goal of the participating transplant physicians and immunogeneticists was to define national standards for the above issues.

Bone Marrow Transplantation↗
PubMed1997

Transfusion of autologous, hydroxyethyl starch-cryopreserved red blood cells.

UNLABELLED: In this prospective, randomized study, we investigated the safety and efficacy of the transfusion of hydroxyethyl starch (HES) cryopreserved red blood cells (RBC) compared with the transfusion of liquid-stored RBC in patients undergoing major orthopedic or urologic surgery. Thirty-six patients donated autologous blood 35 +/- 6 days before elective surgery. Only the first of 3.5 +/- 1.3 donated units of RBC was randomly assigned to be stored in the liquid state at 4 degrees C in phosphate/adenine/guanosine/glucose/saline-Mannitol or frozen below -130 degrees C by means of liquid nitrogen after the addition of HES (molecular weight 200,000 Dalton, degree of substitution 0.5, final concentration 11.5% wt/wt) as a cryoprotectant. After induction of anesthesia, patients donated 900 mL of autologous blood before they received one unit of liquid-stored RBC in Group 1. In Group 2, one unit of cryopreserved autologous RBC was transfused after removal of the cryoprotectant HES. In Group 3, patients received one unit of cryopreserved RBC without any manipulation after thawing. Patients in Groups 1 and 2 received additional 500 mL of 10% HES. Hemodynamic variables, arterial blood gases, plasma hemoglobin, and arterial lactate concentrations were measured after the induction of anesthesia, after hemodilution, and at 10-min intervals after transfusion of the respective RBC concentrate over a period of 40 min. Skeletal muscle tissue oxygen tension was measured in the quadriceps muscle using an automatically stepwise-driven oxygen partial pressure electrode. We found no differences among groups concerning demographics, arterial blood gas values, and lactate concentrations and observed no adverse reactions after transfusion of the conventionally stored or cryopreserved RBC. Hemodynamic variables did not differ among groups, with the exception of an increased mean arterial blood pressure after the transfusion of cryopreserved unwashed RBC. In all groups, the skeletal muscle tissue oxygen tension remained constant after hemodilution and increased after transfusion of either washed or unwashed cryopreserved RBC. Although the free plasma hemoglobin concentration remained constant after the transfusion of liquid-stored RBC (26 +/- 8 mg/dL), the plasma hemoglobin concentration increased twofold after the transfusion of cryopreserved washed RBC (60 +/- 12 mg/dL) and threefold after transfusion of cryopreserved unwashed RBC (98 +/- 20 mg/dL). The authors conclude that transfusion of one unit of RBC after cryopreservation with HES is safe and well tolerated by patients. Intravascular volume replacement and skeletal muscle oxygenation characteristics by erythrocytes did not differ between liquid-stored and cryopreserved RBC. IMPLICATIONS: This study examined whether a colloid should be used to store blood. Our data suggest that the transfusion of one unit of red blood cells after cryopreservation with hydroxyethyl starch is safe and well tolerated by patients. The effects of intravascular volume replacement and skeletal muscle oxygenation provided by red blood cells after liquid storage or cryopreservation were not different.

Adult↗
PubMed1997

Cryopreservation of peripheral blood progenitor cells: characteristics of suitable techniques.

The influence of 6 different cooling rates (1.4, 5, 10, 15, 20, 160 K/min) and 5 different compositions of the suspensions to be frozen (% DMSO/% HES: 10/0, 7.5/2.5, 5/6, 2.5/7.5, 0/10) were investigated in 30 aliquots from each of 12 peripheral blood progenitor cells (PBSC) products which had been obtained by leukapheresis from 11 patients with hematological malignancies and solid tumors and 1 healthy donor treated with 5-24 micrograms/kg body weight/day granulocyte colony stimulating factor (G-CSF) over 5 days. The MNC concentration in the products obtained amounted to 4.51 +/- 1.55 x 10(7)/ml (mean +/- SEM), range: 2.10-7.65 x 10(7)/ml). For freezing, cooling rates were generated by means of a liquid nitrogen(LN2)-operated, computer-controlled freezer, a mechanical -80 degrees C freezer, in the vapor phase over LN2, and by submerging into LN2. The statistical analysis of the results clearly indicates that optimum results compared with the prefreeze values for numerical recovery (80.6 +/- 20.1%, Coulter counter), viability (membrane integrity by Trypan blue exclusion 91.6 +/- 4.1%), and colony-forming potential (56.2 +/- 45.8% erythroid burst-forming units [BFU-E], 63.4 +/- 72.8% myeloid colonies [CFU-GM]) were achieved at a cooling rate of 1.4 K/min with 10% DMSO being present. The values obtained at a cooling rate of 5 K/min (-80 degrees C mechanical refrigerator) in the presence of 5% DMSO and 6% HES did not differ significantly (i.e., membrane integrity 91.8 +/- 3.9%, BFU-E 53.0 +/- 37.4%, CFU-GM 47.8 +/- 58.2%). At cooling rates above 5 K/min and DMSO concentrations lower than 5% (in spite of higher HES concentrations) there was a significant drop of CFU recovery (CFU-GM plus BFU-E) to almost 0%. In parallel, numerical recovery and viability dropped as well, but less pronounced, indicating that both methods are unsuitable for the prediction of CFU recovery when different freezing protocols are applied. We need at least 5% DMSO (in the presence of 6% HES) in spite of the undesirable histamine-releasing effect of this compound. The cooling rate is not critical in the range from 1 to 5 K/min and can easily be achieved by -80 degrees C freezers.

Blood Preservation↗
PubMed1996

Increase of mobilized CD34-positive peripheral blood progenitor cells in patients with Hodgkin's disease, non-Hodgkin's lymphoma, and cancer of the testis.

G-CSF (filgrastim) can effectively mobilize peripheral blood progenitor cells (PBPC) when administered during steady-state hematopoiesis. In this single center study, we compared the effectiveness of two different doses of G-CSF on the mobilization of peripheral blood stem cells in patients with Hodgkin's disease, non-Hodgkin's lymphoma, and cancer of the testis. A first group including 33 patients received 10 micrograms G-CSF/kg BW per day (group A), whereas a second group comprising 34 patients was treated with 24 (2 x 12) micrograms G-CSF/kg body weight (BW) per day (group B) prior to the leukapheresis. A significant difference (P = 0.015) in the total number of CD34+ cells between group A: 11.32 x 10(7) (range 0.34-110.2) and group B: 48.25 x 10(7) (range 1.33-447.4) has been observed in the first leukapheresis product. Moreover, the total number of CFU-GM increased significantly from 34.79 x 10(4) (range 1.07-300.9) to 147.69 x 10(4) (range 1.03- 1204.0) (P < 0.005), and the number of MNC increased from 1.35 x 10(10) (range 0.41-3.09) group A) to 2.93 x 10(10) (range 0.66-9.7) (group B) (P < 0.001). Comparable results were obtained in the second leukapheresis. Our data indicate, that the application of higher doses of G-CSF can significantly improve the effectiveness of mobilizing PBPC during steady-state conditions, and thereby considerably contribute to a safe and fast engraftment as well as a reduced number of leukapheresis procedures to achieve sufficient number of PBPC.

Adolescent↗
PubMed1996

[HLA typing of donor corneas with extended post mortem time].

In case of high-risk patients cornea transplantation should be carried out using HLA-matched donor corneas to minimize the risk of rejection. HLA typing using blood lymphocytes of the donor is impossible because of the long post-mortem times. Alternatively, HLA typing can be performed using retinal pigment epithelium (RPE). Nevertheless, this method is limited by increasing post-mortem times. The aim of the present study was the optimization of culture conditions for RPE cells isolated from donor eyes with long post-mortem times. The HLA type should be evaluated during an acceptable period of organ preservation of the corresponding cornea. In different steps the method for isolation of the cells was optimized and a growth medium for RPE cells was established. Various supplements, including uvea-conditioned medium, were assessed using growth assays. The optimization of culture conditions led to an increase in the estimation of the complete HLA I and HLA II antigens from 36% to 74%. The time needed for the typing-procedure could be reduced from 38 to 17 days (average). At present 30% of the donor tissue with long post-mortem times can be typed in less than 14 days.

Cell Division↗
PubMed1996

Rapid detection of IgG subclasses on DAT positive RBC membranes by flow cytometry (FC).

UNLABELLED: Red blood cell (RBC)-bound IgG can cause hemolysis resulting, e.g. in severe cases of autoimmune hemolytic anemias (AIHA) or in hemolytic disease of the newborn (HDN). Serologic detection and differentiation of these antibodies are often difficult in cases of low antibody titers. We investigated 36 cases of poly- and monospecific IgG-positive DATs by flow cytometry. The RBC samples were washed, diluted, and incubated with monoclonal antibodies directed against IgG1, IgG2, IgG3, and IgG4, respectively. Analysis was performed on a flow cytometer. Nine cases were negative for all 4 IgG subclasses, 8 cases were positive for IgG2, 5 for IgG1, 5 for IgG3, and 3 for all four subclasses. In 6 patients we found combinations of 2 or 3 subclasses (2 for IgG1 and IgG2, 1 for IgG1 and IgG3, 1 for IgG3 and IgG4, 1 for IgG1, IgG2 and IgG4, 1 for IgG1, IgG3 and IgG4). Serological differentiation revealed specific anti-bodies only in 3 cases (anti-Lea, anti-Leb, anti-P1). The type of IgG subclass and the amount of RBC-bound IgG is relevant for the degree of in vivo RBC destruction. Flow cytometry provides a rapid, highly sensitive, cost efficient, and specific tool for IgG detection, including subgroups. CONCLUSIONS: We therefore recommend flow cytometric analysis to be integrated into the serological decision process as an additional method for serological problem cases.

Adult↗
PubMed1996

Tantalum stents induce expression of GMP140 and GP53 on platelets after contact in an in vitro model.

Endovascular implantation of stents has become increasingly important to reduce acute complications and restenosis after percutaneous transluminal coronary angioplasty (PTCA). Nevertheless, the risk of stent related thrombosis still appears to be a limitation in this therapeutic approach. As platelets play a major role in thrombus formation, we examined platelets in an in vitro model by flow cytometry to assess the expression of activation-dependent epitopes caused by shear stress and contact to artificial surfaces of stents. Tantalum wire stents (n = 12) were placed in one of two parallel silicon tubing systems. Both systems were filled with citrated platelet-rich plasma (PRP) of healthy and drug-free volunteers. After recalcification, aliquots of PRP were drawn via a three-way faucet over a course of 10 min. For flow cytometric analysis monoclonal antibodies CD41a, CD42b, CD62p and CD63 were applied. Within 2 min after onset, the expression of CD62p (GMP140) and CD63 (GP53) increased in the tubing system with the stent. Over the course of 10 min, platelet activation progressively increased (CD62p p < 0.05 vs. control system without stent; CD63 p < 0.005 vs. control). Antigens CD41 a and CD42b did not show significant changes in both systems. Artificial surfaces and shear forces of stent meshes may contribute to the activation of platelets, thereby promoting the thrombotic potential. As an utmost sensitive diagnostic tool, flow cytometry may attribute to further improvement of material and design of stents.

Angioplasty, Balloon, Coronary↗
PubMed1996

Storage of peripheral blood stem cell samples alters flow cytometric CD34+ results.

With the use of monoclonal antibodies against the CD34 antigen, flow cytometry (FC) permits rapid assessment of the quality of hematopoietic grafts. We examined PBSC samples (n = 40) to investigate possible influences of storage time and temperature on FC results. After cytapheresis, a sample of the PBSC product was collected and divided into 4 aliquots. Immediate analysis was performed on one aliquot. The other 3 specimen were stored for a) 24 h at room temperature (RT, 20 +/- 2 degrees C), at room temperature with agitation and c) at +4 degrees C. For flow cytometric analyses, samples were labeled with two CD34 markers (HPCA-2, Becton Dickinson Corp. [BD], USA; QBend-10, Immunotech Corp. [IT], Germany). After 24 h CD34+ signals had decreased by 25.4% (BD) and 27.0% (IT) in average, when samples were stored at room temperature in comparison to the results obtained directly after cytapheresis (p < 0.05). At RT in combination with agitation, there was an increase in signal rates compared to baseline values, probably due to binding of CD34 antibodies to myeloid or non-viable cells (+ 9.2% [BD] and 11.2% [IT]). At a storage temperature of +4 degrees C, CD34+ events did not decrease significantly (-0.7% [BD] and -0.2% [IT]). Our data demonstrate that FC results may be influenced by temperature, agitation and storage time.

Adolescent↗
PubMed1996

[Elimination of hydroxyethyl starch after autologous retransfusion of cryopreserved erythrocytes].

In the case of the biodegradable cryoprotectant hydroxyethyl starch (HES) no deglycerolization process is required prior to the transfusion of frozen red blood cells (RBC). In a first study the elimination of an HES 200,000/0.62 from the plasma of 6 dogs was investigated by means of a novel HPLC-GFC method. 16% of the blood volume were replaced by autologous HES protected frozen/thawed RBC. In a second study the HES concentration in the plasma of 7 healthy volunteers was determined following the substitution of 8% of the blood volume, but a washing step has been performed to reduce the concentration of the cryoprotectant (HES 200,000/0.5). In a third study, however, this step was omitted. The elimination of the HES followed always first order kinetics. In the case of the transfusions without postthaw washing in dogs and humans, the initial plasma concentrations amounted to 2.11 +/- 0.15 g/dl and 0.75 +/- 0.26 g/dl, respectively. The corresponding value for the washed preparations was less than 0.03 g/dl. Within 4-5 h the concentrations dropped to less than 50% of the initial values. The 9-hour value was less than 35% (dogs), the 20-hour value about 15% (humans) of the initial concentration. As HES is primarily eliminated via the kidneys, within this period the concentrations of HES in the urine dropped from 4.3 +/- 2.11 g/dl to less than 0.03 g/dl (humans, no washing step). In conclusion, the elimination of the accompanying cryoprotectant HES was no problem in the concentrations applied. A simple washing step with isotonic saline, however, effectively reduced the concentration of the extracellular cryoprotectant HES far below critical levels.

Animals↗