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.

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.

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.

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.

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.

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.

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.

Detection of HLA antibodies by an enzyme-linked photometric immune phagocytosis inhibition test.

The immune phagocytosis inhibition test (IPI) has been described as a sensitive method for the detection of cytotoxic and non-cytotoxic HLA antibodies. We performed a photometric IPI and compared this technique with the conventional microscopic IPI. In further investigations we used the photometric IPI in comparison with the lymphocytotoxicity test (LCT) for the detection of HLA antibodies. The photometric IPI showed a high correlation to the microscopic IPI. In tests with different known HLA antibodies the photometric IPI reached a sensitivity of 85.1% versus 80.5% in the LCT, and a specificity of 92.3 versus 100% in the LCT. Diluted patient sera showed a higher sensitivity in the photometric IPI. We conclude that the photometric IPI can be used as a convenient and sensitive technique for the detection of HLA antibodies.

[Comparison of leukocyte filters for erythrocyte concentration]. / Vergleich von Leukozytenfiltern für Erythrozytenkonzentrate.

UNLABELLED: We compared the following leukocyte filter blood systems (BBS): Biotrans Bio R01 Plus, Diamed Sepacell RS-200, Pall BPF4 (log 4 filters) and Biotrans Bio R01 (log 3 filter). We evaluated the leukocyte removal, handling, the increase of free hemoglobin and the loss of red blood cells (RBC) by filtration. A total of 54 buffy-coat-reduced RBCs were suspended in 200 ml PAGGS-M, stored at +4 degrees C for 24 h and filtered subsequently. Samples were taken before storage, immediately before and after filtration to measure total and free Hb and the white blood cell (WBC) contamination. RESULTS: (1) All log 4 filters showed a comparable reduction of the WBC content per RBC concentrate. (2) Differences were observed in total filtration time and loss of RBCs: Biotrans Bio R01 Plus and Pall BPF4 yielded significantly better results than Diamed Sepacell RS-200. (3) The log 3 filter did not meet the criteria of < 5 x 10(6) WBCs per filtered unit of RBCs (as recommended by the Council of Europe).

[Measurement of alloreactive anti-recipient helper T-cell precursors after allogenic bone marrow transplantation]. / Messung der alloreaktiven Anti-Rezipienten-Helfer-T-Zell-Vorstufen nach allogener Knochenmarktransplantation.

Acute graft-versus-host disease (GvHD) is a severe complication after allogenous bone marrow transplantation (BMT). The compatibility of major histocompatibility complex antigens (MHC) is the strongest stimulus for GvHD, which also occurs in patients with a genetically MHC-identical sibling donor. In such cases it would be helpful to recognize anti-recipient (interleukin-2-producing) T-lymphocyte precursors to detect a minor histocompatibility antigen (mH), which escapes from typing methods. To quantitate the alloreactive immune response initiating acute GvHD, we established the helper T-lymphocyte precursor test (HTL-p) as described by Theobald et al. in 'GvHD direction', using a limiting dilution assay. Serial dilutions of the donor peripheral blood mononuclear cells (PBMCs) were cultured for 14 days with constant numbers of stimulator PBMCs from the recipient, followed by an unspecific restimulation step with phytohemagglutinin (PHA) or specific restimulation with EBV-transformed blast cells from the recipient. The Il-2 production of specific T cells was assessed by addition of CTLL-16 cells, whose proliferation was measured by an ELISA. We suppose that the HTL-p test is a good tool for measuring the number of anti-recipient T-lymphocyte precursors as a predictive value for the intensity of GvHD.

[Cryopreservation of erythrocytes using hydroxyethyl starch: in vivo results of an autologous retransfusion model in humans]. / Kryokonservierung von Erythrozyten mit Hydroxyethylstärke: In-vivo-Ergebnisse im autologen Retransfusionsmodell beim Menschen.

The cryopreservation of human red blood cells (RBC) can be greatly beneficial in certain situations such as for rare blood groups, problems due to multiple antibodies, and as an interim aid during temporary shortages. Additionally, cryopreserved erythrocytes may be useful in cases of civil or military disasters. Frozen/thawed autologous RBC are of particular interest as a supplement to liquid storage for elective operations (e.g. orthopedics, vascular and transplantation surgery) to extend the preoperative collection period, which is otherwise limited to 7 weeks. In this study using 7 healthy volunteers, 500 ml of whole blood was replaced by a suspension of cryopreserved autologous RBC [2 aliquots of 216 ml each, hematocrit (HCT) 43 +/- 2% (v/v), HES (hydroxyethyl starch) concentration 11.5% (w/w)]. No washing step was performed after thawing. Viability of the red cells after thawing in terms of saline stability reached 91.9 +/- 0.7% (n = 4). In all 7 cases the frozen/ thawed autologous RBC were tolerated very well. No adverse reactions could be detected. A slight posttransfusional leukocytosis as well as a moderate increase in LDH and bilirubin were observed, but these effects disappeared within 20 h. The concentrations of platelets, electrolytes, urea, protein and creatinine within their physiological ranges. The activities of the liver enzymes and the coagulation parameters investigated remained unchanged. Initially the level of free plasma hemoglobin increased by a factor of 2. Then it decreased within 20 h, accompanied by a restoration of haptoglobin. More than 85% of the HES was eliminated from the plasma within the 1st day.

[HIV antigen test of blood donors]. / HIV-Antigen-Test bei Blutspendern.

The importance of an optimal diagnostic standard for the prevention of transfusion-associated AIDS (TAA) was recently enforced by a decision of our Supreme Court. Although only 18 transfusion-associated HIV infections following transfusion of approximately 30 million blood units were officially reported since 1985, HIV-1 p24 antigen testing of blood donors was introduced in Hamburg and by the Bavarian Red Cross in summer 1992. So far, no p24 antigen-positive, HIV antibody-negative blood donor has been found (May 1993).

[Transfusion-induced virus infections: how great is the risk?]. / Transfusionsbedingte Virusinfektionen: Wie gross ist das Restrisiko?

The risk of infection by blood transfusions contaminated with the human immunodeficiency virus (HIV) and/or the hepatitis C virus (HCV) was dramatically reduced after the introduction of blood donor screening using specific and sensitive 2nd- or 3rd-generation enzyme immunoassays for virus antibody detection. In addition, donors selection provides the greatest safety. The strategy for safe blood supply includes medical examination and self-exclusion of donors. For example, in German blood donors, the current detection rate of HIV is between 1.36 and 1.82 confirmed positive results per 100,000 blood donations. For hepatitis C the rate of anti-HCV-positive donors is between 0.27 and 0.49%. The overall risk of HIV infection ranges from 1 in 500,000 to 1 in 3 million and that of a transfusion-associated HCV infection from 1 in 20,000 to 1 in 40,000 per transfused blood unit. From the observed virus load among German blood donors, the transfusion-associated mortality was calculated to be 1 in 260,000 per transfused blood unit. Implications are discussed resulting from this low risk of HIV and/or HCV infection by blood transfusions.

A rapid and sensitive immunoassay for antibodies against alloantigens on human platelet glycoproteins (BIPA).

A rapid and sensitive enzyme immunoassay has been developed for the detection and specification of alloantibodies against human platelet antigens (HPA) on platelet membrane glycoproteins. Human platelet glycoprotein complexes were coupled by monoclonal antibodies to immunomagnetic beads with sheep anti-mouse IgG directed antibodies. These beads served as solid-phase target antigens for human alloantibodies in a modified MAIPA assay. This test provided high sensitivity, permitting the identification of antibodies against membrane receptors and required only 1 h of assay time. The specificity and sensitivity of this assay were evaluated in serum samples from polytransfused patients and compared with standard immunoassays employing antigen coated immunowells.

[Initial experiences with HCV detection and confirmation tests of the 3rd generation]. / Erste Erfahrungen mit Anti-HCV-Such- und Bestätigungstests der 3. Generation.

We compared the reactivity of a new 3rd-generation anti-HCV enzyme immunoassay (EIA) using an additional, nonstructural antigen from the NS5 region, with a currently used 2nd-generation anti-HCV EIA, by investigating 419 serum specimens from healthy blood donors with normal liver function and 256 samples from patients at risk for hepatitis C infection; 2 of 419 blood donors and 1 of 256 patients were reactive to the NS5 region antigens only (later confirmed by Western blot and RP-2 RIBA), whereas 1 patient was reactive with the 2nd-generation EIA only (antibodies against c33c). We further evaluated the recently developed anti-HCV RP-2 RIBA by testing samples, that were indeterminate by the 2nd-generation RIBA. Nine of 15 patients were identified as reactive by RP-2 RIBA, 6 remained indeterminate. Of the 6 blood donor samples tested, 2 were found reactive, 1 remained indeterminate, and 3 were negative. These test results suggest a higher sensitivity and specificity of the RP-2 RIBA, whereas the relevance of an isolated NS5 EIA reactivity remains to be established.

[Prevalence of hepatitis C virus in poly-transfused patients with hematologic and oncologic diseases]. / Prävalenz des Hepatitis-C-Virus bei polytransfundierten Patienten mit hämatologischen und onkologischen Erkrankungen.

Patients with hematological and oncological diseases often require intensive, supportive hematotherapy due to the underlying disease or chemo-/radiotherapy. As a consequence, they had an increased transfusion-associated risk of hepatitis C virus infections (non-A, non-B-posttransfusion hepatitis) until 1989-1990, when specific and sensitive HCV antibody tests became available. This is confirmed by our study of 'first' and 'second' generation (1.0 and 2.0) anti-HCV EIAs against structural and non-structural (NS) antigen-determinants. Ten of 101 patients (10.9%) were anti-HCV positive in 2.0 tests. HCV antibodies were detected more often by 2.0 EIAs and the new HCV-immunoblot (4-RIBA), than by 1.0 EIAs. In this respect, the patients' serological HCV profile differs from that of healthy blood donors, which display a prevalence of NS-antibodies.

[Hepatitis C virus antibodies in patients and the normal population]. / HCV-Antikörper bei Patienten und in der Normalbevölkerung.

With regard to the controversial issue of a reduction of transfusion-associated infections by non-remunerated donations, epidemiological data on the prevalence of HIV-1, HIV-2 and hepatitis C virus (HCV) are of particular interest in our country. We investigated four sample categories: (1) healthy employees and workers from Hamburg; (2) hemodialysis patients; (3) hemato-oncological patients, and (4) blood donors, and tried to differentiate between the three disputed vectors of community-acquired (sexually or pregnancy-transmitted), nosocomial and transfusion/transplantation-associated HCV infections. We conclude from our results that--prior to the implementation of blood screening--our carefully selected 'paid blood donors' conferred no higher HCV risks than the general (working) population (0.66 vs. 0.82% HCV antibody prevalence). Besides transfusions/transplantations, significant nosocomial risks apparently exist in hemodialysis units (21.0 vs. 9.5% HCV seroprevalence in polytransfused patients). Preventive measures, e.g. separate dialysis machines for HCV-positive patients, seem to be advisable.