An international study of the performance of sample collection from patients.

BACKGROUND AND OBJECTIVES: Collection of a blood sample from the correct patient is the first step in the process of safe transfusion. The aim of this international collaborative study was to assess the frequency of mislabelled and miscollected samples drawn for blood grouping. MATERIALS AND METHODS: Hospitals in 10 countries provided data on sample error rates during a period of at least 3 months, including the last quarter of 2001. Mislabelled samples were defined as those not meeting local criteria for acceptance by the laboratory. Miscollected samples [wrong-blood-in-tube (WBIT)] were defined as samples in which the blood group result differed from the result on file from prior testing. WBIT rates were corrected for the proportion of repeat samples and for undetectable errors occurring as a result of chance collection of blood from the wrong patient with the same ABO group. Participants also completed a questionnaire on current policies regarding sample collection. RESULTS: A total of 71 hospitals completed surveys describing policies related to sample collection. Sixty-two hospitals provided usable data on the frequency of mislabelled and miscollected samples. Mislabelled and miscollected samples were common. Based on results from over 690,000 samples, the median hospital performance resulted in a rate for mislabelling of 1 in every 165 samples (6.1 per 1000; interquartile range 1.2-17 per 1000). The presence of national patient identification systems in Sweden and Finland was associated with rates of miscollected samples that were too low to estimate. Outside these nations, miscollected samples demonstrating WBIT occurred at a median rate of 1 in every 1986 samples (0.5 per 1000; interquartile range <0.3-0.9 per 1000). There was great variation worldwide in the reported frequency of mislabelled samples, probably resulting from variation in policies for sample acceptance. Miscollected samples occurred at a more constant rate. CONCLUSIONS: The rate of mislabelled samples and miscollected samples is 1000-10,000-fold more frequent than the risk of viral infection. Rates of mislabelled samples and WBIT can be tracked as key indicators of performance of an important step in the clinical transfusion process. WBIT episodes represent important 'near-miss' errors. By providing baseline performance data for the collection of patient blood samples, this study may be useful in formulating future national standards of performance for sample collection from patients.

In vitro megakaryocyte expansion in patients with delayed platelet engraftment after autologous stem cell transplantation.

Increasing the number of megakaryocytic cells in stem cell transplants by ex vivo expansion culture may provide an approach to accelerate platelet engraftment after high-dose chemotherapy. However, it is unknown if a relationship exists between the expansion potential of progenitor cells and the time to platelet engraftment in vivo. Therefore, we questioned if those patients who potentially would benefit most from expanded cell supplements are able to generate megakaryocytic cells efficiently in vitro. The in vitro megakaryocyte proliferation was analyzed from 19 leukapheresis samples from a group of multiple myeloma patients who all showed rapid neutrophil engraftment, but varied from 7 to 115 days post-transplant to achieve platelet levels >20x10(9)/l. CD34+ cells were isolated and analyzed for their potential to form megakaryocytic colonies (CFU-Mk) in colony assays and megakaryocytic (CD61+) cells in suspension cultures. The frequency and size of CFU-Mk and the expansion potential of CD61+ cells varied eightfold between individual patients. A similar range was found with CD34+ cells isolated from normal bone marrow (n=9). Rapid platelet engraftment occurred in patients receiving both high or low CFU-Mk doses and with high and low expansion of CD61+ cells. Four patients who experienced prolonged (>3 weeks) thrombocytopenia received low CFU-Mk doses, but the expansion potential was around median values or higher. Therefore, we conclude that the megakaryocyte proliferation is not impaired and that in vitro expansion could increase the number of megakaryocytic cells, although other factors could be more relevant in platelet engraftment in this group of patients.

Risk management: an important tool for improving quality.

Risk management implies that one has identified and analysed the root cause of the risk. In blood transfusion public and political opinion on the perceived risks are mainly related to product stigmatisation, to cognitive aspects of risk. Therefore this perception is affective, and has a negative connotation. In order to manage risk in an optimal manner, we need to understand how people think about it, and recognise that thoughts, feelings and behaviour are determined not only by psychological factors, but also by social, cultural and political influences. Perception of risk is always situated within a context, which may differ. Therefore people (i.e., public and political opinion) seem to act inconsistently from one risk context to another. Crucial for understanding the logic behind different risk perceptions is how people think about a hazard and organise information about it. The blood supply system has aspects that make it very vulnerable to crises of confidence, as the subject of blood can easily become stigmatised. The impact of the latter on the perception of blood transfusions and their recipients as well as the willingness of the public to accepttransfusions can be dramatic. Risk perception needs to be monitored in order to anticipate and adequately deal with public and political acceptance. We know that risk and stigmatisation are closely interconnected, and that the costs are likely to be high both for human health and for the maintenance of the healthcare system. Thus there is a global need to carefully monitor the safety of the blood supply systems and communicate risk information in a way that both informs people and builds up public and political confidence. It is therefore not sufficient to simply state that the blood supply is safe; it must also be made safe. So risk management becomes an integral part of quality management, as it deals with the public perception of the blood supply system and its respective elements: procurement and use.

Variable leukocyte composition of red blood cell concentrates prepared in top-bottom systems: possible implications for pre-transplant blood transfusion.

BACKGROUND AND OBJECTIVES: The beneficial effect of blood transfusion on kidney graft survival requires the presence of leukocytes in the transfusate, but a minimal dose has not been defined, nor has the role of individual leukocyte subsets been investigated. In the Netherlands, a standard pre-transplant blood transfusion consists of a buffy coat (BC)-depleted red blood cell concentrate (RBCC) containing a maximum of 1.2x10(9) residual leukocytes per unit. However, leukocyte subset composition is not standardized. MATERIALS AND METHODS: Using FACS analysis, this study compared the residual leukocyte composition of RBCCs produced by Compomat((R)) and Optipress((R)), two currently used top-bottom systems. RESULTS: While the total leukocyte content of the RBCCs was equivalent in both press types (0.5x10(9)), the percentage of mononuclear cells (lymphocytes and monocytes) was significantly higher in the Compomat as compared with the Optipress system (p < 0. 0001), resulting in significantly higher numbers of transfused T cells, B cells, HLA-DR-positive cells, NK cells and stem cells. CONCLUSIONS: The leukocyte composition of a pre-transplant blood transfusion depends on the BC depletion method used; this might differentially affect the tolerizing or immunizing potential of a pre-transplant blood transfusion.

Total quality management in blood transfusion.

Quality management is an ongoing development resulting in consistency products and services and ever increasing customer satisfaction. The ultimum is Total Quality Management. Quality systems and quality management in transfusion medicine have gained considerable attention since the outbreak of the AIDS epidemic. Where product orientation has long been applied through quality control, Good Manufacturing Practice (GMP) principles were introduced, shifting the developments in the direction of process orientation. Globally, and particularly in the more industrialised world people and system orientation has come along with the introduction of the ISO9001 concept. Harmonisation and a degree of uniformity are needed to implement a universally applicable Quality System and related Quality Management. Where the American Association of Blood Banks (AABB) is the professional organisation with the most extensive experience in quality systems in blood transfusion, the European Union and the Council of Europe now are in the process to design a quality system and management applicable to a larger variety of countries, based on a hybrid of current GMP and ISO9001 principles. The International Federation of Red Cross and Red Crescent Societies has developed a more universally to implement Quality Manual, with a pilot project in Honduras. It is recommendable to harmonise the various designs and bring the approaches under one common denominator.

Preparation of leukocyte-poor platelet concentrates via a short, hard spin of a pool of buffy coats.

BACKGROUND AND OBJECTIVES: A new method for the preparation of leukocyte-poor platelet concentrates was developed, based on a short, hard spin of a pool of 5 buffy coats (BCs) combined with automated collection of the platelets. MATERIALS AND METHODS: The characteristics of platelet concentrates (PCs) were studied as a function of the total g force applied to a pool of 5 BCs. Pools of BCs were centrifuged for 1 min with a total g force ranging from about 3,300 to 5,000 gmin (n = 7-9 per applied g force). Deceleration took place without the means of a brake. The total centrifugation time was about 11 min. The platelet-rich plasma (PRP) fraction above the cell layer was separated by an automated component preparation device. RESULTS: A short, hard spin with a total g force of between 3,400 and 4,600 gmin resulted in PCs that contained on average more than 290x10(9) platelets and less than 5x10(6) leukocytes without the use of a leukocyte filter, provided that the transfer of PRP was electronically checked and terminated. The cell concentrations in the PCs are a function of the total g force. Both the platelet and leukocyte levels in the concentrate decreased with an increase in the total g force applied to the pool. CONCLUSION: The preparation of PCs via a short hard, spin of BCs, combined with automated collection of the PRP, may be an alternative method for the preparation of leukocyte-poor PCs.

Autologous stem cell transplantation in multiple myeloma after VAD and EDAP courses: a high incidence of oligoclonal serum Igs post transplantation.

Thirty-seven patients with multiple myeloma (stage II and III, 65% increased beta2-microglobulin level) were prospectively treated with a median of 3.7 VAD courses (range 2-8) followed by cyclophosphamide (6 g/m2) in conjunction with G-CSF (5 microg/kg filgrastrim (n = 14), or 3.5 microg/kg lenograstrim (n = 22)), and peripheral stem cell (PSC) isolation. After regeneration this was followed by one EDAP course and high-dose melphalan (HDM, 200 mg/m2) in combination with re-infusion of PSC. Adequate stem cell mobilization was obtained with both G-CSF regimens. A median of 41x10(6) CD34+ cells/kg (range 4.5-161) was collected in a median of 1.6 leukapheresis procedures following filgrastrim (n = 14) and 24x10(6) CD34+ cells/kg (range 2. 3-80) in a median of 1.7 leukapheresis procedures following lenograstrim (n = 22) which indicated no significant difference (P = 0.24) between both G-CSF regimens. A rapid hematological recovery was obtained after HDM with reinfusion of a median of 9.3x10(6) CD34+ cells/kg. After the total courses the overall response was 84% with a complete remission rate of 30%. Currently the median overall survival is 44.0 months (95% CI 38.9-49.1) with a median follow-up of 33 months (range 3-51) and a median event-free survival of 29.0 months (95% CI 25.3-32.7) (n = 33). Post transplantation a high incidence of oligloclonal serum immunoglobulins (Igs) was observed. In 73% of the patients new oligoclonal or monoclonal serum bands were noticed 3 months post transplantation. IgG-lambda and IgG-kappa bands predominated. In 48% of the cases the oligoclonal Igs disappeared after a median follow-up of 22 months (range 8-36), whereas in 52% of the cases the oligoclonal Igs persisted with a median follow-up of 31 months (range 21-45), which did not correlate with a significant difference in overall, and event-free survival between both subgroups.

Immune effects of blood transfusion.

Blood is a complicated tissue that has been routinely applied over the past century. Since the recognition of the principles of species specificity (1818) and major compatibility for red cell antigens (1900), considerable attention has been given in the second half of this century to leukocyte-determined immune effects following transfusion. Most reactions are febrile and nonhemolytic and show, in limited situations, true relation to immune activity of transfused white blood cells. The complete picture is not yet finished; however, the more important immune effects are stimulation and modulation, tolerance and suppression. Here most work done is ex vivo and in animal experiments. Little clinical evidence has been obtained or assessment done. Most publications therefore relate to laboratory work, extrapolating results to the bedside. The most interesting publication over the review period with regard to the core immune effects of blood transfusion relates to measured concentrations of active, soluble molecules like HLA class I and II and Fas ligand molecules and to their in vitro immunomodulating effect as well as effects on induction of apoptosis. Stored red cell preparations show an increase in soluble molecules and death of viable leukocytes. Platelet preparations show similar phenomena, although information on numbers of white blood cells and storage conditions are lacking. The authors propose selective use of these aged components in specific clinical settings to allow immune suppression, induction of cell anergy, and apoptosis, whereas fresher products not showing increased levels of soluble molecules could be applied in clinical settings in which immunosuppression is not wanted. No reference is found to scientifically support and substantiate the global universal leukodepletion movement.

Effects of peripheral stem cell or bone marrow reinfusion on peripheral serotonin metabolism.

Reinfusion of autologous hematopoietic peripheral blood stem cells (PBSC) or bone marrow is often accompanied by flushing, dyspnea, abdominal cramping, nausea and diarrhea. These symptoms and the observation that they can be prevented by ondansetron, a selective 5-HT3 receptor antagonist, led to the assumption that these side-effects are due to infusion of free serotonin during the reinfusion of PBSC or bone marrow. Twenty-five patients with solid tumors received, after myeloblative chemotherapy, a total of 30 reinfusions of PBSC and/or bone marrow. In 17 patients, serotonin levels in the bags containing the PBSC were measured. In all patients, platelet serotonin levels were determined before and 1 h post-reinfusion. In addition, before and 24 h after reinfusion urine was collected for determination of 5-hydroxyindole acetic acid (5-HIAA) and serotonin concentrations. Mean (+/- s.d.) total serotonin concentration in the bags was 2404 +/- 1555 nmol/l. Mean total volume reinfused was 471 +/- 185 ml. After reinfusion, the mean (+/- s.d.) levels of serotonin in platelets in patients increased from 3.2 +/- 1.4 nm/10(9) at baseline to 3.8 +/- 2.0 nm/10(9) (P = 0.02). Neither 24 h urinary 5-HIAA nor serotonin levels were affected. These results indicate that reinfusion of PBSC or bone marrow is accompanied by substantial infusion of free serotonin, which might explain the observed side-effects and justify the use of 5-HT3 receptor antagonists as pre- medication for this procedure.

Saponin, an inhibitory agent of carbon dioxide production by white cells: its use in the microbiologic examination of blood components in an automated bacterial culture system.

BACKGROUND: Blood components with a white cell count > 100 x 10(9) per L may cause false-positive results when the BacT/Alert system is used for the microbiologic examination. The effects of different concentrations of saponin on bacterial growth and on carbon dioxide production by blood fractions with a high white cell count, in particular peripheral blood progenitor cells and buffy coats, were investigated. STUDY DESIGN AND METHODS: The effect of saponin on carbon dioxide production was studied by adding different fractions of white cell-rich material (buffy coat or leukapheresis material) to BacT/Alert culture bottles with or without saponin and incubating these bottles. Five bacterial strains were used to inoculate the culture bottles at four levels ranging from about 1 colony-forming unit per mL to about 10(3) colony-forming units per mL. Aerobic and anaerobic bottles with and without saponin were used. RESULTS: It was demonstrated that the addition of 0.5 percent saponin to BacT/Alert culture bottles effectively inhibited carbon dioxide production, without affecting bacterial growth. CONCLUSION: Saponin at a concentration of 0.5 percent is a valuable additive to BacT/Alert culture media because it prevents false-positive results in the examination of white cell-rich blood components.

[Subspecialty blood transfusion medicine]. / Aandachtsgebied bloedtransfusiegeneeskunde.

The unification of Europe, the related principle of self-sufficiency and the prevention of blood banks turning into bureaucratic institutes that lose connection with bedside medicine underscores the need for training in transfusion medicine and its international organization. In most European countries transfusion medicine is now recognized as a specialty in its own right. In the Netherlands it was decided to recognize transfusion medicine as subspecialty of Internal Medicine. This new initiative led to a training programme of 6 years in all, of which the last 18 months are devoted to transfusion medicine exclusively. The importance of continuous education and practice in both fields is recognized.

Prevalence of delta F508 cystic fibrosis carriers in The Netherlands: logistic regression on sex, age, region of residence and number of offspring.

An average cystic fibrosis (CF) carrier frequency of 1 in 25 in Europe is cited in numerous reports, although a great variability in estimated prevalences has been found in different European populations. The estimates of these frequencies were based on numbers of CF patients before identification of the gene in 1989. Here we report the results of a study to determine the carrier frequency of the delta F508 mutation in The Netherlands by analyzing mouthwashes and matched blood samples from 11654 blood donors all over the country. We analyzed possible relationships between a number of theoretically explanatory variables and the delta F508 carrier frequency by means of univariate and multivariate logistic regression. These variables were: distance of the blood banks from the northeastern part of the country (distance); whether the blood donors knew that we were looking for a CF mutation; sex and age of the donor; and number of children of the donor (family size). We detected a delta F508 carrier frequency of 1 in 42 (95% CI 1/37-1/47) in The Netherlands. If we assume that the relative frequency of the delta F508 mutation among carriers and patients is comparable in The Netherlands, this gives an estimated overall CF carrier frequency of 1 in 32 (95% CI 1/28-1/36), significantly less than 1 in 25. The univariate logistic regression analysis of the effects of the explanatory variables on the carrier frequency revealed no significant relationships, except for an increase in carrier frequency with increasing distance from the northeastern region. In the multivariate analysis with all five independent variables, distance, age and family size were significantly related to the carrier frequency, but sex and CF information were not. There was a significant interaction between age and family size. In our final model, distance, age and family size were positively related to the carrier frequency, while the interaction of age with family size showed a negative relation. These results confirm that there is a gradient in gene frequency with low frequencies in the northeastern part of the country and high frequencies in the southern part. They also suggest a relation of age and family size with carrier frequency. This relation, however, is too complex to be explained by heterozygote advantage.

[The use of blood and blood products in total hip arthroplasty in 31 European hospitals, 1990/'91]. / Het gebruik van bloed en bloedproducten bij totale heupartroplastieken in 31 Europese ziekenhuizen, 1990/'91.

OBJECTIVE: To compare the use of blood products and artificial colloids during total hip arthroplasty in European hospitals. DESIGN: Descriptive. SETTING: Europe. METHODS: During the period October 1990-October 1991 transfusion data were obtained about patients who underwent a planned total hip replacement for the first time. The investigations were performed in 31 teaching hospitals in ten countries of the EC, as a part of the ¿Safe and good use of blood in surgery' (SANGUIS)-project. RESULTS: Red cells were ordered preoperatively in 97.4% of the 1647 cases and transfused in 81% (ranges among hospitals: 29-100). Hospitals in the Mediterranean area used more auto-transfusion than those in Central and Northern Europe. Plasma was transfused in 6% of the patients, predominantly in hospitals in southern European countries. Albumin was used especially in Central and Northern European countries. The reasons for red cell transfusion were stated in the medical records in 23% of the cases, for plasma transfusions in 7% and for albumin in 1%. Averaged transfusion-related costs were 192 ecu per patient (ranges per hospital: 60-383 ecu). CONCLUSION: Differences between European hospitals in the use of blood products for total hip arthroplasty are considerable.

Validation of the determination of deltaF508 mutations of the cystic fibrosis gene in over 11 000 mouthwashes.

Mouthwashes can be used as a DNA resource for mutation detection and, because collection and DNA isolation is simple and cheap, they could in particular, be used for large numbers of samples. To determine the failure rate (the proportion of mouth samples in which no PCR product was obtained) and the specificity of buccal epithelial cell mutation detection in large numbers of samples, we collected mouthwashes and blood samples from 11 413 blood donors and tested the mouthwashes for the deltaF508 mutation, which has an estimated frequency Of 75% among cystic fibrosis chromosomes in The Netherlands. Blood samples were tested for the deltaF508 mutations only if the mutation was identified in the mouthwash or in the case of a failure to obtain PCR products. The sensitivity of the test was determined in mouthwashes of 75 deltaF508 carriers known from earlier family studies. These samples were offered blindly between the mouthwashes of the blood donors. Both specificity and sensitivity of the mouthwash procedure were 100%. The overall failure rate was 5.6%. This large figure was caused mainly by insufficient rinsing of the mouth in one particular blood bank. Exclusion of the results of this blood bank reduced the failure rate to 1.8%. Our results also confirmed that for a large number of samples the mouthwash procedure is suitable for mutation detection and, with proper instructions, can be used in community screening.

Factor VIII:C assay: influence of buffer components used in immunoaffinity chromatography for purification of factor VIII/von Willebrand factor.

A monoclonal antibody purified factor VIII concentrate containing FVIII/vWF complex has been assayed by one-stage clotting (CA) and chromogenic substrate (CSA) methods. The influences of potassium iodide (KI) and albumin in combination with predilution buffers, standards and storage of samples have been examined. These components are compared for their effect on FVIII potencies in final product and in-process controls. FVIII:C purified by immunoaffinity chromatography can not be measured reliably by CA or CSA, because of KI which interfere on the assay. Overall yield of FVIII, efficiency of IAC step and purity of FVIII can be determined by assaying the desalted samples.

Intensive chemotherapy with autologous bone marrow transfusion as primary treatment in women with breast cancer and more than five involved axillary lymph nodes.

Patients with breast cancer and a high number of involved axillary lymph nodes have a poor prognosis, despite adjuvant chemotherapy. The 5-year disease-free survival (DFS) in this group amounts to 30-40% and the 10-year DFS is only 15-20%. Therefore, new treatment modalities are being sought for this group of patients. The aim of the present study was the evaluation of the efficacy of high-dose chemotherapy combined with autologous bone marrow support. 24 patients with a primary breast cancer with more than five involved axillary lymph nodes received, after surgery, six courses of induction chemotherapy followed by ablative chemotherapy and reinfusion of autologous bone marrow. All patients were premenopausal or less than 2 years postmenopausal. Induction chemotherapy consisted of methotrexate (MTX) 1.5 g/m2 intravenous (i.v.) and 5-fluorouracil (5-FU) 1.5 g/m2 i.v. on day 1, prednisone 40 mg/m2 orally on days 2-14, doxorubicin 50 mg/m2 i.v. and vincristine 1 mg/m2 i.v. on day 14. Courses were repeated six times every 4 weeks. 10 patients received cyclophosphamide 7 g/m2 i.v. and etoposide 1.5 g/m2 i.v. as intensive regimen, in 14 patients this comprised mitoxantrone 50 mg/m2 i.v. and thiotepa 800 mg/m2 i.v. Reinfusion of autologous marrow followed on day 7. Finally, patients received locoregional radiotherapy for extranodal disease and tamoxifen 40 mg daily orally over a period of 2 years. The median age of patients was 42 years, range 29-54. The median number of involved nodes was 10. During induction therapy, fever requiring i.v. antibiotics occurred in 4% of 144 courses, 14% of patients suffered from mucositis WHO grade 2-3, and the other patients had mucositis grade 1. During the ablative chemotherapy, 1 patient died, 6 developed septicaemia, 5 showed mucositis grade 3-4 and the other patients had mucositis grade 1 or 2. In the follow-up, 1 patient died from acute cardiac failure. Reversible radiation-induced pneumonitis occurred in 7 out of 14 irradiated patients; symptoms started directly following radiotherapy and lasted for several weeks, but disappeared in due course. During follow-up, 2 patients with six and > 10 positive nodes, respectively, have relapsed after 18 and 36 months, both in the cyclophosphamide/etoposide regimen. Median observation is 3 years, disease-free survival at 5 years is predicted to be 84%. Intensive treatment in these patients with high numbers of involved axillary lymph nodes is a toxic regimen, but may improve the chance of surviving free of disease.

Recovery of fibrinogen in cryoprecipitate pasteurized in the presence of sucrose and glycine.

The effect of sucrose (60% w/w) and 1 M glycine as thermal stabilizers for fibrinogen in cryoprecipitate was studied. Sucrose (9.2 g) and glycine (0.9 g) were dissolved in 6 g of cryoprecipitate and the solution was pasteurized at 60 degrees C for 10 h. The preparation was then dialyzed for 20 h in phosphate buffered saline (PBS), lyophilized, stored for one week at -40 degrees C and resuspended in distilled water. The recovery of total proteins and fibrinogen in the final product averaged 66.4 +/- 4.1% and 43.8 +/- 6.4% of the initial contents, respectively (mean +/- SEM, N = 9). The pasteurization of cryoprecipitate in the presence of PBS (control experiments) produced extensive precipitation, which is characteristic of protein denaturation. Thus, this method partially protected fibrinogen and other proteins in cryoprecipitate from inactivation by prolonged exposure to heat during pasteurization.

Recovery of fibrinogen in cryoprecipitate pasteurized in the presence of sucrose and glycine

The effect of sucrose (60 per cent w/w) and 1 M glycine as thermal stabilizers for fibrinogen in cryoprecipitate was studied. Sucrose (9.2 g) and glycine (0.9 g) were dissolved in 6 g of cryoprecipitate and the solution was pasteurized at 60 degrees C for 10 h. The preparation was then dialyzed for 20 h in phosphate buffered saline (PBS), lyophilized, stored for one week at -40 degrees C and resuspended in distilled water. The recovery of total proteins and fibrinogen in the final product averaged 66.4 +/- 4.1 per cent and 43.8 +/- 6.4 per cent of the initial contents, respectively (mean +/- SEM, N = 9). The pasteurization of cryoprecipitate in the presence of PBS (control experiments) produced extensive precipitation, which is characteristic of protein denaturation. Thus, this method partially protected fibrinogen and other proteins in cryoprecipitate from inactivation by prolonged exposure to heat during pasteurization