Reproducible scoring of CFU-GM and BFU-E grown in collagen-based semisolid medium after a short (3 h) training.

Colony counting remains an important source of variation in colony-forming unit-granulocyte-macrophage (CFU-GM) assays performed in methylcellulose or agar. We studied the reliability of colony scoring of CFU-GM assays carried out with collagen, a matrix that allows gel collection on glass slides and in situ cellular morphology. Fourteen slides were exchanged among laboratories, and two rounds of colony (CFU-GM and burst-forming units-erythrocyte [BFU-E]) counting were performed by 11 (first counting), then 8 (second counting) different laboratories, the majority of which had no previous experience of collagen gel cultures and reading. Two-way analysis of variance (ANOVA) of the first round of colony counting showed significant differences among centers in CFU-GM counts (p = 0.023) but not in BFU-E counts (p = 0.163). Coefficients of variation for the 14 slides ranged from 22% to 50% (median 28%) for CFU-GM counts and from 12% to 74% (median 23%) for BFU-E counts. After a 3 h session of collective colony reading attended by members of 8 laboratories, a second round of colony counting was performed. This time, ANOVA showed no significant difference among centers for CFU-GM (p = 0.533) and BFU-E (p = 0.328) counts, and coefficients of variation were significantly improved, with medians of 17% for CFU-GM counts and 20% for BFU-E counts. In addition, when data from the second round of readings were analyzed without the 2 centers counting consistently low (center 8) or consistently high (center 5), variance among centers was further improved for both CFU-GM (p = 0.798) and BFU-E (p = 0.619). In summary, this study shows for the first time that reproducible BFU-E and CFU-GM scoring can be achieved using collagen-based semisolid medium (now commercially available) as long as adequate training in colony identification is provided.

Transfusion-associated or nosocomial hepatitis G virus infection in patients undergoing surgery.

BACKGROUND: Despite blood donor screening, there are still cases of transfusion-associated hepatitis. From 1988 to 1992, a prospective study was conducted on the incidence of non-A, non-B posttransfusion hepatitis (PTH). STUDY DESIGN: The present investigation was designed to determine if transfusion recipients with PTH who are negative for hepatitis C virus (HCV) were positive for hepatitis G virus (HGV). Patients admitted for surgery who had normal liver tests and no transfusions during the previous 6 months were enrolled. Alanine amino transferase levels were determined monthly for 6 months after surgery and for 1 year in the case of PTH (defined as alanine aminotranferase twice the upper limit of normal in two consecutive assays). HGV RNA and E2 antibodies were tested for in samples from transfusion recipients with or without PTH and from nontransfused patients. RESULTS: Of the 308 blood recipients who were enrolled in the study, 21 (6.8%) had PTH. HGV RNA was detected at the onset of hepatitis in 3 patients with PTH (14%), 2 of whom were also anti-HCV and HCV RNA positive. One patient developed E2 antibodies without detectable HGV RNA. Three (10.7%) of 28 recipients of an allogeneic transfusion without PTH developed HGV infection. HGV RNA was also found in two nontransfused patients, which suggests nosocomial transmission of HGV. CONCLUSION: Some cases of PTH are associated with HGV; most cases of postoperative HGV infection are not associated with liver abnormalities; and most PTH cases are not associated with known hepatotropic viruses.

[Viral attenuation of labile blood products]. / Atténuation virale des produits sanguins labiles.

Viral inactivation is one of the possibilities to reduce the residual risk of blood products. It is now applied to all plasma derived products (PDP). Application of such techniques to labile blood products (LBP) is difficult for two main reasons: any method should inactivate cell-associated viruses and should avoid any injury of the cells constituting the active ingredient. Physical techniques may reduce the viral content of cellular BPL (leucodepletion, washing, gamma irradiation), but none of them is active enough to comply with the present requirements for efficacy. An important work has been dedicated to the development of virus photoinactivation techniques. They consist of the addition of a photoreagent followed by illumination at an appropriate wavelength which results in a photochemical reaction responsible for the viral inactivation. Treatment of platelet concentrates by psoralen derivatives and UV-A illumination significantly inactivate in vitro enveloped and naked viruses, free and cell-associated viruses and also sequences integrated in the viral genome. Recent progresses have led to these results without detectable functional alteration of platelets and mutagenicity. Viral inactivation of red blood cells yet did not reach the same level because hemoglobin does not allow the use of the photoreagent compounds applicable to platelet concentrates. Viral decontamination of fresh frozen plasma by solvent and detergent, active on enveloped viruses, has been used in France since 1992. Other techniques of comparable efficacy, have received an agreement in other countries. The research on viral inactivation of LBP could prove to be of great importance in the near future in bringing additional safety to patients not only for the residual viral risk but maybe also for the residual bacterial risk of LBP.

Collagen matrix: an attractive alternative to agar and methylcellulose for the culture of hematopoietic progenitors in autologous transplantation products.

Autografts using untreated or in vitro manipulated bone marrow and peripheral blood stem cells represent promising approaches to the treatment of malignant diseases. In this work, the collagen gel culture technique was compared with agar and methylcellulose for its capacity to permit the growth of human granulomonocytic (day 14 CFU-GM; collagen vs agar or MTC) or erythroblastic (day 7 CFU-E and day 14 BFU-E; collagen versus methylcellulose) colonies in autologous transplantation products. Our results show that the collagen culture system always gave as many or more colonies than the other techniques. It also allowed harvesting of gels onto glass slides and subsequent May-Grünwald-Giemsa, cytochemical or immunocytochemical staining. We suggest that the collagen assay represents an interesting alternative to the widely used agar or methylcellulose systems for the culture of hematopoietic progenitors because of the equal or higher number of colonies detected, the easy phenotypical identification of colonies in stained gels, and the ability to store high-quality documentation. This technique is particularly attractive for use in the quality control of autologous bone marrow transplantation procedures.

Seropositivity to hepatitis C virus in Tunisian haemodialysis patients.

The prevalence of anti-hepatitis C virus (anti-HCV) antibodies and of hepatitis B markers (HBs antigen, anti-hepatitis B core antigen) was assessed in 63 haemodialysis patients from the Tunisian Sahel. As measured by second generation ELISA assays (Ortho and Organon), the frequency of anti-HCV antibodies was 42% (27/63), while 4 patients (6.3%) were HBs Ag positive and 30 (47.6%) anti-HBc positive. Anti-HCV seropositivity was significantly correlated with duration of dialysis (p = 0.007) and number of blood transfusions (> 10 units, p = 0.0004). Among 12 subjects with a history of abnormal ALAT levels, 10 were anti-HCV positive (p = 0.0016) and the results suggest hepatitis C viral infection to be the main cause of liver disease in haemodialysis patients in Tunisia.

[Allergic reactions to transfusion]. / Les accidents allergiques transfusionnels.

Frequent allergic reactions following transfusion are observed. Usually, they are benign but sometimes we observe severe allergic reactions. Adverse reactions may be brought about by least two mechanisms. First, immediate-type hypersensibility reactions due to IgE. Secondly, anaphylactic-type reactions due to interaction between transfused IgA and class specific anti IgA in the recipient's plasma. They are characterized by their severest form (anaphylactic shock). The frequency of severe reactions following the transfusion blood plasma is very low. These transfusion reactions are complement-mediated and kinins-mediated. Prevention of allergic reactions is necessary among blood donors and recipients.

[Initial personal results of a technic of removing leukocytes by filtration on cotton fibers (Imugard Ig 500, Terumo)]. / Premiers résultats personnels d'une technique de déleucocytation par filtration sur fibres de coton (ImugardR IG 500, TerumoR).

The technical parameters of blood filtration through cotton Imugard IG 500 filters were evaluated. Sixty units of red cell concentrates were filtered. The mean value of the residual leukocytes indicated a leukocyte depletion greater than 98%, disregarding the unit age and the filtration temperature. The number of residual leukocytes was below 1 X 10(8) in 94% of the filtered units and less than 0.3 X 10(8) in 68%. The number of residual platelets was less than 1 X 10(10) in 92% of these units.

Filtration pressure and red blood cell deformability: evaluation of a new device: erythrometre.

The authors have tested a new device that records the filtration pressure of a suspension of red blood cells (R.B.C.) passing through a 3 micron pore diameter filter. As the flow rate is constant, the pressure increase allows to appreciate an index of the R.B.C. deformability. In order to test the performances of this new equipment, the authors have studied the influence of the variation of the following parameters. Haematocrit from 0.5 to 2%, Buffer composition; time elapsing from drawing to measurement: from hour 0 to hour 3. This analysis has been performed on donors; on packed red cells preserved in CPD, from D0 to D21 and on patients specimens and the results have been compared to the whole blood filtration according to the Reid and Dormandy method. The following parameters were simultaniously measured: Whole blood viscosity by means of a Low Shear 30 viscosimeter, Intraerythrocyte ATP by bioluminescence, Membrane lipid composition (cholesterol, phospholipids), Scanning electron microscopy. This new apparatus, measuring the filtration pressure seems to be a more sensitive and reproductible method allowing to approach the RBC deformability, and quite usefull in clinical haemorheology.

[Accidental case delayed by anti-D allo-immunization in a patient suffering from multiple traumatic injuries. One case (author's transl)]. / Accident hémolytique retardé par allo-immunisation anti-D chez une polytraumatisée.

An accidental case of delayed haemolytic reaction due to an anti-D allo-immunization in a patient suffering from multiple traumatic injuries allows us to define a procedure of emergency treatment towards non iso-Rhesus transfusions and the problems posed by the detection of acquired allo-antibodies.

Unchanged electrophoretic mobility in human cryopreserved lymphocytes.

Cell surface charge, assessed by the analytical electrophoresis of fresh and cryopreserved human peripheral blood lymphoctes, is changed in any perceivable manner by freezing and thawing. This was confirmed by different membrane markers (E, EA and EAC rosettes).

[Changes in the electric charge of blood lymphocyte populations after secondary immunization with tetanus antitoxin in man]. / Modifications de la chargeélectrioue des populations lymphocytaires sanguines après immunisation secondaire par l'anatoxine tétanique chez l'homme.

The electrophoretic mobility of circulating lymphocytes has been studied in normal human subjects after immunization by tetanus toxoid. The mean migration speed was shown to increase, particularly two and three days after secondary immunization. This increase appeared to be due to the elevation of percentage of T cells migrating at 1.20 and 1.35 micrometer. sec.-1v.-1 cm. (active rosettes-forming cells), with a decrease of the percentage of B cells and T lymphocytes migrating at 1.10 micrometer. sec.-1v.-1 cm. The return to the anterior status was observed between day 4 and 8 after immunization.