Microbial contamination of cord blood stem cells.

BACKGROUND AND OBJECTIVES: After storage, low levels of contaminating bacteria in standard blood components can reach bacteraemic levels, causing severe transfusion-associated sepsis. For cord blood (CB), the significance of low levels of contaminating bacteria and the optimal detection method is unknown and not supported by available guidelines. MATERIALS AND METHODS: Spiking experiments and testing of various subfractions of CB units were used to determine the behaviour of bacteria during centrifugation, freezing and thawing. For routine testing of CB, different volumes were compared for the detection of potential pathogens and micro-organisms of low pathogenicity. RESULTS: Centrifugation, as applied to CB fractionation, does not show concentration of bacteria in any particular fraction and supports the possibility of culture of waste fractions. Dimethylsulphoxide (DMSO) and freezing does not affect the viability of bacteria under the conditions used in this study. Owing to the low contamination level, a large sample volume of 20 ml was more sensitive than a 10-ml sample volume. Eighty five per cent of the isolated strains can be considered to be of low pathogenicity. CONCLUSION: When an optimal waste fraction sample volume of 20 ml was cultured, the contamination rate of CB was found to be approximately 13%, with low levels of < 1 colony-forming unit (CFU)/ml. Such levels of bacteria of low pathogenicity are expected to be of clinical importance only when CB is expanded in vitro for a prolonged period of time.

Optimization of cryopreservative procedures for human articular cartilage chondrocytes.

Procedures are being developed which use isolated articular cartilage (AC) chondrocytes to restore damaged articular surfaces. The availability of isolated human chondrocytes for transplantation may be increased by low-temperature storage (banking). At present, no single method of freezing chondrocytes has been proven to be optimal. In this project, two different freezing protocols, I and II, were compared. Protocol I used freezing rates of -1 degrees C/min down to a temperature of -40 degrees C. Protocol II used a freezing rate of -1 degree C/min down to -10 degrees C and faster rates thereafter. Cells were stored for 2 weeks at -196 degrees C. Survival and function of the cells after thawing were evaluated by histological examination and determination of 35S- and 3H-thymidine incorporation after 1 and 2 weeks of high-density monolayer culture. Cells frozen with protocol I showed better function and survival (99.75%) than cells frozen with protocol II (85%). Both groups showed slowing of metabolism and replication after freezing when compared with controls. We conclude that controlled freezing of adult human chondrocytes at rates of -1 degrees C/min improves survival. Banking of human AC chondrocytes may be feasible using protocol I, although some questions regarding the long-term behaviour of human AC chondrocytes after cryopreservation remain to be answered.

Preparation of random donor leukocyte-poor platelets by cytapheresis.

Platelet refractoriness in treatment of multitransfused patients with haematologic malignancies can be delayed or prevented by transfusing leukocyte-poor platelet concentrates. Absence of consensus about the number of residual leukocytes that leads to a delay or prevention of HLA-antibody formation, may be based on a lack of sampling for determination of leukocyte contamination levels in platelet concentrates. From data presented in this study we conclude that if every preparation of platelets is also tested for platelet count, it reduces costs when the pheresis platelets can be split. Transfusion of platelets in patients can also be better evaluated.

Prevention of primary cytomegalovirus infection after allogeneic bone marrow transplantation by using leukocyte-poor random blood products from cytomegalovirus-unscreened blood-bank donors.

Cytomegalovirus infection was studied in 59 seronegative recipients of bone marrow depleted of lymphocytes by counterflow centrifugation. Eighteen patients died within 3 months after bone marrow transplantation without evidence of CMV infection, and they were excluded from analysis. Twenty-eight valuable seronegative patients received marrow from a seronegative donor, and 13 from a seropositive donor. All but 2 patients received acyclovir orally (4 x 400 mg/day) from days -9 to +60. CMV prophylaxis with immunoglobulin preparations was not given. All blood products were prepared from random, CMV-unscreened blood-bank donors. The red cell concentrates were depleted of leukocytes by filtration, and leukocytes were removed from the platelet concentrates by centrifugation. None of the patients with seronegative donors showed any clinical sign compatible with CMV infection. Two nonfatal primary CMV infections occurred in the recipients of bone marrow from CMV-positive donors. One of the 59 patients developed interstitial pneumonia, in this case caused by Pneumocystis carinii. Leukocyte depletion of blood products from random CMV-unselected blood donors appeared to prevent primary infection in CMV-seronegative BMT recipients. We conclude that prophylactic use of immunoglobulin preparations is not necessary to prevent CMV primo-infection in patients receiving leukocyte-depleted blood products and acyclovir prophylaxis during the first 2 months postgrafting.

A study on the dimeric structure of creatine kinase (EC 2.7.3.2).

The generally accepted dimeric structure of creatine kinase (CK) is one in which M subunits and B subunits can occur. This paper reports the existence of three MM-bands and two MB-bands in human sera with increased CK-activity. These findings and supplementary data gathered from hybridisation experiments have led to the conclusion that there are two different M subunits, which can both occur in the enzyme in vivo. Further, findings are reported on the different CK-isozyme patterns of extracts from the cerebellum and from the cortex and the medulla of the cerebrum.