Learning from incidents in medically assisted reproduction: the Notify Library as a learning tool.

Biovigilance is the systematic monitoring of serious adverse reactions and events (SARE) that ensures the quality and safety of tissues and cells for human application in medically assisted reproduction (MAR). The Notify Library is an open access database launched by the World Health Organization and supported by the Italian National Transplant Centre (CNT) that has collected information on documented adverse occurrences in transplantation, transfusion and MAR. It is not a SARE register, but rather a collection of SARE types identified primarily by review of published articles and case reports from national or regional vigilance programmes. The Notify Library includes many well-documented records of adverse occurrences in MAR treatment, representing a useful tool for MAR operators in the evaluation of the risks associated with the clinical application of reproductive tissues and cells. It is updated with new records when a new type of incident is reported for the first time. All incident types described might have teaching value during the risk management carried out by a MAR centre. Sharing lessons learned from these incidents represents an important didactic opportunity that can help MAR centres to improve their processes and to achieve higher standards of quality and safety.

Tissue banking, biovigilance and the notify library.

This issue is dedicated to the contributions of Professor Glyn O. Phillips to the field of tissue banking and the advancement of science in general. The use of ionizing radiation to sterilize medical products drew the interest of the International Atomic Energy Agency (IAEA). A meeting in 1976 in Athens Greece to present work on the effects of sterilizing radiation doses upon the antigenic properties of proteins and biologic tissues was my first introduction of Professor Phillips and the role that he was to play in Tissue Banking (Friedlaender, in Phillips GO, Tallentine AN (eds) Radiation sterilization. Irradiated tissues and their potential clinical use. The North E. Wales Institute, Clwyd, p 128, 1978). The IAEA sponsored subsequent meetings in the Republic of Korea, Czechoslovakia and Rangoon, the later including a visit to the tissue bank by Professor Phillips. His advocacy resulted in multiple workshops and teaching opportunities in a variety of countries, one of which led to the establishment of the Asia Pacific Surgical Tissue Banking Association in 1989 (Phillips and Strong, in Phillips GO, Strong DM, von Versen R, Nather A (eds) Advances in tissue banking, vol 3. World Scientific, Singapore, pp 403-417, 1999).

Coding and traceability for cells, tissues and organs for transplantation.

Modern transplantation of cells, tissues and organs has been practiced within the last century achieving both life saving and enhancing results. Associated risks have been recognized including infectious disease transmission, malignancy, immune mediated disease and graft failure. This has resulted in establishment of government regulation, professional standard setting and establishment of vigilance and surveillance systems for early detection and prevention and to improve patient safety. The increased transportation of grafts across national boundaries has made traceability difficult and sometimes impossible. Experience during the first Gulf War with mis-identification of blood units coming from multiple countries without standardized coding and labeling has led international organizations to develop standardized nomenclature and coding for blood. Following this example, cell therapy and tissue transplant practitioners have also moved to standardization of coding systems. Establishment of an international coding system has progressed rapidly and implementation for blood has demonstrated multiple advantages. WHO has held two global consultations on human cells and tissues for transplantation, which recognized the global circulation of cells and tissues and growing commercialization and the need for means of coding to identify tissues and cells used in transplantation, are essential for full traceability. There is currently a wide diversity in the identification and coding of tissue and cell products. For tissues, with a few exceptions, product terminology has not been standardized even at the national level. Progress has been made in blood and cell therapies with a slow and steady trend towards implementation of the international code ISBT 128. Across all fields, there are now 3,700 licensed facilities in 66 countries. Efforts are necessary to encourage the introduction of a standardized international coding system for donation identification numbers, such as ISBT 128, for all donated biologic products.

Development of a transplantation transmission sentinel network to improve safety and traceability of organ and tissues.

The US lags behind other developed countries in creating a system to monitor disease transmission and other complications from human allograft use, despite a pressing need. The risks of transmission are amplified in transplantation, since at least 8 organs and more than 100 tissues can be recovered from a single common organ and tissue donor. Moreover, since many allografts collected in the US are distributed internationally, tissue safety is a global concern. In June 2005, participants of a US government-sponsored workshop concluded that a communication network for the tracking and reporting of disease transmissions for tissues and organs was critically needed. The United Network for Organ Sharing (UNOS) entered into a cooperative agreement with the Centers for Disease Control and Prevention (CDC) in 2006 to develop a system prototype. Over the following 3 years, the Transplantation Transmission Sentinel Network (TTSN) was developed and piloted with the participation of organ procurement organizations, tissue banks and transplant centers. The prototype centered around three elements of data entry: (1) donation, (2) tissue implantation, and (3) adverse event. The pilot proved that a system can be built and operated successfully, but also suggested that users may be hesitant to report adverse events. CDC has requested further input on scope and cost to build a transplant surveillance infrastructure for a fully functional national system. For tissues however, in contrast to organs, tracking from recovery to implantation will be necessary before a system is operable, requiring common identifiers and nomenclature. Until a US sentinel network is operational, future transmission events that are preventable may result nationally and globally due to its absence.

In vitro culture and differentiation of osteoblasts from human umbilical cord blood.

It is well accepted that human umbilical cord blood (UCB) is a source of mesenchymal stem cells (MSCs) which are able to differentiate into different cell phenotypes such as osteoblasts, chondrocytes, adipocytes, myocytes, cardiomyocytes and neurons. The aim of this study was to isolate MSCs from human UCB to determine their osteogenic potential by using different kinds of osteogenic medium. Eventually, only those MSCs cultured in osteogenic media enriched with vitamin D(2) and FGF9, were positive for osteocalcin by RT-PCR. All these cells were positive for alizarin red, alkaline phosphatase and Von Kossa. The results obtained from RT-PCR have confirmed that osteogenesis is complete by expression of the osteocalcin marker. In conclusion, vitamin D(2), at least in vitro, may replace vitamin D(3) as an osteogenic stimulator factor for MSC differentiation.

Organ and tissue safety workshop 2007: advances and challenges.

A workshop in June 2005 ("Preventing Organ and Tissue Allograft-Transmitted Infection: Priorities for Public Health Intervention") identified gaps in organ and tissue safety in the US. Participants developed a series of allograft safety initiatives. "The Organ and Tissue Safety Workshop 2007: Advances and Challenges" assessed progress and identified priorities for future interventions. Awareness of the challenges of allograft-associated disease transmission has increased. The Transplantation Transmission Sentinel Network will enhance communication surrounding allograft-associated disease transmission. Other patient safety initiatives have focused on adverse event reporting and microbiologic screening technologies. Despite progress, improved recognition and prevention of donor-derived transmission events is needed. This requires systems integration across the organ and tissue transplantation communities including organ procurement organizations, eye and tissue banks, and transplant infectious disease experts. Commitment of resources and improved coordination of efforts are required to develop essential tools to enhance safety for allograft recipients.

Performance of an algorithm for the reentry of volunteer blood donors deferred due to false-positive test results for antibody to hepatitis B core antigen.

BACKGROUND: Blood donor testing for antibody to hepatitis B core antigen (anti-HBc) has been used in the United States for more than 20 years as a surrogate to prevent transmission by transfusion of non-A, non-B hepatitis, as a human immunodeficiency virus surrogate, and to reduce transmission of hepatitis B virus (HBV). Nonspecific anti-HBc assays have caused deferral of hundreds of thousands of otherwise qualified donors. A more specific anti-HBc test and a sensitive HBV DNA test should permit donor reentry after false-positive anti-HBc. STUDY DESIGN AND METHODS: A total of 1324 otherwise eligible volunteer donors, deferred for anti-HBc reactivity on more than one occasion, were recruited from four collection facilities. They were tested using a licensed, more specific anti-HBc test, a licensed hepatitis B surface antigen (HBsAg) test, and a licensed HBV DNA assay with a 95 percent limit of detection of not more than 10 copies per mL. RESULTS: From 11 to 32 percent of donors contacted by participating sites entered the study. Overall, 488 (37%) of the donors were negative on the more specific anti-HBc test. The proportion of putative false-positive samples varied according to the test responsible for the original deferral. A single donor, negative for the presence of anti-HBc and HBsAg, was positive for the presence of HBV DNA in one of three replicates. Repeat testing of this donor 10 months later was negative for the presence of all markers of HBV infection, and the donor had a history of HBV vaccination with documented postimmunization anti-HBs seroconversion 10 years before her anti-HBc deferral, and was considered HBV DNA false positive. CONCLUSION: These data support reentry of donors with false-positive anti-HBc results on the relatively nonspecific assays that have been in use in the United States for more than 20 years.

Probability of viremia with HBV, HCV, HIV, and HTLV among tissue donors in the United States.

BACKGROUND: Tissue-banking organizations in the United States have introduced various review and testing procedures to reduce the risk of the transmission of viral infections from tissue grafts. We estimated the current probability of undetected viremia with hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and human T-lymphotropic virus (HTLV) among tissue donors. METHODS: Rates of prevalence of hepatitis B surface antigen (HBsAg) and antibodies against HIV (anti-HIV), HCV (anti-HCV), and HTLV (anti-HTLV) were determined among 11,391 donors to five tissue banks in the United States. The data were compared with those of first-time blood donors in order to generate estimated incidence rates among tissue donors. The probability of viremia undetected by screening at the time of tissue donation was estimated on the basis of the incidence estimates and the window periods for these infections. RESULTS: The prevalence of confirmed positive tests among tissue donors was 0.093 percent for anti-HIV, 0.229 percent for HBsAg, 1.091 percent for anti-HCV, and 0.068 percent for anti-HTLV. The incidence rates were estimated to be 30.118, 18.325, 12.380, and 5.586 per 100,000 person-years, respectively. The estimated probability of viremia at the time of donation was 1 in 55,000, 1 in 34,000, 1 in 42,000, and 1 in 128,000, respectively. CONCLUSIONS: The prevalence rates of HBV, HCV, HIV, and HTLV infections are lower among tissue donors than in the general population. However, the estimated probability of undetected viremia at the time of tissue donation is higher among tissue donors than among first-time blood donors. The addition of nucleic acid-amplification testing to the screening of tissue donors should reduce the risk of these infections among recipients of donated tissues.

Detection of HIV-1 and HCV infections among antibody-negative blood donors by nucleic acid-amplification testing.

BACKGROUND: Testing of blood donors for human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) RNA by means of nucleic acid amplification was introduced in the United States as an investigational screening test in mid-1999 to identify donations made during the window period before seroconversion. METHODS: We analyzed all antibody-nonreactive donations that were confirmed to be positive for HIV-1 and HCV RNA on nucleic acid-amplification testing of "minipools" (pools of 16 to 24 donations) by the main blood-collection programs in the United States during the first three years of nucleic acid screening. RESULTS: Among 37,164,054 units screened, 12 were confirmed to be positive for HIV-1 RNA--or 1 in 3.1 million donations--only 2 of which were detected by HIV-1 p24 antigen testing. For HCV, of 39,721,404 units screened, 170 were confirmed to be positive for HCV RNA, or 1 in 230,000 donations (or 1 in 270,000 on the basis of 139 donations confirmed to be positive for HCV RNA with the use of a more sensitive HCV-antibody test). The respective rates of positive HCV and HIV-1 nucleic acid-amplification tests were 3.3 and 4.1 times as high among first-time donors as among donors who gave blood repeatedly. Follow-up studies of 67 HCV RNA-positive donors demonstrated that seroconversion occurred a median of 35 days after the index donation, followed by a low rate of resolution of viremia; three cases of long-term immunologically silent HCV infection were documented. CONCLUSIONS: Minipool nucleic acid-amplification testing has helped prevent the transmission of approximately 5 HIV-1 infections and 56 HCV infections annually and has reduced the residual risk of transfusion-transmitted HIV-1 and HCV to approximately 1 in 2 million blood units.

Prestorage leukoreduction does not increase hemolysis of stored red cell concentrates.

BACKGROUND: In December 2004, Pall Corporation initiated voluntary recall of certain filters used for leukocyte-reduction of blood products. Although our center had not used the implicated lots, certain customers reported observing increased hemolysis in the red-cell units (RC) provided by us. The purpose of this study was to determine the level of hemolysis seen in RC produced by our center. METHODS: In the first-phase, we evaluated 20 leukocyte-reduced (LR)-RC, those judged by one of our hospitals to have the highest degree of hemolysis (age: 10-30 days; average=16 days). Results were compared to ten randomly selected non-LR-RC (age: 10-19 days; average: 15 days). Samples obtained directly from the RC were tested for hemoglobin (Hb), hematocrit (Hct) and supernatant-Hb. Percent-hemolysis (% hemolysis) was calculated. In the second-phase, the above measurements were made on 70RCs. Ten RCs were studied before and after leukofilteration on day-2 after collection. Ten units each (LR & non-LR) were selected randomly from inventory at days: 15, 30 and 40 after collection (LR-units filtered within 48 h). RESULTS: In the first-phase LR-RCs exhibited an average 0.06% hemolysis vs. 0.02% for non-LR units. In the second-phase the average % hemolysis before and after filteration on day-2 (LR: 0.04% & non-LR: 0.04%) was similar. While on days: 15 (LR: 0.09%, non-LR: 0.05%) and 30 (LR: 0.16%, non-LR: 0.13%) % hemolysis was slightly more in LR as compared to non-LR. It was the opposite for day 40 (LR: 0.19%, non-LR: 0.31%). However, none of these differences were statistically significant. CONCLUSIONS: The % hemolysis increased as the age of the unit increased. There was no significant statistical difference between LR-RC and non-LR-RCs. This data did not confirm our hospitals' concerns regarding increased hemolysis following LR.

WITHDRAWN: Prestorage leukoreduction does not increase hemolysis of stored red cell concentrates.

The Publisher regrets that this article was an accidental duplication of an article that has already been published in Transfus Apher Sci, 36 (1) 17 - 22, doi:10.1016/j.transci.2006.09.007. The duplicate article has therefore been withdrawn.

Blood-bank testing for infectious diseases: how safe is blood transfusion?

Remarkable progress has been made in transfusion safety from infection over the past three decades. Donor deferrals for at-risk behaviors, the introduction of more-sensitive viral-screening assays and the recent introduction of nucleic-acid amplification technology have nearly eliminated transmission of HIV and hepatitis C virus (HCV) by blood transfusion in North America. Nevertheless, risks of other infectious agents for which such robust screening tools have not been developed, such as bacteria and parasites, still remain. As a result of these successes, the non-infectious risks such as misidentification of patients and inadequate and inappropriate transfusion have become the primary sources of transfusion risk.

Increased oxygen consumption rates in response to high glucose detected by a novel oxygen biosensor system in non-human primate and human islets.

In this study, we investigated the use of a novel oxygen biosensor system to detect changes in oxygen consumption rates (OCRs) by islets in response to glucose. Islets from non-human primate and human pancreata were seeded into an oxygen biosensor system microplate and exposed to basal (2.8 or 5.6 mM) or high (16.7 or 33.3 mM) glucose over either a long-term or a short-term culture. Our data clearly demonstrated that non-human primate islets cultured in high glucose conditions exhibited significant increases in OCRs over a 168 h extended culture period (P<0.05), which indicates an accelerated rate of beta-cell metabolism triggered by glucose over time. Significant increases in OCRs (P<0.01) were also attained in both non-human primate and human islets exposed to high glucose conditions in a 120 min short-term incubation period. OCRs exhibited by human islets exposed to different glucose concentrations correlated with insulin secretion (r(2)=0.7681, P<0.01). Moreover, the OCR stimulation index (i.e. OCR at high glucose/OCR at basal glucose) was significantly greater in human islets displaying high viabilities as opposed to islets exhibiting low viabilities (P<0.05). Together these data demonstrate that this novel oxygen biosensor system documents significant increases in islet oxygen consumption upon acute and chronic exposure to high glucose concentrations. Importantly, this methodology rapidly and robustly detects changes in OCRs by islets in response to high glucose stimulation that correlate well with the metabolic activities and functional viability of islets and clearly delineates significant differences in OCR stimulation index between high and low viability human islets, and therefore may prove to be an effective approach for quickly assessing the functional viability of islets prior to transplantation.

Efficacy of human islet isolation from the tail section of the pancreas for the possibility of living donor islet transplantation.

BACKGROUND: Islet transplantation is on the rise for the treatment of type 1 diabetes. Apparent donor shortages could be alleviated through use of living donor pancreata. A critical issue for using a section of pancreas from living donors is whether islet yields would be sufficient for transplantation. METHODS: After obtaining human pancreata, islets were isolated from the head section (n=20, head group), tail section (n=23, tail group) or whole pancreas (n=24, whole group). Islets were isolated by enzymatic digestion followed by purification, then assessed for yields, purity, morphology, functionality, and insulin content. RESULTS: Fifteen of twenty cases (75%) in the head group, all cases (100%) in the tail group, and 23 of 24 cases (96%) in the whole group were successfully completed for islet isolation. Islet yield per gram pancreas was significantly higher in the tail group compared with both the head and whole groups (head, 1,472+/-326 IE/g; tail, 4,256+/-574 IE/g; whole, 2,424+/-506 IE/g). Total islet yield from the head group was significantly lower compared with both tail and whole groups (head, 75,016+/-18,933 IE; tail, 197,469+/-28,236 IE; whole, 208,207+/-43,414 IE), and the tail group showed similar islet yield to the whole group. The whole group showed significantly lower purities and the head group showed significantly lower morphologic scores. There were no significant differences in viability, function, and insulin content among the three groups. CONCLUSIONS: The tail section of the human pancreas is suitable for islet isolation. The living donor islet transplantation may be feasible using only this section of the pancreas for the first transplantation to reduce hypoglycemic unawareness for small recipients, which might be followed by the second islet transplantation from cadaveric donor.

Effect of the two-layer (University of Wisconsin solution-perfluorochemical plus O2) method of pancreas preservation on human islet isolation, as assessed by the Edmonton Isolation Protocol.

BACKGROUND: Current techniques for isolating islets require that pancreata stored with University of Wisconsin solution (UW) are processed within 12 hours of cold storage. In this study, we hypothesized that the two-layer method (TLM) could extend the acceptable preservation period of pancreata before islet isolation and increase islet yields. METHODS: In the first experimental set, eight pancreata were maintained for an average of 8.3+/-1.2 hours in UW and transferred into the TLM for an additional 14.3+/-1.1 hours for a total cold ischemic period of 22.6+/-1.6 hours (prolonged TLM). Four pancreata were maintained as a control group in UW alone for a total of 21.3+/-2.0 hours. In the second experimental set, six pancreata were maintained for an average of 6.4+/-1.8 hours in UW followed by 4.8+/-0.8 hours with the TLM for a total preservation time of 11.3+/-2.5 hours (short TLM). The control organs for the short TLM group were stored for an average of 9.5+/-1.3 hours in UW alone. Islets were isolated and evaluated according to the Edmonton protocol. RESULTS: Between each group of the two experimental sets, there was no significant difference in donor-related factors (i.e. gender, age, body mass index [BMI], etc.). The TLM as compared with UW preservation resulted in a significant increase in islet yields postpurification for both short (3,353+/-394 islet equivalents [IE] vs. 2,027+/-415 IE; mean+/-SEM) and prolonged (2,404+/-503 IE vs. 514+/-180 IE) periods of storage. Furthermore, islet yields after prolonged storage with the TLM were not significantly different from organs maintained for only a short period with UW (P=0.17). The quality of islets as assessed by size, postculture viability, survival rates, insulin content, and insulin secretion were similar for each of the four groups. CONCLUSION: In comparison with UW organ preservation, exposure of pancreata to the TLM result in greater islet yields and extended preservation times.

Donor age and gender are the strongest predictors of marrow recovery from cadaveric vertebral bodies.

The purpose of this retrospective analysis was to determine whether there were donor factors that were useful for predicting the yield of nucleated cells from marrow derived from cadaveric vertebral bodies. An analysis of 132 donors over a 6-year period was performed. The average number of vertebral bodies procured from each donor was 10.2 +/- 1.6 (range 5-14). The total number of nucleated cells recovered per donor ranged from 24 x 10(9) to 160 x 10(9) with an average recovery of 69 +/- 28 x 10(9) cells. The cell viability of the recovered cells was > 95%. The average age of the donors was 33 +/- 14 years (mean +/- SD; range 12-65) with an average weight of 169 +/- 41 lb (range 82-308 lb). Males comprised 68% of the donor population. The average number of days from admission to death was 1.9 +/- 1.7 with a range of 1-11.4 days and the interval between asystole and procurement averaged 3.1 +/- 2.3 h (range (0.1-14.7 h). The majority of donors died from head trauma due to an intracranial bleed, gunshot wound, or closed head injury. Regression analysis of the data indicated that the total nucleated cell yield tended to decrease with increasing time between hospital admission and death. The data also indicated that in general female donors yielded lower cell numbers independent of age and male donors under 30 years of age yielded the highest number of cells.

Polyphenol, an extract of green tea, increases culture recovery rates of isolated islets from nonhuman primate pancreata and marginal grade human pancreata.

Investigations indicate that an extract of green tea, polyphenol, can significantly increase the culture survival rate of rat islets without deteriorating their functionality. In this study, we examined the effect of adding polyphenol to islets isolated from human pancreata and nonhuman primate pancreata. Islets were isolated from human pancreata that did not meet criteria for clinical transplantation (n = 6) and from nonhuman primate pancreata (n = 5). The islets were cultured in CMRL-1066 + 10% FCS with the addition of 0, 30, 60, 125, 250, or 500 microg/ml of polyphenol. After 24 or 48 h of culture, islet yield, viability, purity, morphology, and stimulation index was assessed. RT-PCR and Western blot analysis were also performed to assess the expression levels of the apoptotic related genes, Bcl-2 and BAX. After 24 h of culture, islet yields were significantly higher in cultures supplemented with 30-250 microg/ml of polyphenol than in cultures without polyphenol. After 48 h of culture, significant differences in islet numbers were observed with polyphenol concentrations of 125 microg/ml (p < 0.01) and 250 microg/ml (p < 0.01). However, no significant differences were noted in islet viability, purity, morphology, and stimulation index at each time point with or without polyphenol. RT-PCR and Western blot analysis of the islets indicated that Bcl-2 levels increased by 2.5-fold and BAX levels decreased by twofold in cultures supplemented with polyphenol. This resulted in BAX/Bcl-2 ratios that were lower in polyphenol-supplemented cultures than with control cultures. Polyphenol increases culture recovery rates by precluding islet apoptosis.

A comparative evaluation of culture conditions for short-term maintenance (<24 hr) of human islets isolated using the Edmonton protocol.

Once human islets are isolated, they are typically transplanted into type 1 diabetic recipients within 2 h of isolation. This time restriction makes it difficult for patients to travel from distant locations to receive an islet transplant and it also makes it difficult to complete pre-release quality control assessments (i.e., endotoxin and gram stain) before the expiration of the islet product. Therefore, there were two goals for this study. The first was to measure the stability of islets after a 24 h culture period using CMRL media 1066 (CMRL) supplemented with either fetal bovine serum (FBS); albumin or insulin transferrin and selenium (ITS). The second was to determine the impact of cell concentration and media depth on islet stability. The results of the study indicated that culture recoveries at 37 degrees C with CMRL + ITS (also known as Memphis media) were higher (64.1 +/- 8.3%) than with CMRL supplemented with FBS (38.7 +/- 9.7%) or albumin (47.6 +/- 8.2%) and that post-culture islet viabilities, post-culture purities and stimulation indexes (SIs) were comparable. In the second series of experiments, the results showed that islets recoveries and SIs in cultures with low islet concentrations (300 IE/ml) were significantly better than cultures at high islet concentrations (1500 IE/ml). Additionally, at a shallow media depth (1.4 vs. 7 mm of media) the SI of the islets improved, and this effect was independent of the additive (i.e., FBS, albumin and ITS).

Banking of osteochondral allografts. Part I. Viability assays adapted for osteochondrol and cartilage studies.

The aim of this study was to adapt a reliable, reproducible and simple viability assay for cartilage and osteochondral studies. The previous assays (radioisotope uptake, assessment of matrix components, histological methods, oxygen consumption etc.) were complex, laborious, time consuming or suffer from difficulty of interpretation. MTT assay was chosen because it has been widely and successfully used in different cell and tissue studies, but has not been published on human solid articular cartilage. Fresh intact cartilage samples of human tali were tested to investigate the assay. The reliability of the MTT assay was also tested by an fluorescent dye combination. The MTT assay is based on the production of purple formazan pigment from methyltetrazolium salt by the mitochondrial enzymes of viable chondrocytes. The enzyme kinetics of the reaction was also investigated because it was unknown in the case of cartilage. The amount of pigment formed can be measured by spectrophotometry after extraction by methyl cellosolve. The color density is proportional to mitochondrial enzyme activity, reflecting the number of viable chondrocytes. The optimal reagent concentration, biopsy size, and incubation period were established. There is a linear relationship between the cartilage weight and the pigment production activity. A 9.8% nonspecific raction was observed in the negative controls. The enzyme kinetics of the reaction was also investigated. The MTT clevage up to 0.1% (w/v) follows the Michaelis kinetics. We calculated the Michaelis constant (2835 +/- 130 microM), the maximal velocity (36 +/- 3.2 x 10(-5)microMsec(-1)) and the velocity constant (1.27 +/- 0.2 x 10(-7)sec(-1)) of the reaction. The latter is a significant marker for each tissue type. The viability of cartilage was also assessed and calculated by a fluorescent dye combination comprising 1 microg/ml propidium iodide (PI) and 4 microM/ml SYTO-16 stains. The PI stains dead cells (red fluorescence), the SYTO-16 stains live cells (green fluorescence). The staining can be visualised simultaneously, and the live/dead ratio can be calculated by image analysis software from saved image files. The MTT assay is a simple, non-expensive, efficient, reliable, reproducible, sensitive viability test for cartilage studies. The MTT reduction assay and the staining method were corrobative.