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PubMed1983

Microbiological investigations on cryopreserved human semen.

Primary mycological contamination was studied in 565 random human ejaculates. After sperm preservation at low temperature, secondary mycological contamination of 55 of the ejaculates was investigated. 25 ejaculates were cryopreserved with and without antibiotics and examined bacteriologically before and after different phases of cryopreservation. Furthermore, semen samples of several donors were inoculated with suspensions of different concentrations of yeasts and with the test bacteria Escherichia coli K 12 and Staphylococcus aureus SG 511. Microbiological results were then compared before and after cryopreservation. The following results were obtained: 1.9.8% of samples showed primary mycological contamination (1.9% with Candida albicans). 2. Cryopreservation reduced the concentration of fungi by more than 90% on average. 3. Only one semen sample was contaminated with bacteria (Staphylococcus aureus haemolyticus and Streptococcus nonhaemolyticus). Non-pathogenic bacteria were found in 11 ejaculates (Micrococcus luteus in 5 samples and Staphylococcus epidermidis in 6 samples), and contamination was largely unaffected by cryopreservation, even in the presence of antibiotics. 4. Test bacteria inoculated into semen were not influenced by the addition of antibiotics to the cryoprotective medium.

Anti-Bacterial Agents↗
PubMed2002

Prospective, randomized, blinded evaluation of donor semen quality provided by seven commercial sperm banks.

OBJECTIVE: To evaluate variability in donor semen quality between seven commercial donor sperm banks, within sperm banks, and between intracervical insemination and intrauterine insemination. DESIGN: Prospective, randomized, blind evaluation of commercially available donor semen samples. SETTING: An academic andrology laboratory. PATIENT(S): Seventy-five cryopreserved donor semen samples were evaluated. INTERVENTION(S): Samples were coded, then blindly evaluated for semen quality. MAIN OUTCOME MEASURE(S): Standard semen quality parameters, including concentration, motility parameters, World Health Organization criteria morphology, and strict criteria morphology. RESULT(S): Significant differences were observed between donor semen banks for most semen quality parameters analyzed in intracervical insemination samples. In general, the greatest variability observed between banks was in percentage progressive sperm motility (range, 8.8 +/- 5.8 to 42.4 +/- 5.5) and normal sperm morphology (strict criteria; range, 10.1 +/- 3.3 to 26.6 +/- 4.7). Coefficients of variation within sperm banks were generally high. CONCLUSION(S): These data demonstrate the variability of donor semen quality provided by commercial sperm banks, both between banks and within a given bank. No relationship was observed between the size or type of sperm bank and the degree of variability. The data demonstrate the lack of uniformity in the criteria used to screen potential semen donors and emphasize the need for more stringent screening criteria and strict quality control in processing samples.

Cervix Uteri↗
PubMed1999

Diagnostic testicular biopsy and cryopreservation of testicular tissue as an alternative to repeated surgical openings in the treatment of azoospermic men.

Between May 1996 and May 1998, 64 azoospermic patients underwent an investigative testicular biopsy combined with the cryopreservation of spermatozoa which were retrieved from a simultaneously examined fresh sample. Testicular tissue cryopreservation was carried out in 43 cases (67%) for late intracytoplasmic sperm injection (ICSI) attempts. In all, 23 couples underwent 26 assisted conception cycles; the fertilization rate was 64% with spermatozoa (139/218, 24 cycles), 40% with round spermatids (2/5, one cycle), and 69% with elongated spermatids (9/13, one cycle). The embryo cleavage rate was 84%. A mean number of 2.7 +/- 0.7 embryos were replaced in 24 patients. In two cases, embryo quality was very poor and they were not transferred. Eight clinical pregnancies resulted (35% per patient and 33% per transferred cycle) with an implantation rate of 14.1%: two patients have already delivered and six are ongoing. In conclusion, the cryopreservation of testicular tissue during the first diagnostic biopsy is an alternative to repeated surgical openings and permits patients to initiate an ovarian stimulation cycle with the certitude of having spermatozoa available. Moreover, since only one straw is routinely used for each ICSI cycle, the frozen tissue remains as a sperm source for multiple attempts.

Adult↗
PubMed2000

Testicular sperm extraction combined with cryopreservation of testicular tissue in the treatment of azoospermia.

BACKGROUND: The aim of the present study was to verify the feasibility of cryopreserving testicular tissue during the first diagnostic biopsy and then using thawed sperm to inseminate the partner's oocytes. The expected advantages are: (i) minimal risk of not having spermatozoa available at the time of intracytoplasmic sperm injection; (ii) no repeated surgical interventions, and (iii) programming the treatment cycle at the couple's convenience. MATERIALS AND METHODS: Between May 1996 and May 1998, 64 azoospermic patients underwent investigative testicular biopsy combined with cryopreservation of spermatozoa which were retrieved in a simultaneously examined fresh sample. Testicular tissue cryopreservation was carried out in 43 cases (67%) for later intracytoplasmic sperm injection attempts. RESULTS: In all, 23 couples underwent 26 assisted conception cycles; the fertilization rate was 64% with spermatozoa (139/218, 24 cycles), 40% with round spermatids (2/5, 1 cycle), and 69% with elongated spermatids (9/13, 1 cycle). The embryo cleavage rate was 84%. The mean number of embryos replaced in 24 patients was 2.7 +/- 0.7. In 2 cases, embryo quality was very poor, and they were not transferred to the patients. Eight clinical pregnancies resulted (35%/patient and 33%/transferred cycle) with an implantation rate of 14.1%; 2 patients have already delivered and 6 pregnancies are ongoing normally. CONCLUSIONS: Testicular tissue cryopreservation during the first diagnostic biopsy is an alternative to repeated surgical interventions. Patients can initiate an ovarian stimulation cycle, confident of having spermatozoa available. Moreover, since only one straw is routinely used for each intracytoplasmic sperm injection cycle, the frozen tissue remains as a sperm source for multiple attempts.

Adult↗
PubMed2002

Electromagnetic re-warming of cryopreserved tissues: effect of choice of cryoprotectant and sample shape on uniformity of heating.

A method that has been proposed for the cryopreservation of tissues and organs is to add a cryoprotective agent (CPA) in sufficient concentration to allow vitrification, and to use rapid electromagnetic heating to prevent the formation of ice crystals during the re-warming. We have compared the physical and biological properties of four CPAs, measuring the speed and uniformity of heating in a 36 mm sphere placed in a 434 MHz applicator, and the toxicity to ECV304 endothelial cells. Ethanediol and dimethyl sulfoxide were found to be suitable for rapid, uniform heating but toxic to the endothelial cells at vitrifying concentrations. Butane-2,3-diol was less toxic, but the heating patterns were unacceptably non-uniform. Propane-1,2-diol was not significantly more toxic than butane-2,3-diol, and did allow uniform heating. It is therefore the best choice of CPA for the vitrification of tissues. We have shown that the uniformity of heating correlates with the dielectric properties of the perfusate. Furthermore, we have shown that uniform heating is feasible in non-spherical samples provided they are approximately ellipsoidal.

Butylene Glycols↗
PubMed2003

Rabbit and pig ear skin sample cryobanking: effects of storage time and temperature of the whole ear extirpated immediately after death.

The post-mortem temporal and thermal limits within which there will be ample guarantees of rescuing living skin cells from dead specimens of two species, rabbit and pig, were studied. Post-mortem extirpated whole ears were stored (in non-aseptic conditions) either at 4 degrees C or at room temperature (from 22 to 25 degrees C) or at 35 degrees C for different time lapses after animal death. In both species, the post-mortem maximum time lapses where cell viability was not significantly reduced were 240, 72, and 24 h post-mortem (hpm) for 4, 22-25 and 35 degrees C, respectively. Once the post-mortem temporal limits for each tested thermal level at which cells from skin samples are able to grow in culture were defined, the survival ability of skin samples submitted to these temporal limits and cryopreserved were tested. In the pig, skin samples stored at the three tested thermal levels survived after vitrification-warming, reaching confluence in culture. In rabbit, only tissue samples from ears stored at 35 degrees C for 24 hpm did not survive after vitrification-warming. In conclusion, we should remark that cell survival rates obtained according to the assayed post-mortem time lapses and thermal levels are sufficient to collect and to cryopreserve skin samples from the majority of dead specimens.

Animals↗
PubMedDate not supplied

Influence of artificial stimulation on unprocessed and Percoll-washed cryopreserved sperm.

The ability of pentoxifylline and 2-deoxyadenosine to stimulate sperm motility and motion characteristics was assessed in unprocessed and processed (Percoll-separated) cryopreserved specimens. Specimens from 12 healthy volunteers were obtained, and the motion characteristics were analyzed; half the sample was immediately cryopreserved and the other was washed using the Percoll gradient technique. To study stimulation, samples were thawed and divided into four aliquots: One was used as a control, and the others were incubated with 2.5 mM 2-deoxyadenosine, 2.5 mM pentoxifylline, or 5.0 mM pentoxifylline for 60 min. Sperm characteristics were analyzed on a sperm motion analyzer at 0 and 60 min incubation. In both unprocessed and processed samples, percentage motility improved significantly after stimulation with 2-deoxyadenosine or pentoxifylline (p = .003 or p = .0002, respectively); other characteristics improved to varying extent after 2-deoxyadenosine or pentoxifylline stimulation. Comparison after stimulation revealed that sperm motion characteristics in the unprocessed specimen were similar to sperm motion characteristics in the processed group.

Cryopreservation↗
PubMed1998

No induction of chromosome aberrations in human spermatozoa exposed to extremely low frequency electromagnetic fields.

Clastogenic effects of extremely low frequency electromagnetic fields (ELF-EMFs) on human sperm chromosomes were studied using an interspecific in vitro fertilization system with zona-free golden hamster oocytes. Semen samples from healthy men were exposed to ELF-EMFs (50 Hz, 20 mT) for 2 h at 37 degreesC under 5% CO2 in air. The samples were then cryopreserved in liquid nitrogen for shipment to a cytogenetic laboratory. After thawing the samples, motile spermatozoa were collected using a continuous Percoll density gradient centrifugation and then capacitated for in vitro fertilization with hamster oocytes. Sperm-derived chromosomes were analyzed at first cleavage metaphase. The present experiment was performed twice using semen samples from two different donors. In test-1, incidence of spermatozoa that displayed structural chromosome aberrations was 17.0% (35/206) in the exposed group and 20.8% (55/264) in the control group. In test-2, structural chromosome aberrations were observed in 11.1% (13/117) of exposed spermatozoa and 13.8% (13/94) of spermatozoa in the control group. In both tests, there was no significant difference in the incidence of chromosomally abnormal spermatozoa between the exposed group and the control group. Types of aberrations observed and their incidences per spermatozoon in the exposed group were similar to those of the control group. Despite the small sample size, the present results suggest that ELF-EMFs have no clastogenic effect on human sperm chromosomes.

Animals↗
PubMed1999

Amphotropic retrovirus transduction of hematopoietic stem cells.

Mice treated with cytokines for 5 days have large numbers of hematopoietic stem cells (HSCs) in their peripheral blood and bone marrow at 1 and 14 days after the last injection. We fractionated the HSCs from the bone marrow of these mice using elutriation at flow rates of 25, 30 and 35 ml/min. The subpopulations of HSCs from cytokine-treated mice show a 3- to 8-fold higher level of mRNA encoding the amphotropic retrovirus receptor (amphoR) compared with the corresponding HSC subpopulation from untreated mouse bone marrow. In an earlier study with mouse HSCs we showed a direct correlation between high levels of amphoR mRNA and efficient retrovirus transduction. We have now utilized our gene transfer protocol to assay amphotropic retrovirus transduction efficiency using HSCs from the bone marrow of mice treated with granulocyte-colony stimulating factor/stem cell factor (G-CSF/SCF). To extend these findings to a more clinically relevant protocol we analyzed the amphoR mRNA levels in HSCs from human cord blood and adult bone marrow. The amphoR mRNA level in HSCs from human bone marrow and fresh cord blood was detectable at an extremely low level compared with the HSC population in cryopreserved cord blood samples. The 12- to 22-fold increase in amphoR mRNA in HSCs from cryopreserved cord blood renders these HSCs likely candidates for high efficiency, gene transfer.

Adult↗
PubMed1990

Cryopreservation of semen from unique lines of chicken germ plasm.

Frozen semen is a practical means of preserving valuable germ plasm. Monitored samples of semen cryopreserved with glycerol for heterozygous, dominant marker stocks and for nine chromosomal rearrangement lines had sufficiently high fertility for germ-line retrieval. The results also indicated a potential for the genetic selection of certain lines for the freezability of spermatozoa, since stock and line differences in fertility occurred when previously frozen semen was used for insemination. Freezing the semen of stocks routinely reproduced provides insurance against possible disasters.

Animals↗
PubMed1993

Ca2+ regulation by cryopreserved bull spermatozoa in response to A23187.

The regulation of intracellular Ca2+ by fresh and cryopreserved bull spermatozoa from the same ejaculates (n = 5) was investigated using the fluorescent Ca2+ indicator, indo-1. Relative internal Ca2+ levels of the spermatozoa were monitored for 30 min prior to the addition of phosphate-buffered saline (PBS), 1 mM Ca2+, the Ca2+ ionophore A23187 (0.1 microM), or Ca2+ + A23187; during the additions the levels of intracellular Ca2+ were observed in detail. After these additions, changes in internal Ca2+ levels were monitored for 120 min. The initial intracellular Ca2+ levels in the cryopreserved spermatozoa were greater than those in fresh (1.05 +/- 0.03 vs 0.97 +/- 0.03 Ca2+ units, P = 0.0001). The addition of Ca2+ + A23187 induced elevated cellular Ca2+ in both fresh (P = 0.0177) and cryopreserved spermatozoa (P < or = 0.0588) within 5 s. Ca2+ alone did not differ from Ca2+ + A23187 in increasing Ca2+ in cryopreserved spermatozoa (P = 0.2225); fresh spermatozoa were slower to respond to exogenous Ca2+ alone (P = 0.0438). At the start of the post-treatment 120 min, cryopreserved spermatozoa had more internal Ca2+ than the corresponding fresh samples (P < or = 0.0088) except for those exposed to both Ca2+ + A23187 (P = 0.2918). All spermatozoa increased internal Ca2+ over the post-treatment time except for the cryopreserved PBS controls, and these cryopreserved controls differed from the fresh controls in accumulation of Ca2+ (P = 0.0620). None of the Ca2+/A23187 treatments induced different rates of change in internal Ca2+ over time in fresh cells (P = 0.3142). The rate of Ca2+ accumulation by cryopreserved spermatozoa in the presence of exogenous Ca2+ exceeded controls (23.12 +/- 6.75 vs 8.11 +/- 6.75, P = 0.0005), but A23187, with or without Ca2+, had no effect (P > or = 0.0907). Following Ca2+ measurements, the viability of the cryopreserved samples was reduced (P < or = 0.015) in all but the Ca(2+)-treated spermatozoa (P = 0.1474); no viability differences were noted for fresh spermatozoa (P > or = 0.2298). The cryopreservation process did not affect acrosomal morphology of the indo-1-exposed spermatozoa (P > or = 0.1147). These data indicate that Ca2+ regulation by Ca(2+)- and A23187-challenged bull spermatozoa differs following cryopreservation procedures, possibly relating to the reduced fertilization capacity of commercially cryopreserved semen.

Animals↗
PubMed2002

Sperm evaluation in cryopreserved bovine semen recovered by two selection methods.

Previous experiments have established that various semen manipulation techniques are able to increase the qualitative features of the spermatozoa used in different techniques of assisted reproduction, but practically no comparative data on frozen-thawed bovine semen have been found. The aim of this study was to compare the efficacy of two sperm selection methods: centrifugation on Percoll gradient and filtration through a Sephadex ion-exchange column, to improve the recovery of motile and morphologically normal spermatozoa, without inducing sperm damage, from cryopreserved bovine semen samples. Semen samples were thawed and centrifuged on a discontinuous Percoll gradient, or were filtered through a Sephadex G-15-120 column with the addition of ion exchangers. Sperm concentration, percentages of motile spermatozoa, acrosome integrity, superoxide dismutase activity and lipid peroxidation were evaluated in recovered samples and controls. The motility of spermatozoa obtained by Sephadex ion-exchange filtration (88.87 +/- 6.37%) and by Percoll gradient centrifugation (83.00 +/- 6.21%) were significantly greater than that of control samples (60.14 +/- 8.44%). Other results disclosed that both sperm selection methods significantly increased the percentage of intact acrosome and superoxide dismutase activity. In both cases, the number of recovered spermatozoa diminished significantly versus untreated samples. Although the number of recovered spermatozoa was low, these methods were effective to select viable sperm from cryopreserved bovine semen.

Animals↗
PubMed2006

Human semen refrigeration at + 4 degrees C: bio-kinetic characteristics.

The aim of our study was to evaluate the bio-kinetic characteristics of human semen refrigerated for different periods and to compare the effects of refrigeration at +4 degrees C against cryopreservation of human sperm at -196 degrees C. Semen was obtained from 30 male partners of infertile couples (infertile subjects) with the following semen profile: sperm count >or=10 x 10(6)/ml; progressive motility >or=20%; atypical forms <70% and white blood cells <1.0 x 10(6)/ml. Fifteen normospermic subjects were also selected as controls (control subjects). The following tests were carried out on basal, refrigerated and cryopreserved sperm: a) sperm kinetic properties (by Superimposed Image Analysis System); b) the Hypoosmotic Viability Test (HVT) (combined Hypoosmotic Swelling and Viability Test). The results of the study showed that the percentage recovery of kinetic properties and of HVT were optimum for up to 48 h. After refrigeration for 72 h, a drastic decrease in straight motility recovery was observed. No significant differences were observed between cryopreservation and refrigeration at +4 degrees C for 48 h for motility or HVT recoveries in samples from control subjects. However, in infertile subjects, a significant decrease in straight progressive motility and HVT recoveries was observed in cryopreserved samples compared to those refrigerated for 48 h. Neither refrigeration nor cryopreservation led to the growth of pathogenic bacteria in any of the cases studied. Based on the above results, refrigeration could represent a useful alternative to the cryopreservation method.

Case-Control Studies↗
PubMed2001

Transplantation of cryopreserved cardiomyocytes.

BACKGROUND: The present study examined the survival and rate of contraction of (1) cardiomyocytes cultured from cryopreserved fetal rat myocardium and (2) cryopreserved cultured cardiomyocytes. In addition, the effects of transplantation of cryopreserved fetal cardiomyocytes were evaluated. METHODS: Segments of fetal rat myocardial tissue (0.2, 2.0, and 6.0 mm(3) mince size) and cultured cardiomyocytes were cryopreserved in liquid nitrogen for 1, 2, and 4 weeks. After cryopreservation, the tissue samples and cultured cardiomyocytes were thawed at 37 degrees C and cultured, and cell proliferation and rate of contraction were determined. Cultured cryopreserved (n = 5) and noncryopreserved (control, n = 5) fetal cardiomyocytes were transplanted into the subcutaneous tissue and into a transmural left ventricular free wall scar of Sprague-Dawley rats (n = 3). The survival and rate of contraction of these transplanted cells were also examined. RESULTS: Cryopreservation of cultured fetal cardiomyocytes resulted in viable and functional cardiomyocytes although the cell number and percentage of beating cells were diminished. Survival of cardiomyocytes isolated from cryopreserved fetal myocardium was a function of tissue size before cryopreservation; the lowest survival was recorded in tissues with the largest mince size (6.0 mm(3)). The subcutaneous transplants contracted spontaneously and regularly with an idioventricular rhythm. In addition, the transplanted cardiomyocytes were elongated and formed a myocardium-like pattern with blood vessels present within the contractile tissue. In the transmural left ventricular scar, both control and experimental fetal cardiomyocyte transplants formed myocardium-like tissue. CONCLUSIONS: The present study uncovers the following key observations: (1) cryopreservation of fetal cardiomyocytes and cardiomyocytes isolated from cryopreserved myocardial tissue results in viable and functional cells, (2) cryopreserved fetal cardiomyocytes can be successfully transplanted into subcutaneous and myocardial scar tissue, and (3) improvements in cryopreservation techniques are required to augment the rates of cardiomyocyte survival observed in the study.

Animals↗
PubMed2001

Properties of human free apolipoprotein(a) and lipoprotein(a) after either freezing or lyophilization in the presence and absence of cryopreservatives.

Apolipoprotein(a), apo(a), the specific multikringle glycoprotein constituent of lipoprotein(a), Lp(a), occurs in the plasma mostly bound to apoB100-containing lipoproteins but also in a free form. Often the properties of these products are determined after storage in the cold; yet limited information is available on their stability at low temperatures. To shed light on this subject, we examined the effect of two parameters, freezing and lyophilization, in either the absence or the presence of cryopreservatives. Lp(a)s each having a single apo(a) size isoform containing either 14 or 17 kringle (K) IVs were isolated from the plasma of healthy donors by combining density gradient ultracentrifugation and lysine-Sepharose column chromatography using solutions containing both antioxidants and proteolytic inhibitors. Apo(a) was obtained from parent Lp(a) by a mild limited reductive procedure. Either freezing at -20 degrees C or lyophilization in the presence of 5% sucrose did not change the electrophoretic, immunochemical, and lysine-binding properties of Lp(a) including its ability to generate free apo(a). Irrespective of source, apo(a) remained stable when either frozen at -20 and -80 degrees C or lyophilized in the presence of 125 mM trehalose. In all cases, the absence of cryopreservatives caused the samples to aggregate irreversibly. Thawed or reconstituted samples of both free and bound apo(a) kept at 4 degrees C under sterile conditions in the presence of antioxidants, proteolytic inhibitors, and cryopreservative exhibited no significant changes in properties within the time of observation. Both apo(a) isoforms gave comparable results. We conclude that apo(a), either free or bound, can be kept stable at low temperatures in the presence of appropriate cryopreservatives.

Apolipoproteins A↗
PubMed2003

Biological sample collection and processing for molecular epidemiological studies.

Molecular epidemiology uses biomarkers and advanced technology to refine the investigation of the relationship between environmental exposures and diseases in humans. It requires careful handling and storage of precious biological samples with the goals of obtaining a large amount of information from limited samples, and minimizing future research costs by use of banked samples. Many factors, such as tissue type, time of collection, containers used, preservatives and other additives, transport means and length of transit time, affect the quality of the samples and the stability of biomarkers and must be considered at the initial collection stage. An efficient study design includes provisions for further processing of the original samples, such as cryopreservation of isolated cells, purification of DNA and RNA, and preparation of specimens for cytogenetic, immunological and biochemical analyses. Given the multiple uses of the samples in molecular epidemiology studies, appropriate informed consent must be obtained from the study subjects prior to sample collection. Use of barcoding and electronic databases allow more efficient management of large sample banks. Development of standard operating procedures and quality control plans is a safeguard of the samples' quality and of the validity of the analyses results. Finally, specific state, federal and international regulations are in place regarding research with human samples, governing areas including custody, safety of handling, and transport of human samples, as well as communication of study results.Here, we focus on the factors affecting the quality and the potential future use of biological samples and some of the provisions that must be made during collection, processing, and storage of samples, based on our experience in the Superfund Basic Research Program and Children's Environmental Health Center, at the University of California, Berkeley.

Humans↗
PubMed1995

Cryopreservation modifies flow-cytometric analysis of hemopoietic cells.

Although cryopreservation of human bone marrow has become very common in modern medicine, the knowledge on the effects of this procedure on hemopoietic cells is still limited. We herein have investigated whether the process of concentrating of human bone marrow to its buffy coat, the exposure to the cryoprotectant dimethyl sulfoide (DMSO), and/or the rate of controlled freezing/thawing procedures modifies the flow cytometric analysis of human bone marrow cells. We found that both the exposure of marrow cells to DMSO and/or the freezing procedure significantly modifies both the relative proportions of hemopoietic cell subsets and the intensity of expression of certain surface antigens. Thus, percentages of cells expressing CD7, CD13, CD33 and CD34, were found to be lower in both cryopreserved buffy coat and buffy coat merely exposed to DMSO, in comparison to those in untreated coat samples. Moreover, the intensity of the surface expression of CD33 decreased in cells exposed to DMSO, and further in cryopreserved samples. By contrast, the intensity of the CD19 antigen was higher in the last groups of samples than in the buffy coat or the unfractionated human bone marrow. Our present results suggest that flow-cytometric analysis of cryopreserved human bone marrow cells is not fully equivalent to that corresponding to fresh bone marrow or its fractionated buffy coat.

Antigens, CD↗
PubMed1997

Biomonitoring of possible human exposure to environmental genotoxic chemicals: lessons from a study following the wreck of the oil tanker Braer.

In January 1993 the oil tanker Braer ran aground in the Shetland Islands, Scotland. Approximately 80,000 tons of crude oil were released. Exceptionally high winds caused extensive pollution and exposure of the local population to crude oil. We describe the study which was immediately set in place to examine the exposed population for evidence of genotoxic exposure. Blood samples were taken and primary DNA damage was measured in the mononuclear cell fraction by the butanol modification of the 32P-postlabelling method. Mutation was measured at the hprt locus in T lymphocytes. No evidence of genotoxicity was obtained for either end point, but nevertheless, we believe that useful lessons were learnt, which should be incorporated into the design of future studies: (1) A rapid response is essential, and even if sufficient funds are not immediately available, it is still worth attempting to obtain samples quickly and use cryopreservation, also to attempt to estimate exposure. (2) Adequate numbers of volunteers must be sought, together with enough controls, not just to allow meaningful analysis but to overcome loss of samples and failure of things to go according to plan. (3) Points concerning laboratory practice include: (i) samples should be coded, (ii) clearly defined and proven protocols should be used, (iii) irreplaceable samples should not be used for method development, (iv) should a problem become apparent during the study, work on such samples should cease immediately until the problem is solved, (v) all critical experimental components should be pretested against a laboratory standard. (4) The study design should include replicate experiments to monitor experimental variability and reproducibility, as well as internal standards and cryopreserved "in house" samples. Care must be taken that samples from any one exposure group are spread between a number of independent experiments and that each experiment includes samples from a number of exposure groups. (5) A computerised data base should be maintained with full details of experimental variables, donor attributes, and raw data so that any contribution of experimental artefacts to "outlier" results can be monitored. (6) Because of the nature of the statistical variation for many environmental genotoxicity end points, only a large-scale study is likely to be capable of yielding useful information.

Accidents, Occupational↗