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PubMed2003

Field and in vitro assay of three methods for freezing ram semen.

Glycerol has been the most widely used cryopreservation agent for spermatozoa and a wide range of factors affect its action on sperm viability and fertilizing capacity. We tested three methods for freezing ram semen packed in 0.25 ml straws (final cellular concentration: 100 x 10(6) spz/ml). Method M1: Two-thirds of the final volume of diluent was added as solution A (without glycerol) to the pure semen at 35 degrees C. The sample was cooled to 5 degrees C (-0.30 degrees C/min), one-third of final diluent volume was added as solution B (final concentration of glycerol 4%) and the sample was maintained at 5 degrees C for 2h. It was then frozen in a programmable biofreezer (-20 degrees C/min down to -100 degrees C). Method M2: The sample was diluted with a specific solution at 35 degrees C (final concentration of glycerol 3%), cooled to 5 degrees C (-0.20 degrees C/min) and left for 2h. After that, it was frozen in nitrogen vapours. Method M3: Semen was diluted 1:1 in a specific solution (concentration of glycerol 2%) and cooled to 5 degrees C (-0.25 degrees C/min). The sample was then diluted again in the same solution to the final cellular concentration (final concentration of glycerol 4%). It was left for 1h at 5 degrees C and then frozen in a programmable biofreezer (-20 degrees C/min down to -100 degrees C). Best total motility (TM) and progressive motility (PM) (75.8 and 55.18%) were obtained using Method M3. Methods M1 and M3 gave significantly higher values (P<0.05) for kinetic parameters: average path velocity (VAP) (81.3 and 85.2 microm/s), straight-line velocity (VSL) (72.8 and 77.3 microm/s) and linearity (LIN) (66.6 and 68.8%). Method M2 showed the lowest kinetic parameters of motility (VAP 74.4, VSL 67.3 and LIN 62.5) and the highest percentage of cells with damaged plasma membrane (53.8%). Method M1 gave the worst results in viability and acrosome status assessed using fluorescence probes (31.3%-dead cells with damaged acrosomes-versus 25.4% in M2 and 23.3% in M3). A field trial carried out on fertility showed a significantly higher percentage of pregnant or lambing ewes (P<0.05) with Method M3 (67.3% versus 51.1% for M1 and 58.8% for M2). We concluded that the use of a simple dilution medium (test-fructose-glycerol-egg yolk) with the addition of glycerol (to 2% at 35 degrees C and to 4% at 5 degrees C) in two steps together with a programmable biofreezer was a productive method for freezing ram semen.

Acrosome↗
PubMed2000

Leukapheresis components may be cryopreserved at high cell concentrations without additional loss of HPC function.

BACKGROUND: The aim of this study was to assess the feasibility of freezing mobilized peripheral blood progenitor cell (PBPC) components at higher cell concentrations than are classically recommended for bone marrow. This approach might have potential benefits, such as lower cost of processing and storage and less risk of the complications associated with the transfusion of large component volumes and large quantities of DMSO. STUDY DESIGN AND METHODS: In the first phase, small aliquots of 19 apheresis components were cryopreserved at standard and higher cell concentrations (Aliquots A and B, respectively). In the second phase, 21 apheresis components were split into two bags each and frozen at standard (Bag A) and high (Bag B) cell concentrations. The differences in viability, cloning efficiency, and nucleated cell recovery in Bags A and B were examined. Finally, the hematologic recovery of 10 patients who underwent autologous transplantation with PBPC components frozen at high cell concentrations was analyzed. RESULTS: The median cell concentration at freezing was 94 (57-100) x 10(6) per mL and 291 (220-467) x 10(6) per mL for Aliquots A and B, respectively, and 90.9 (45.4-92) x 10(6) per mL and 332 (171-582) x 10(6) per mL for Bags A and B, respectively. The viability was significantly lower in samples frozen at higher cell concentrations: 92 versus 83 percent (p = 0.001) and 87 versus 77 percent (p<0.001) for Aliquots and Bags A and B, respectively. Significant differences were not observed in the recovery of total nucleated cells (102 vs. 101% and 98 vs. 105%) or the cloning efficiency after thawing (13 vs. 16% and 27 vs. 23%) for Aliquots and Bags A and B, respectively. The time to granulocyte engraftment >0.5 x 10(9) per L and platelet engraftment >20 x 10(9) per L was 9 (8-11) and 10.5 (7-21) days, respectively. CONCLUSION: The cryopreservation of PBPC components at standard concentrations and 3.3 (1.8-6.2)-fold cell concentrations has no adverse effect on the function of HPCs after thawing.

Blood Preservation↗
PubMed2006

Methods for reverse genetic screening in zebrafish by resequencing and TILLING.

Animal models provide an in vivo system to study gene function by transgenic and knockout approaches. Targeted knockout approaches have been very successful in mice, but are currently not feasible in zebrafish due to the inability to grow embryonic stem cells. As an alternative, a reverse genetic approach that utilizes screening by resequencing and/or TILLING (Targeting Induced Local Lesions INGenomes) of mutagenized genomes has recently gained popularity in the zebrafish field. Spermatogonia of healthy males are mutagenized using ENU (N-ethyl-N-nitrosourea) and F1 progeny is collected by breeding treated males with healthy wild type females. Sperm and DNA banks are generated from F1 males. DNA is screened for ENU-induced mutations by sequencing or TILLING. These mutations can then be studied by in vitro fertilization (IVF) from the cryopreserved sperm of the corresponding F1 male followed by breeding to homozygosity. A high-throughput method of screening for rare heterozygotes and efficient recovery of mutant lines are important in identification of a large number of mutations using this approach. This article provides optimized protocols for resequencing and TILLING based on our experiences. We performed a pilot screen on 1235 F1 males by resequencing 54 exons from 17 genes and analyzed the sequencing data using multiple programs to maximize the mutation detection with minimal false positive detection. As an alternative to sequencing, we developed the protocols for TILLING by capillary electrophoresis using an ABI Genetic analyzer 3100 platform followed by fragment analysis using GeneScan and Genotyper softwares. PCR products generated by fluorescently labeled universal primers and tailed exon-specific primers were pooled 4-fold prior to heteroduplex formation. Overall, our pilot screen shows that a combination of TILLING and sequencing is optimal for achieving cost-effective, high-throughput screening of a large number of samples. Amplicons with fewer common SNPs are ideal for TILLING whereas amplicons with multiple SNPs and in/del polymorphisms are best suited for sequencing followed by analysis with SNPdetector.

Animals↗
PubMed2006

Sperm characteristics and DNA integrity of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa frozen in the presence of enzymatic and nonenzymatic antioxidants.

The main goal of this study was to investigate the potential protective effects of enzymatic and nonenzymatic antioxidants on cryopreservation injuries to red deer epididymal spermatozoa. In Experiment 1, the effects on sperm freezability of the enzymatic antioxidants catalase, superoxide dismutase, and a combination thereof were studied. In Experiment 2, sperm cryoresistance was evaluated when different nonenzymatic antioxidants, such as vitamin E, vitamin C, and butylated hydroxytoluene (BHT), were added to the freezing extender. Sperm quality was judged in vitro by microscopic assessments of individual sperm motility (SMI), viability, and acrosome (ie, spermatozoa with normal apical ridges; % NAR) and membrane (by means of the HOS test) integrity. To address fully these topics, we incorporated a new set of functional sperm tests for mitochondrial function, membrane phospholipid disorder, and sperm chromatin stability. Samples were evaluated after freezing and thawing, and after a 2-hour period of incubation at 37 degrees C. The present study demonstrates that the addition of enzymatic antioxidants to freezing extenders improves sperm viability after cooling, and improves sperm motility, acrosome integrity, and mitochondrial status (P<.05) after thawing. After a 2-hour incubation period at 37 degrees C in the presence of enzymatic antioxidants, an improvement in membrane integrity (P<.05) was observed. However, when nonenzymatic antioxidants were present in the freezing diluents, no positive effects on thawed sperm parameters were noted. The chromatin stability test did not show significant differences between the treatments. We conclude that enzymatic antioxidants should be present in the early steps of cryopreservation of epididymal spermatozoa from red deer, so as to improve motility and acrosome integrity.

Animals↗
PubMed1997

Meniscal allografts: cryopreservation vs deep-frozen technique. An experimental study in goats.

Meniscal transplantation was performed in two groups of 15 adult goats each, using cryopreserved (group I) and deep-frozen (group II) allografts. Animals were killed at 2 weeks, 1, 3, 6 and 12 months, and a gross, histological and biochemical (water and glycosaminoglycan) evaluation of the menisci was performed. The allografts of both groups showed a normal gross appearance and had in most cases healed at the horn attachments and at the peripheral capsular tissue with a dense scar tissue and no signs of rejection. Histological analysis showed that at 2 weeks in group I the cell number was decreased compared with the controls, and the cells were mainly distributed in the superficial layers. In group II at 2 weeks, only a few cells were present at the peripheral attachment of the menisci. At 1 month in both groups, the cell repopulation can be seen extending from the peripheral area to the superficial layers. Cell proliferation and vascularization are particularly evident in both groups in the 3-month samples. At 6 months and 1 year the grafts can be seen to be completely remodelled and morphologically similar to normal menisci in both groups. Biochemical analysis showed in both groups an increase in water content and a progressive decrease in the concentration of glycosaminoglycans. At 1 year in both groups, there were moderate degenerative changes in the articular cartilage of the tibial plateau, which were more evident in the area of exposed cartilage than in that covered by the meniscus. These results suggest that there are no significant differences between the cryopreserved and deep-frozen grafts, and that even if cryopreservation makes it possible to maintain a partial cell viability in the tissue, this does not seem to improve the morphological and biochemical characteristics of the graft.

Animals↗
PubMed1996

Effect of biopsy and vitrification on in vitro survival of ovine embryos at different stages of development.

The objective of the present study was to assess the in vitro viability of ovine embryos at different stages of development after combining cell sampling and vitrification. Precompacted morulae, compacted morulae and blastocysts were obtained from superovulated Sarda ewes at 4, 5 or 6 d following insemination. Embryo cell biopsy was carried out in a 100-microl drop of PBS + 10% fetal calf serum (FCS) with 10 micromol nocodazole and 7.5 microg/ml cytochalasin-b by aspiration (3-5 cells). Embryos were cryopreserved at room temperature after exposure of 2 solutions for 5 min, transferred into a vitrification solution, loaded into the center of 0.25-ml straws separated by air bubbles from 2 columns of sucrose 0.5 M and plunged immediately into liquid nitrogen. In Experiment 1, the in vitro viability of manipulated or vitrified embryos after in vitro co-culture in TCM 199 medium with 10% FCS and sheep oviductal epithelial cells (SOEC) in 5% CO2 humidified atmosphere in air at 39 degrees C was significantly lower (P < 0.05 and P < 0.01, respectively) at precompacted morula (60 and 30%) and compacted morula (62 and 39%) stages than intact embryos at the same stages (87 and 88%). No differences were found at the blastocyst stage. In Experiment 2, the in vitro survival rate of precompacted morulae which were manipulated and immediately vitrified was lower (P < 0.05) than in those manipulated and, after a temporary period of culture, vitrified at blastocyst stage (21 vs 48%); while no differences were found at compacted morula and blastocyst stages. The results show that 1) the stage of development influences the subsequent in vitro viability of manipulated and vitrified ovine embryos, 2) temporary culture after manipulation and before vitrification improves the in vitro viability of embryos, and 3) the hole in the zona pellucida resulting from biopsy does not affect blastocyst survival after subsequent vitrification.

Journal Article↗
PubMedDate not supplied

Enumeration of previously frozen platelets using the Coulter Counter, phase microscopy, and the technicon optical system.

The Coulter Counter, phase microscopy, and the Technicon optical system were used to enumerate platelets in samples of whole blood, platelet-rich plasma, platelet concentrates before and after addition of DMSO, and on platelet concentrates that had been frozen, thawed, and washed. We observed agreement among the three counting methods when platelet counts were determined in whole blood, platelet-rich plasma, and platelet concentrate before and after DMSO addition. Enumeration after the cryopreservation process, however, showed highly significant differences among the counting systems. Platelet counts on platelet concentrates after freezing the thawing performed with the Coulter Counter were 25 per cent greater than with phase microscopy and 55 per cent greater than with Technicon. Counts with phase microscopy were 30 per cent greater than Technicon values. These data indicate that the method used to enumerate previously frozen platelets affects the apparent platelet count.

Cryoprotective Agents↗
PubMed2000

Suppression of LH during ovarian stimulation: effects differ in cycles stimulated with purified urinary FSH and recombinant FSH.

There has been much debate about the role of luteinizing hormone (LH) during follicle stimulating hormone (FSH)-treated ovarian stimulation for assisted reproduction, where the endogenous LH is suppressed using a gonadotrophin-releasing hormone analogue. The requirement for LH in oestradiol biosynthesis is established, but other effects of 'insufficiency' are less clear, and little attention has been paid to the specific origin of the FSH used. The aim of this study was to examine the roles of profoundly suppressed circulating LH concentrations in cycles of ovarian stimulation for IVF, which were affected in two large separate cohorts of patients undergoing assisted reproduction. They were stimulated by either purified urinary FSH (MHP) or recombinant human FSH (rFSH). Within each dataset, outcomes were examined with respect to the circulating concentrations of LH in the mid-follicular phase, as plasma samples were stored prospectively, and assayed retrospectively. Patients with profoundly suppressed LH showed much reduced oestradiol concentrations at mid-follicular phase and at human chorionic gonadotrophin administration in cycles treated with either MHP or rFSH. However, gross ovarian response, as became evident by FSH dose demands, duration of stimulation, and also oocyte and embryo yields and embryo cryopreservation were influenced only in cycles treated with MHP. Furthermore, no effect upon pregnancy survival was observed. Thus, it is concluded that there is a demand for additional exogenous LH treatment only in cycles treated with purified urinary FSH where the LH is profoundly suppressed.

Abortion, Spontaneous↗
PubMed1993

Leishmania donovani parasites in the nasal secretions, tonsillopharyngeal mucosa, and urine centrifugates of visceral leishmaniasis patients in Kenya.

In the early 1930s, investigators of visceral leishmaniasis stated that Leishman-Donovan bodies are found in body fluids of kala-azar patients, for example, in urine, feces, semen, and nasal and pharyngeal secretions. Based on this finding, we investigated the diagnostic potential of nasal secretions, tonsillopharyngeal mucosal swabs, and urine centrifugates inoculated into Schneider's Drosophila Medium (containing antibiotics and antifungal agents) as well as with Giemsa-stained smears. Consequently, 64 randomly selected patients with visceral leishmaniasis from Kenya (59 who were splenic culture or Giemsa stain positive and five who were culture negative but Giemsa stain positive) were tested by three noninvasive methods. These tests were all performed before the patients were treated with Pentostam. Cultures of nasal and tonsillopharyngeal swabs and urine centrifugates produced 28 positive samples representing 24 patients (37.5%). Moreover, a set of 25 Giemsa-stained slide smears made from the nasal and tonsillopharyngeal mucosa of 25 patients with visceral leishmaniasis who had not tested positive in cultures produced nine positives. Therefore, the overall total of patients who tested positive by all of the above methods was 33 or 51.6%. The cryopreserved Leishmania isolates were characterized by cellulose acetate electrophoresis using 20 enzyme systems. The isoenzyme profiles produced by the parasites were represented in five different L. donovani s.l. zymodemes. Representatives of these isolates were also characterized by DNA Southern blotting analysis, which corroborated the isoenzyme results.

Adolescent↗
PubMed2005

Effect of semen collection method on pre- and post-thaw Guirra ram spermatozoa.

In this study, we evaluated the potential effect of the method of recovery (artificial vagina or electroejaculation) on the production and quality of Guirra ram spermatozoa cryopreserved for the possible constitution of a sperm bank. In order to address this question, we evaluated the effect of semen collection method on fresh semen quality parameters, including: volume, concentration, production, microscopic analysis (abnormal sperm and intact apical ridge) and sperm motility parameters determined by CASA system. For frozen-thawed semen, we evaluated motility parameters by CASA and intact apical ridge, acrosomal status, assessed by dual staining by IP and FITC-PNA and capacitation status, assessed by M540 and Yo-pro1, using flow cytometry. The main findings from this study were: (i) that electroejaculation resulted in a lower recovery efficiency (80% of the cases), as a consequence of contamination with urine or lack of response to the electrical stimulation; (ii) the fresh seminal quality was not significantly different between recovery methods, except for the concentration of spermatozoa, but total number of spermatozoa and the consequent number of possible seminal doses for artificial insemination were similar; and, (iii) a higher number of stable and functional spermatozoa (higher number of live non-capacitated cells, higher live acrosome intact cells and live acrosome reacted cells) were found for frozen-thawed spermatozoa collected by electro ejaculation than by artificial vagina. According to our results, we are able to develop both methodologies in the creation of the Guirra sperm bank. Assuming the advantages and limitations of both methodologies, in Guirra breed, would enable the rapid constitution of a sperm bank including samples from a large number of non-trained rams in a short period of time, which will increase the genetic variability, and so guarantee the conservation of this breed.

Acrosome↗
PubMedDate not supplied

Control of Campylobacter fetus in artificially contaminated bovine semen by incubation with antibiotics before freezing.

Fresh, diluted semen containing 1.55 X 10(6) cfu/ml of Campylobacter fetus subsp. venerealis was incubated with 500 iu of penicillin, 500 micrograms of streptomycin, 160 micrograms of lincomycin and 300 micrograms of spectinomycin per ml at 35 degrees C for 0, 1, 2, 5, 10, 20 or 40 minutes. The semen was cooled to 5 degrees C, packaged in 0.25 ml French straws and then frozen in liquid nitrogen for 2 weeks. Immediately after thawing and removal of the antibiotics by centrifugation semen samples from each of the seven treatment groups were cultured as for C. fetus. Semen samples were also examined by in-vitro tests for sperm motility prior to and post-freezing. Incubation with the antibiotics for 5, 10, 20 or 40 min prior to freezing reduced the numbers of C. fetus in the semen to non-detectable levels in 38%, 69%, 88% and 100% of samples respectively. The incubated semen showed no significant reduction of sperm motility although fertility trials have not been done.

Animals↗
PubMed1989

The detection of group-specific component from urine samples.

The detection of group-specific component (Gc) from serum and bloodstains has been widely used in the forensic laboratory. A recent increase in substituted or adulterated urine samples in various drug screening programs has necessitated methods to determine the donor. This paper discusses the detection of Gc from urine samples. The samples were concentrated and applied to ultrathin polyacrylamide gel and focused for 150 min. This method separates the samples into the three common phenotypes found in all human populations. A nitrocellulose membrane blotting technique was used to detect the Gc bands. Serum and urine samples were collected from each individual and were typed for Gc. Urine samples tested after 6 months of storage (4 degrees C) were still readable. This method provides the forensic laboratory with an additional test from a body fluid which, until recently, provided little information.

Cryopreservation↗
PubMed2005

Bio-banking in microbiology: from sample collection to epidemiology, diagnosis and research.

Millions of biological samples, including cells of human, animal or bacterial origin, viruses, serum/plasma or DNA/RNA, are stored every year throughout the world for diagnostics and research. The purpose of this review is to summarize the resources necessary to set up a bio-banking facility, the challenges and pitfalls of sample collection, and the most important techniques for separation and storage of samples. Biological samples can be stored for up to 30 years, but specific protocols are required to reduce the damage induced by preservation techniques. Software dedicated to biological banks facilitate sample registration and identification, the cataloguing of sample properties (type of sample/specimen, associated diseases and/or therapeutic protocols, environmental information, etc.), sample tracking, quality assurance and specimen availability. Bio-bank facilities must adopt good laboratory practices and a stringent quality control system and, when required, comply with ethical issues.

Animals↗
PubMed2003

Vitrification of ECV304 cell suspensions using solutions containing propane-1,2-diol and trehalose.

In this paper, we report on the suitability of solutions containing propane-1,2-diol (propylene glycol, PD), sugars, and salts for the vitrification of the human cell line, ECV304. Cooling (at 10 degrees C/min) and rewarming (at 80 degrees C/min) were at rates that are practicable for the tissues to be studied later. Under these conditions, 45% PD in phosphate-buffered saline (PBS) sometimes froze during cooling and always devitrified during rewarming but both events were avoided if the PBS salts were replaced by an osmotically equivalent concentration of sucrose or trehalose. The effect of such solutions on cells was evaluated using a cell culture assay in which the number of cells recovered after 3 days of culture was divided by the number cells plated, giving a cell multiplication factor or CMF. In the absence of PD the cells tolerated a low-salt concentration in solutions that were made isotonic with sugars, but they recovered poorly when 45% PD was also present. Trehalose gave significantly better recovery than sucrose. When 39% PD and 15% trehalose were included in a low-salt vehicle solution (LSV) that contained approximately 5% of the total salt concentration of PBS (this solution was designated LSV/39/15), the cells exhibited approximately 40% of untreated control CMF following exposure for 9min. LSV/39/15 vitrifies with a glass transition temperature of -102 degrees C, does not devitrify when warmed at 80 degrees C/min, and has suitable dielectric properties for uniform and rapid dielectric heating. An improved method for adding and removing LSV/39/15 gave a CMF of approximately 55% of untreated controls. Using this method, 1.0ml suspensions of ECV304 cells was cooled to, and stored briefly at, -120 degrees C and then rewarmed by immersion in a 37 degrees C water bath ( approximately 75 degrees C/min). The CMF of the cooled samples was similar to that of the exposure-only controls, approximately 50% of the untreated control CMF in both cases.

Calorimetry, Differential Scanning↗
PubMed1991

Storage and preparation of samples for erythropoietin radioimmunoassay.

The evaluation of a radioimmunoassay for erythropoietin developed using recombinant material as immunogen and radiotracer is presented. A series of serum samples prepared and stored under varying conditions showed that immunoreactive erythropoietin levels were stable at room temperature for at least 10 days and at -20 degrees C for 5 months. The optimum time for separating sera from samples was between 6 and 24 h after venepuncture. Serum EPO values were significantly higher than those measured in heparin or potassium EDTA plasma.

Antibodies↗
PubMed2003

Anti-hepatitis A virus immunoglobulin M antibodies in urine samples for rapid diagnosis of outbreaks.

The main goal of this study was to test the feasibility of using urine for diagnosing hepatitis A virus (HAV) infections. A correlation of 90.78% between the test results of urine and serum samples was obtained. Four outbreaks of hepatitis A were confirmed by testing only urine samples. The levels of anti-HAV immunoglobulin M (IgM) antibodies in urine samples remained stable during 6 months of storage at -70 degrees C but decreased when the samples were stored at 4 degrees C. The results of tests of samples obtained 2 and 6 months after infection suggested that IgM levels decline more rapidly in urine than in serum.

Case-Control Studies↗
PubMed1995

P-glycoprotein expression in acute myeloblastic leukemia analyzed by immunocytochemistry and flow cytometry.

Using the APAAP technique, we assessed the reactivity of five monoclonal antibodies, JSB1, C219, Mab 57, 2F8 and MRK16, to gp 170 in 60 cases of de novo acute myeloid leukemia (AML) and 13 relapses. Reactivity, varied between the five antibodies, and positivity was obtained with 2F8 > JSB1 > MRK16 > Mab57 > C219. Sixteen of the 60 cases were also studied by flow cytometry. In 10 cases, the results with the two techniques corresponded, in the other 6 cases, flow cytometry proved more sensitive than APAAP in detecting small amounts of gp170. In the flow cytometry analysis, the cells fixed in methanol and paraformaldehyde were more fluorescent than unfixed samples or those fixed in paraformaldehyde or methanol alone. Our results thus reveal that positivity for gp 170 depends on various factors, including the specificity of the monoclonal antibodies, the techniques used and the preservation of the samples. This suggests the need for a clear standardization of the methods to detect gp170.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Safety of cryopreservation straws for human gametes or embryos: a preliminary study with human immunodeficiency virus-1.

The aim of this preliminary experimental study was to test the stability of cryopreservation straws to human immunodeficiency virus-1 (HIV-1). Three kinds of straws were tested: four polyvinyl chloride (PVC), four polyethylene terephthalate glycol (PETG) and 20 high-security ionomeric resin (IR). The PVC and PETG straws were sealed ultrasonically, and the IR straw by thermosoldering. Each sealed straw was cut in half to produce two demi-straws and then filled with 100 microl of HIV-1-containing supernatant (reverse transcriptase activity: 15 000 c.p.m./50 microl). The unsealed cotton end of PVC and PETG straws and the two halves of the IR straws (cotton and plastic plug ends) were tested. Each demi-straw was two- thirds submerged in RPMI medium at 37 degrees C, and RPMI samples were withdrawn on days 3, 7 and 11. Viral RNA was extracted from the medium and then amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) followed by nested PCR using primers specific to HIV-1 protease. On day 7, no HIV-1 RNA was detected in any of the different samples of medium that had surrounded the unsealed PVC and PETG straws with cotton ends, but three IR specimens were positive. On day 11, PVC and PETG remained negative but HIV-1 RNA was detected in RPMI samples for two more IR demi-straws (n = 5). In conclusion, under these experimental conditions (at 37 degrees C), the unsealed cotton end PVC, PETG and thermosoldered cotton end IR demi-straws appeared to be safe for HIV-1, while IR straws, sealed or unsealed with a plastic plug and with unsealed cotton ends, leaked.

Cryopreservation↗