The pain symptoms and mass recurrence rates after ovarian cystectomy or uni/bilateral oophorectomy procedures in patients over 40 years old with endometriosis.

OBJECTIVES: To evaluate the rates of pain and mass recurrence of the patients over 40 years old with endometriosis who underwent ovarian cystectomy or uni/bilateral oophorectomy. MATERIAL AND METHODS: A retrospective study was conducted with 98 patients undergoing laparoscopic surgery for endometriosis in a tertiary referral center between the time period July 2015 and July 2019. All the patients followed every 3 months and requested to fill the Visual Analogue Scale (VAS) for evaluation of pelvic pain and an ultrasound scan was performed. The inclusion criteria for this study were as follows, patients with ages over 40, with regular menstrual periods, and who denied hysterectomy and any postoperative hormonal medical treatments. RESULTS: When the groups were compared in terms of age, body mass index, cyst diameter, CA-125 serum concentrations, preoperative and after surgical pelvic pain scores, mean follow up periods, postoperative hospital stay. However, each of the mean numbers of gravidity and parity were significantly higher than bilateral salpingo-oophorectomy (BSO) groups compared to the other groups (p = 0.04 and p = 0.03, respectively). The laterality, the recurrence rates, and the type of recurrence did not have a significant effect in the group comparison. CONCLUSIONS: The ovarian tissue preserving procedures could be offered for the women over 40 years old suffering from endometriosis with no significant increase in pain symptom or mass recurrence rates considering beneficial effects of estrogen on cardiovascular system, vasomotor symptoms, and bone mineral density.

Frequency of Complications During Preparation of Corneal Lamellae Used in Posterior Lamellar Keratoplasty Using the Pneumodissection Technique (Big Bubble).

PURPOSE: To determine the frequency of formation of various types of bubbles and the potential impact of donor and lamella parameters on this frequency, and to identify possible risk factors of unsuccessful "big-bubble" creation in preparation of pre-Descemet endothelial keratoplasty and Descemet membrane endothelial keratoplasty with peripheral stromal support. METHODS: Donor age and sex, death to preservation time (DPT), storage time, presence of corneal scars (mainly a condition after cataract surgery), and endothelial cell density of 256 donor corneas were assessed before Descemet membrane endothelial keratoplasty with peripheral stromal support or pre-Descemet endothelial keratoplasty lamella preparation using the big-bubble technique. RESULTS: Mean donor age was 62.3 ± 8.5 years (28.3% women and 71.7% men). Mean endothelial cell density of the donor graft was 2866 ± 255 cells/mm. Mean DPT was 10.12 ± 4.88 hours, and mean storage time of the transplant before surgery was 6.5 ± 4.8 days. Corneal scars were present in 17 donor grafts (6.6%) after cataract surgery. Eleven corneas were devalued because of Descemet membrane rupture during preparation (4.3%). In 182 corneas, standard bubble type I was created (71.7%); in 27 corneas, bubble type II was created; eventually, both types of bubbles formed simultaneously (10.5%); in 47 corneas, no bubble was created (18.4%). CONCLUSIONS: We identified higher endothelial cell density, shorter DPT, and the presence of corneal scars after cataract surgery as risk factors threatening successful bubble formation. The only risk factor for creating type II bubbles was higher donor age in our study.

Hypothermic continuous machine perfusion enables preservation of energy charge and functional recovery of heart grafts in an ex vivo model of donation following circulatory death.

OBJECTIVES: Cardiac transplantation using hearts from donors after circulatory death (DCD) is critically limited by the unavoidable warm ischaemia and its related unpredictable graft function. Inasmuch as hypothermic machine perfusion (MP) has been shown to improve heart preservation, we hypothesized that MP could enable the use of DCD hearts for transplantation. METHODS: We recovered 16 pig hearts following anoxia-induced cardiac arrest and cardioplegia. Grafts were randomly assigned to two different groups of 4-h preservation using either static cold storage (CS) or MP (Modified LifePort© System, Organ Recovery Systems©, Itasca, Il). After preservation, the grafts were reperfused ex vivo using the Langendorff method for 60 min. Energetic charge was quantified at baseline, post-preservation and post-reperfusion by measuring lactate and high-energy phosphate levels. Left ventricular contractility parameters were assessed both in vivo prior to ischaemia and ex vivo during reperfusion. RESULTS: Following preservation, the hearts that were preserved using CS exhibited higher lactate levels (57.1 ± 23.7 vs 21.4 ± 12.2 µmol/g; P < 0.001), increased adenosine monophosphate/adenosine triphosphate ratio (0.53 ± 0.25 vs 0.11 ± 0.11; P < 0.001) and lower phosphocreatine/creatine ratio (9.7 ± 5.3 vs 25.2 ± 11; P < 0.001) in comparison with the MP hearts. Coronary flow was similar in both groups during reperfusion (107 ± 9 vs 125 ± 9 ml/100 g/min heart; P = ns). Contractility decreased in the CS group, yet remained well preserved in the MP group. CONCLUSION: MP preservation of DCD hearts results in improved preservation of the energy and improved functional recovery of heart grafts compared with CS.

[Ovocyte vitrification: a tool for the future]. / La vitrification ovocytaire : un outil d'avenir.

Ovocyte vitrification was finally authorized by the new law voted in July 2011 upon the revision of the French bioethics law. Expected for 30 years, cryopreservation of female gametes had a major impact on Assisted Reproductive Technologies (ART) practice worldwide and in our country. It brought tremendous changes in the field of reproductive biology, from intraconjugal infertility management to gamete donation, through autologous cryopreservation for fertility preservation. Although it appears to be "obvious", ovocyte vitrification seems to be barely used as a routine technique in French IVF laboratories. We will discuss the events that led to the present situation. We will also tackle the expected benefits of ovocyte vitrification especially as an alternative to embryo freezing.

Donor and tissue profile of a community eye bank in Eastern India.

PURPOSE: The purpose of this study is to analyze the donor and tissue profile of a community eye bank in Eastern India. MATERIALS AND METHODS: Eye bank records were analyzed for the period July 2007-June 2011. Variables analyzed included donor demographics (age, gender, and ethnicity), donor cause of death, consent for recovery, death-to-preservation interval, preservation-to-utilization interval, endothelial cell density (ECD), corneal suitability for transplantation, and corneal tissue utilization. RESULTS: During this study period, 743 corneal tissues were retrieved from 373 donors (male:female = 263:110). The mean age of donors was 52 ± 21 years (range: 3-95 years). The most common donor age group was 41-50 and 71-80 years. Most of the donors belonged to one religious faith (99%). The most common causes of death were cardiorespiratory failure (34%) followed by road traffic accident (30%). Majority donors were motivated (n = 320; 86%), and remaining (n = 53; 14%) were voluntary. Most of the consents were given by sons or daughters of the deceased (45%) followed by siblings (18%). Mean death-to-preservation interval was 3.9 ± 1.9 h. Mean preservation-to-utilization interval was 56.0 ± 24.4 h. The mean ECD of donor corneal tissue was 2857 ± 551 cells/mm 2 and the median value was 2898 cells/mm 2 . Of harvested corneas 556 (75%) corneal tissues were utilized. The most common causes of nonutilization were septicemia in donor (n = 56; 30%) and poor quality of tissue (n = 55; 30%). CONCLUSIONS: Although, there is significant corneal tissue utilization, there is a need for increased awareness among people in order to augment voluntary donations.

Cryopreservation versus subcutaneous preservation of autologous bone flaps for cranioplasty: comparison of the surgical site infection and bone resorption rates.

OBJECTIVE: Decompressive craniectomy is performed to treat malignant brain hypertension. Surgical site infection (SSI) and bone resorption are common complications following cranioplasty, and the storage method that minimizes such complication has yet to be identified. METHODS: Over a 10-year period, the details of 290 decompressive craniectomy procedures performed at our trauma and stroke center were recorded. Bone flaps from 110 patients were preserved in subcutaneous pockets (SPs), and 180 were preserved via cryopreservation (CP). RESULTS: SSIs occurred in 20 cases (18.2%) in the SP group and 20 cases (11.1%) in the CP group (P=0.129). After dividing each group according to the traumatic brain injury (TBI) etiologies, we found that in the SP group, the SSI rates in the TBI and non-TBI patients were 17.3% and. 20.7% (P=0.899), respectively, and in the TBI- and non-TBI CP-group patients, the SSI rates were 11.9% and. 9.7% (P=0.864), respectively. The average decrease in bone flap thicknesses were 1.14 mm in the SP group (n=34) and 1.89 mm in the CP group (n=57), and this difference was significant (P=0.039). CONCLUSIONS: In this series, the SSI rates were similar in the SP and CP groups. There was no significant difference when the patients were grouped by TBI etiology. The incidence of bone flap resorption in the CP group was higher than that in the SP group. However, identifying of the method that yields superior results might depend on the individual surgeon's preference and the available equipment.

The influence of Australian eye banking practices on corneal graft survival.

OBJECTIVE: To identify eye banking practices that influence corneal graft survival. DESIGN, SETTING AND PARTICIPANTS: Prospective cohort study of records of 19,254 followed corneal grafts in 15160 patients, submitted to the Australian Corneal Graft Registry between May 1985 and July 2012. MAIN OUTCOME MEASURES: Influence of corneal preservation method (organ culture, moist pot, Optisol, other); death-to-enucleation, death-to-preservation and enucleation-to-graft times; transportation by air; graft era; and indication for graft on probability of graft survival at most recent follow-up. RESULTS: In multivariate analysis, 919 penetrating grafts performed using corneas transported interstate by air exhibited worse survival than 14,684 grafts performed using corneas retrieved and used locally (hazard ratio [HR], 1.44; 95% CI, 1.21-1.73; P = 0.001). This was also the case for traditional lamellar grafts (64 corneas transported by air and 813 used locally; HR, 1.69; 95% CI, 1.03-2.78; P = 0.038). Indication for graft influenced survival of penetrating grafts (4611 keratoconus, 727 emergency or high-risk, 10,265 other indication; global P < 0.001) and traditional lamellar grafts (65 keratoconus, 212 emergency or high-risk, 600 other indication; global P < 0.001). The preservation medium in which corneas used for traditional lamellar grafts were stored exerted a marginal influence on graft survival (global P = 0.047). CONCLUSIONS: Donor corneas transported interstate exhibited poorer survival after transplantation than those retrieved and grafted locally. Higher proportions of emergency procedures involving transported corneas did not account for this difference. Where possible, efforts to avoid transportation of corneal tissue by air freight within Australia may be warranted.

[Effect of multiple freeze-thaw cycles on detection of IgA, IgG and IgM antibodies to selected bacterial antigens]. / Wplyw wielokrotnego, cyklicznego zamrazania i rozmrazania próbki surowicy na poziom przeciwcial klasy IgA, IgG i IUgM dla wybranych antygenów bakteryjnych.

INTRODUCTION: In presented study we investigated the effect of multiple freeze-thaw cycles of human sera on the determination of IgA, IgG and IgM antibodies to selected bacterial antigens. METHODS: A panel of 15 serum samples with elevated levels of antibodies to Mycoplasma peumoniae, Yersinia enterocolitica and Salmonella spp. were used (5 positive sera for each pathogen). One set of aliquots designed as the baseline, was taken and stored at 4-8o C for the remainder for the study. The remaining seven sets of aliquots were divided into two parts and repeatedly frozen respectively at two different temperatures: -65 degrees C and -25 degrees C. Once a day the aliquot sets were removed from the freezer and allowed to stand at room temperature for approximately 1 h until completely thawed. For the determination of the level of antibodies the sera after: 2, 5, 10, 15, 20, 25 and 30 freeze/cycle were used. The measurement of IgA, IgG and IgM antibodies was done using a home-made ELISA with four different antigens: whole-cell antigen of M. pneumoniae FH strain, LPS and Yop antigens of Y. enterocolitica serotype O:3 and LPS extracted by Westphal method from Salmonella serogroup B +D. The results were presented as the arithmetic mean of the antibody titre in five sera which were treated by the same number of freeze-thaw cycles. RESULTS: There was no significant statistic difference between levels of antibodies in unfrozen and frozen sera even after 30 freeze-thaw cycles. Depending of the antigen used in ELISA a slight varations in the level of antibodies were observed but the changes were small and not clinically significant. Examination of the ELISA values does not suggest any consistent nonlinear trend in levels of IgA, IgG and IgM antibodies in sera frozen at -65 degrees C as well at -25 degrees C. CONCLUSIONS: Our study demonstrates that the IgA, IgG and IgM antibody activity levels measured for M. pneumoniae, Y enterocolitica and Salmonella antigens are stable even after 30 freeze-thaw cycles.

Bone flap storage following craniectomy: a survey of practices in major Australian neurosurgical centres.

INTRODUCTION: The resurgence of decompressive craniectomy surgeries for management of intracranial hypertension has led to a parallel increase in cranioplasty procedures for subsequent reconstruction of the resultant extensive skull defects. Most commonly, cranioplasties are performed using the patients' own cryopreserved skull flaps. Currently, there are no standardized guidelines for freeze-storage of bone flaps either nationally or internationally. In this initial study, the authors surveyed major neurosurgical centres throughout Australia to document current clinical practices. METHODOLOGY: Twenty-five neurosurgical centres affiliated with major public, teaching hospitals in all Australian states were included in the current survey study. A standardized survey guide incorporating standardized questions was used for data collection either by phone interviews and/or electronic (email) communication. Details regarding bone flap preparation following craniectomy, temperature and duration of freeze-storage, infection control/micro-contamination detection protocols, pre-implantation procedures were specifically recorded. RESULTS: Cranioplasty using cyropreserved autogenous bone flaps remains the most common (96%) mode of skull defect reconstruction in major neurosurgical centres throughout Australia. Following the initial craniotomy, the harvested skull flaps were most frequently (88%) double- or triple-bagged under dry, sterile conditions. In 16% of hospitals, skull flaps were irrigated either with antibiotic mixed-saline or Betadine prior to cryopreservation. Skull biopsies or swabs were obtained from the skull flaps for micro-contamination studies in accordance with departmental protocol in 68% of hospitals surveyed. Subsequently, the bone flaps were cryopreserved at wide ranging temperatures between -18°C to -83°C, for variable time intervals (6 months to 'until patient deceased'). Twelve neurosurgical centres (48%) elected for bone flap storage to be undertaken at the local bone bank. In the remainder (52%) of the hospitals, bone flaps were cryopreserved in locally maintained freezers. Prior to re-implantation of the skull flaps at subsequent cranioplasty surgeries, six (24%) of the neurosurgical centres had specific thawing procedures involving immersion of the frozen bone flaps in Ringer's solution and/or Betadine. Further pre-implantation bacteriological cultures from bone biopsies or swabs were obtained only in three (12%) hospitals. CONCLUSIONS: This study has documented highly varied skull flap cryopreservation and storage practices in neurosurgical centres throughout Australia. These differences may contribute to relatively high complication rates of infection and bone resorption reported in the literature. The results of the current study argue for the further need of high quality clinical and basic science research, which aims to characterize the effect of current skull flap management practices and freeze-storage conditions on the biological and biomechanical properties of skull bone.

The status of oocyte cryopreservation in the United States.

OBJECTIVE: To determine the current status of oocyte cryopreservation across the United States, and the perceived indications for its use. DESIGN: Cross-sectional survey of all IVF Centers in the United States. SETTING: Telephone and fax based survey of all IVF practice or laboratory directors, conducted March to June of 2009. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Prevalence of oocyte cryopreservation, acceptable indications and age groups, number of oocyte cryopreservation cycles performed and thawed, fertilization and pregnancy rates, number of live births. RESULT(S): Of 442 centers contacted, 282 (64%) responded in 49 states. In these centers 143 (51%) programs currently offer oocyte cryopreservation, with a geographic trend toward the western-located clinics. Of all programs, 36% offer oocyte cryopreservation only for cancer patients or as an alternative to embryo cryopreservation after IVF, whereas 64% of programs offer it electively in women of advancing maternal age. For elective indications, 87% of programs accept patients aged 35-37 years, 49% consider age 38-40 years as acceptable, whereas only 26% of programs cryopreserve oocytes beyond age 40 years. Three hundred thirty-seven live births resulting from 857 thawed cycles (39.3% pregnancy rate [PR]) were reported across all centers. CONCLUSION(S): Oocyte cryopreservation is offered in more than 50% of ART clinics in the United States. Most programs that perform oocyte cryopreservation for cancer indications offer it for elective delay of childbearing as well. These data suggest a growing acceptance for this technology within our field.

Evolving practice of the Helsinki Skin Bank.

The purpose of this study is to review the management and running of the Helsinki Skin Bank between the years 2001 and 2008. Further aims were to assess the microbiological safety of the glycerol-preserved allograft skin and analyse its clinical use. The files of the Helsinki Skin Bank were reviewed for allograft skin harvested from organ donors between 2001 and 2008. Data on harvested skin area and microbiological culture results were collected. The patients receiving allograft were also identified and operation indications analysed. Allograft skin was collected from 115 donors, with a mean of 44,335 cm(2) per year. No skin batches were discarded. Microbiological cultures of the allograft skin batches were negative in 86 (75%) cases. Thirty-five donor skin batches were used in 69 operations. The most common indication was 'Biological dressing on partial-thickness burns', comprising 52% of cases. The cost per cm(2) was 0.81euro. The use of allograft skin in the Helsinki Skin Bank is microbiologically safe and continues to provide a versatile and useful treatment modality in many major burn cases with few observed complications. As compared with synthetically produced temporary dressings currently available, our allograft skin is also more economical.

Técnica de conservación de huesos en peróxido de hidrógeno / Técnica de conservación de huesos en peróxido de hidrógeno

Se presenta la Osteotecnia en peróxido de hidrógeno y ácido acético como una herramienta para la conservacón de material óseo para fines docentes en colegios y universidades o de exhibición en museos o instituciones afines. Esta técnica de conservación de material óseo permite además rescatar piezas y estructuras óseas de difícil adquisición. Es un método rápido y muy baja toxicidad, fácil de aplicar y de un costo relativamente bajo en comparación con otros descritos en la literatura.

An Osteotechnique is presented on hidrogen peroxide and acetic acid as a tool for the conservation of bone material with teaching purposes on high schools and colleges, museum exhibitions or similar institutions. This bone preservation technique allowds to rescue pieces and bone structures that are difficult to adquire. It`s a simple and low toxicity method, easy to apply and with relatively low cost compared to other techniques described on the literature.

Microbiota bacteriana aeróbia da conjuntiva de doadores de córnea / Aerobic bacterial microbiota of the conjunctiva of cornea donors

OBJETIVOS: Determinar a microbiota bacteriana aeróbia da conjuntiva de doadores de córnea e seu padrão de suscetibilidade a antibióticos; verificar o número de córneas utilizadas para transplante e a média de tempo de preservação em solução preservante com gentamicina e estreptomicina; traçar o perfil dos doadores e receptores de córnea. MÉTODOS: Espécimes clínicos foram colhidos de saco inferior da conjuntiva de ambos os olhos, de 40 doadores de córnea. As amostras foram inoculadas em ágar sangue azida, ágar chocolate e ágar MacConkey e o antibiograma foi realizado pelo método de Kirby-Bauer. RESULTADOS: A freqüência de cultura positiva da conjuntiva de doadores de córnea foi de 72,5 por cento, sendo que Gram-positivos totalizaram 81,6 por cento e apenas 18,4 por cento das amostras foram identificadas como Gram-negativos. Vancomicina inibiu 100 por cento dos Gram-positivos, ao passo que a sensibilidade dos Gram-negativos à gentamicina foi de 53,8 por cento e à estreptomicina foi de 30 por cento. O sexo masculino predominou entre os doadores e receptores, a média de tempo entre o óbito e a enucleação foi de 2h e a de preservação em solução preservante com gentamicina e estreptomicina foi de 7 dias. Neoplasia e mais de uma causa associada foram as causas de óbito mais freqüentes. O ceratocone foi a principal indicação para transplante (51,7 por cento). CONCLUSÕES: Staphylococcus coagulase negativo foi o microrganismo com o maior número de isolamentos, apresentando sensibilidade variada aos antimicrobianos. A quantidade de córneas utilizadas para transplante foi bastante inferior em relação ao total de captações. O perfil dos doadores e receptores de córnea mostrou-se heterogêneo para grande parte das variáveis analisadas.

PURPOSE: To determine aerobic bacterial microbiota of the conjunctiva of cornea donors and its patterns of susceptibility to antibiotics; verify the number of corneas used for transplant and the average time of preservation in solutions with gentamicin and streptomycin; trace the profile of donors and receptors of cornea. METHODS: Clinical specimens were collected from the inferior bag of the conjunctiva of both eyes of 40 cornea donors. The samples were inoculated into acid blood, chocolate and MacConkey agars, and the antibiogram was performed by the Kirby-Bauer method. RESULTS: The frequency of positive cultures of the conjunctiva of cornea donors was 72.5 percent, with Gram-positive totalling 81.6 percent and only 18.4 percent of the samples were identified as Gram-negative. Vancomycin inhibited 100 percent of Gram-positive microorganisms, while sensitivity of the Gram-negative to gentamicin was 53.8 percent and to streptomycin 30 percent. Most donors and recipients were men, the average time between death and enucleation was approximately 2 hours and preservation in solution with gentamicin and streptomycin was around 7 days. Neoplasms and more than one associated cause were the most frequently causes of death. Keratoconus was the main indication for transplant (51.7 percent). CONCLUSIONS: Coagulase-negative Staphylococcus was the most frequently isolated microorganism, presenting variable sensitivity to antimicrobians. The amount of corneas used for transplant was below the number of available corneas. Donor and receptor profiles were very heterogeneous regarding most of the analyzed variables.

Comparison of frozen and RNALater solid tissue storage methods for use in RNA expression microarrays.

BACKGROUND: Primary human tissues are an invaluable widely used tool for discovery of gene expression patterns which characterize disease states. Tissue processing methods remain unstandardized, leading to unanswered concerns of how to best store collected tissues and maintain reproducibility between laboratories. We subdivided uterine myometrial tissue specimens and stored split aliquots using the most common tissue processing methods (fresh, frozen, RNALater) before comparing quantitative RNA expression profiles on the Affymetrix U133 human expression array. Split samples and inclusion of duplicates within each processing group allowed us to undertake a formal genome-wide analysis comparing the magnitude of result variation contributed by sample source (different patients), processing protocol (fresh vs. frozen vs. 24 or 72 hours RNALater), and random background (duplicates). The dataset was randomly permuted to define a baseline pattern of ANOVA test statistic values against which the observed results could be interpreted. RESULTS: 14,639 of 22,283 genes were expressed in at least one sample. Patient subjects provided the greatest sources of variation in the mixed model ANOVA, with replicates and processing method the least. The magnitude of variation conferred by processing method (24 hours RNALater vs 72 hours RNALater vs. fresh vs frozen) was similar to the variability seen within replicates. Subset analysis of the test statistic according to gene functional class showed that the frequency of "outlier" ANOVA results within each functional class is overall no greater than expected by chance. CONCLUSIONS: Ambient storage of tissues for 24 or 72 hours in RNALater did not contribute any systematic shift in quantitative RNA expression results relative to the alternatives of fresh or frozen tissue. This nontoxic preservative enables decentralized tissue collection for expression array analysis without a requirement for specialized equipment.

Catastrophic failures of freezing bags for cellular therapy products: description, cause, and consequences.

BACKGROUND: Container integrity is critical for maintaining sterility of cryopreserved cellular therapy products. We investigated a series of catastrophic bag failures, first noticed in early 2001. METHODS: Process records were reviewed for all PBPC and lymphocyte products cryopreserved in bags from January 2000 through April 2002. Patient charts were also reviewed. RESULTS: One thousand two hundred and four bags were removed from storage for infusion to 261 patients. All products had been cryopreserved in Cryocyte poly(ethylene co-vinyl acetate) (EVA) bags in either 10% DMSO or 5% DMSO and 6% pentastarch. Product volumes were 25-75 mL, and bags were stored with overwrap bags in a liquid nitrogen tank. From January 2000 to April 2001, failure occurred in 10 of 599 (1.7%) bags. From May 2001 to April 2002, 58 of 605 (9.6%) bags failed, typically with extensive fractures that were visible before thaw. Of the 58 that failed, 24 were salvaged by aseptic methods and infused to patients under antibiotic coverage; 10 of those 24 (42%) had positive bacterial cultures. Bag failures were not related to product type, cryoprotectant solution, liquid versus vapor storage, or freezer location. Failures were linked to use of four Cryocyte bag lots manufactured in 2000 and 2001. After replacing these lots with a 1999 Cryocyte lot and with KryoSafe polyfluoroethylene polyfluoropropylene (FEP) bags, no more failures occurred in 75 and 102 bags, respectively, thawed through April 2002. DISCUSSION: High rates of bag failure were associated with four Cryocyte bag lots. No serious adverse patient effects occurred, but bag failures led to microbial contamination, increased product preparation time, increased antibiotic use, and increased resource expenditure to replace products.

Viability analysis of cryopreserved rat pancreatic islets using laser scanning confocal microscopy.

We have developed a digital image analysis technique to assay the viability of frozen-thawed pancreatic islets by using laser scanning confocal microscopy (LSCM) in conjunction with double fluorescent staining [acridine orange/propidium iodide (AO/PI)]. Freshly isolated rat pancreatic islets were cultured for 18-24 h and then brought to a 2 M concentration of dimethyl sulfoxide (Me2SO) by serial addition at decreasing temperatures. Ice was nucleated in the islet suspension at a defined temperature (-10 degrees C), followed by a controlled period for equilibration and then cooling in a programmable bulk freezer at rates of 0.3, 1, 3, 10 and 30 degrees C/min to -70 degrees C. Samples were then stored in liquid nitrogen. Subsequent to rapid thawing and serial dilution with sucrose solution to remove Me2SO, AO/PI-stained individual islets were prepared for imaging on the LSCM. A series of optical sections through individual stained islets were obtained and processed to obtain high-contrast images at two different wavelengths; 488 nm and 514 nm for viable and damaged tissue, respectively. Image analysis algorithms consisted of template masking, generation of histograms of the pixel intensity profile, and gray level thresholding to obtain binary images. The total percentages of both types of tissue in the islet were computed by summing the two populations in each serial section. The spatial distributions of viable and damaged tissue were calculated from the three-dimensional (3-D) data base for both cultured (control) and cryopreserved islets. The 3-D spatial distributions of damaged and viable tissue in the islets were computed by determining the normalized distance of each viable/damaged voxel from the centroid of the islet volume to a mathematically estimated 3-D superquadric surface used to estimate the outer boundary of the islet. Further, each isolated damaged cell was identified and its volume determined. These studies indicate that cryopreserved islets exhibit shape distortion and a decrease in the numerical density of cells in comparison to unfrozen controls. Maximal survival was observed at the slower cooling rates. Accordingly, damage was found to occur throughout the 3-D islet volume in distinct spatial distributions for islets frozen at the slower and the faster cooling rates. Further, it was found that the volume of the majority of damaged cells identified was consistent with that of cells ranging in diameter from 5 to 9 micrometers.

Decrease in high density lipoprotein cholesterol during prolonged storage. CELL Study Group.

Different studies on the stability of high density lipoprotein cholesterol (HDL) in frozen serum or plasma have yielded conflicting results, namely increase, decrease, or no change at all during prolonged storage under freezing conditions. As part of a major trial on lipid-lowering strategies we statistically demonstrated a time-related decrease in HDL cholesterol during storage up to 46 months at -20 degrees C. We therefore re-analysed 85 frozen samples that had been analysed fresh and then stored from 26 to 46 months, using the dextran sulphate 500/Mg2+ method. A linear regression analysis of change in HDL cholesterol on time was performed. The slope was significantly negative (p < 0.0005). The regression equation was (decrease in HDL) = 0.05 - 0.008 x (time in months), i.e. after 6 months' storage at -20 degrees C there was almost a 1% decrease in the HDL cholesterol concentration per month of storage.