Cryopreserved Samples Research Articles

Preservação da fertilidade masculina no Centro Hospitalar de Vila Nova de Gaia/Espinho EPE – 17 anos de experiência

ObjectiveA report is presented of 17 years of experience in male fertility preservation due to oncological, genetic, immunological and endocrinological disease, as well as severe spermatogenesis alterations. Material and methodsAn evaluation is made of demographic parameters, sperm characteristics and results of assisted reproduction techniques cycles with thawed samples. ResultsA total of 271 men with a mean age 31.5±6.3 years (16 to 52 years) were referred to sperm banking. The most common cause was oncological disease (34.3%), followed by secretory azoospermia, oligoasthenoteratozoospermia, genetic disease, testicular atrophy/hypogonadism, auto‐immune disease, endocrine‐metabolic disease, and prior to potentially fertility damage surgery.Among oncological disease, testicular tumour was the most common cancer (64.5%), followed by haematological cancer, and other aetiologies. Klinefelter syndrome was the most common (50%) among the genetic diseases.A total of 194 men had samples cryopreserved (1099 total samples; 5.7±3.4 per patient). Men with oncological disease had a significantly higher number of cryopreserved samples (p<.001). Testicular tumour cases had a mean lower sperm concentration relative to other tumours (p<.005).During these 17 years, 58 men (29.9%) asked to have assisted reproduction techniques performed with their thawed samples. The 87 cycles performed resulted in 19 pregnancies (21.8%) and 15 healthy newborns. ConclusionsAlthough the number of men asking to use their cryopreserved samples is low, we cannot undervalue the positive psychological impact of fertility preservation, taking in consideration that it can be the only possibility to biological fatherhood.

Fertility preservation for females receiving gonadotoxic treatment: utilization and disposition choices for cryopreserved samples

Fertility preservation for females receiving gonadotoxic treatment: utilization and disposition choices for cryopreserved samples

In vitro adaptation of Plasmodium falciparum reveal variations in cultivability.

BackgroundCulture-adapted Plasmodium falciparum parasites can offer deeper understanding of geographic variations in drug resistance, pathogenesis and immune evasion. To help ground population-based calculations and inferences from culture-adapted parasites, the complete range of parasites from a study area must be well represented in any collection. To this end, standardized adaptation methods and determinants of successful in vitro adaption were sought.MethodsVenous blood was collected from 33 P. falciparum-infected individuals at Goa Medical College and Hospital (Bambolim, Goa, India). Culture variables such as whole blood versus washed blood, heat-inactivated plasma versus Albumax, and different starting haematocrit levels were tested on fresh blood samples from patients. In vitro adaptation was considered successful when two four-fold or greater increases in parasitaemia were observed within, at most, 33 days of attempted culture. Subsequently, parasites from the same patients, which were originally cryopreserved following blood draw, were retested for adaptability for 45 days using identical host red blood cells (RBCs) and culture media.ResultsAt a new endemic area research site, ~65 % of tested patient samples, with varied patient history and clinical presentation, were successfully culture-adapted immediately after blood collection. Cultures set up at 1 % haematocrit and 0.5 % Albumax adapted most rapidly, but no single test condition was uniformly fatal to culture adaptation. Success was not limited by low patient parasitaemia nor by patient age. Some parasites emerged even after significant delays in sample processing and even after initiation of treatment with anti-malarials. When ‘day 0’ cryopreserved samples were retested in parallel many months later using identical host RBCs and media, speed to adaptation appeared to be an intrinsic property of the parasites collected from individual patients.ConclusionsCulture adaptation of P. falciparum in a field setting is formally shown to be robust. Parasites were found to have intrinsic variations in adaptability to culture conditions, with some lines requiring longer attempt periods for successful adaptation. Quantitative approaches described here can help describe phenotypic diversity of field parasite collections with precision. This is expected to improve population-based extrapolations of findings from field-derived fresh culture-adapted parasites to broader questions of public health importance.

Adipose Derived-Mesenchymal Stem Cells Viability and Differentiating Features for Orthopaedic Reparative Applications: Banking of Adipose Tissue.

Osteoarthritis is characterized by loss of articular cartilage also due to reduced chondrogenic activity of mesenchymal stem cells (MSCs) from patients. Adipose tissue is an attractive source of MSCs (ATD-MSCs), representing an effective tool for reparative medicine, particularly for treatment of osteoarthritis, due to their chondrogenic and osteogenic differentiation capability. The treatment of symptomatic knee arthritis with ATD-MSCs proved effective with a single infusion, but multiple infusions could be also more efficacious. Here we studied some crucial aspects of adipose tissue banking procedures, evaluating ATD-MSCs viability, and differentiation capability after cryopreservation, to guarantee the quality of the tissue for multiple infusions. We reported that the presence of local anesthetic during lipoaspiration negatively affects cell viability of cryopreserved adipose tissue and cell growth of ATD-MSCs in culture. We observed that DMSO guarantees a faster growth of ATD-MSCs in culture than trehalose. At last, ATD-MSCs derived from fresh and cryopreserved samples at −80°C and −196°C showed viability and differentiation ability comparable to fresh samples. These data indicate that cryopreservation of adipose tissue at −80°C and −196°C is equivalent and preserves the content of ATD-MSCs in Stromal Vascular Fraction (SVF), guaranteeing the differentiation ability of ATD-MSCs.

The effects of storing and transporting cryopreserved semen samples on dry ice.

ObjectiveThis study aimed to test the effects on sperm viability of transporting cryopreserved semen samples on dry ice.MethodsTwenty normozoospermic semen samples were cryopreserved and divided into five groups. The samples in Group 1 were immersed in liquid nitrogen throughout the experiment in cryogenic storage tanks; the cryopreserved straws in Group 2 were placed in a Styrofoam box containing dry ice and kept under these conditions for 48 hours; the samples in Group 3 were kept for 48 hours on dry ice under the same conditions as the Group 2 samples, and were then moved to a storage tank filled with liquid nitrogen; Group 4 samples were also kept for 48 hours in dry ice storage, and the Styrofoam box containing the samples was shipped by plane to assess the effects of shipping; the samples in Group 5 were shipped together with the Group 4 samples and were placed in a storage tank with liquid nitrogen after spending 48 hours stored on dry ice. After thawing, sperm parameters were analyzed for viability, vitality, and motility; spermatozoa were also tested for mitochondrial activity.ResultsSignificant decreases in motility recovery rates (P=0.01) and vitality (P=0.001) were observed in all groups when compared to the control group. Mitochondrial activity was significantly decreased only in Group 5 (P=0.04), as evidenced by greater numbers of sperm cells not stained by reagent 3,3'-diaminobenzidine.ConclusionsTransportation did not affect the quality of cryopreserved semen samples, but dry ice as a means to preserve the samples during transportation had detrimental effects upon the sperm parameters assessed in this study.

Preservation of microbial communities enriched on lignocellulose under thermophilic and high-solid conditions.

BackgroundMicrobial communities enriched from diverse environments have shown considerable promise for the targeted discovery of microorganisms and enzymes for bioconversion of lignocellulose to liquid fuels. While preservation of microbial communities is important for commercialization and research, few studies have examined storage conditions ideal for preservation. The goal of this study was to evaluate the impact of preservation method on composition of microbial communities enriched on switchgrass before and after storage. The enrichments were completed in a high-solid and aerobic environment at 55 °C. Community composition was examined for each enrichment to determine when a stable community was achieved. Preservation methods included cryopreservation with the cryoprotective agents DMSO and glycerol, and cryopreservation without cryoprotective agents. Revived communities were examined for their ability to decompose switchgrass under high-solid and thermophilic conditions.ResultsHigh-throughput 16S rRNA gene sequencing of DNA extracted from enrichment samples showed that the majority of the shift in composition of the switchgrass-degrading community occurred during the initial three 2-week enrichments. Shifts in community structure upon storage occurred in all cryopreserved samples. Storage in liquid nitrogen in the absence of cryoprotectant resulted in variable preservation of dominant microorganisms in enriched samples. Cryopreservation with either DMSO or glycerol provided consistent and equivalent preservation of dominant organisms.ConclusionsA stable switchgrass-degrading microbial community was achieved after three 2-week enrichments. Dominant microorganisms were preserved equally well with DMSO and glycerol. DMSO-preserved communities required more incubation time upon revival to achieve pre-storage activity levels during high-solid thermophilic cultivation on switchgrass. Despite shifts in the community with storage, the samples were active upon revival under thermophilic and high-solid conditions. The results suggest that the presence of microorganisms may be more important than their relative abundance in retaining an active microbial community.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0392-y) contains supplementary material, which is available to authorized users.

The effects of cryopreservation on angiogenesis modulation activity of human amniotic membrane

Amniotic membrane (AM), as the innermost layer of placenta, has side dependent effects on the angiogenesis. Cryopreservation is a necessary process to avoid the challenging problems of fresh tissues; a procedure which makes the AM ready-to-use. Since the cryopreservation can influence the AM characteristics for experimental and clinical purposes, in this study the effects of cryopreservation were evaluated on angiogenesis modulation activity of the AM compared to fresh tissues in an animal model. The AM was implanted mesenchymal side up or epithelial side up in a rat dorsal skinfold chamber. The length and number of branches of formed capillaries were measured via intravital microscopy after 7 days. The amount of IL-8 (interleukin-8) and TIMP-2 (Tissue Inhibitor of Matrix Metalloproteinase-2) as two factors in amniotic cells which have great impacts on angiogenesis were evaluated using ELISA assay. The epithelial surface of cryopreserved AM had inhibitory effects on vessel formation. The cryopreserved amniotic mesenchymal side increased the vessel length and sprout. The result of cryopreserved AM on angiogenesis was similar to that of fresh tissues. The levels of IL-8 and TIMP-2 in cryopreserved samples were significantly less than fresh AMs which shows that angio-modulatory properties are not limited to the effects of amnion epithelial and mesenchymal stem cells and the other components such as extracellular matrix may contribute in angio-modulatory effects. These promising results show that inducing and inhibitory effects of the AM, which make it an appropriate candidate for different clinical situations, were maintained after cryopreservation.

Sperm DNA fragmentation in cryopreserved samples from subjects with different cancers.

Sperm cryopreservation is widely used by cancer patients undergoing chemo- or radiotherapy. Evidence suggests that IVF outcome with cryopreserved spermatozoa from cancer patients is less successful. To determine whether sperm DNA fragmentation (SDF) is involved in the lower fertilising ability of cryopreserved spermatozoa of cancer patients, SDF was evaluated in thawed spermatozoa from 78 men affected by different cancers and 53 men with non-cancer pathologies. SDF was assessed by the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL), propidium iodide (PI), flow cytometry procedure, which allows determination of two different cell populations (PIbrighter and PIdimmer) and thus to determine the percentage of DNA fragmented sperm in both. PIdimmer spermatozoa are totally unviable, whereas PIbrighter spermatozoa with SDF may be motile and morphologically normal, having higher biological relevance in the reproductive process. We found that the proportion of DNA fragmented PIbrighter cells was significantly higher in thawed spermatozoa from cancer than non-cancer patients. Moreover, a positive correlation was found between the degree of DNA fragmentation and sperm motility in the PIbrighter population of spermatozoa from cancer patients that wasn't seen in non-cancer patients. The results of the present study suggest that higher SDF levels may contribute to the lower IVF success of cryopreserved spermatozoa from cancer patients and that evaluation of SDF could complement genetic counselling as part of the routine management of cancer patients who seek fertility preservation.

Cryopreservation and cryotherapy of grapevine (Vitis vinifera L.)

This study aimed at testing the efficiency of a droplet-vitrification cryopreservation protocol in eliminating selected grapevine viruses. The cryopreservation protocol led to approximately 50 % recovery with cultivar 'Portan' and five international cultivars tested, but very low recovery was noted with Croatian cultivars. GFLV and GLRaV-3, two (economically important grapevine) viruses were eliminated in a high percentage (up to 100 %) of plants regenerated from cryopreserved shoot tips. Virus sanitation was observed as well in samples before liquid nitrogen exposure. Genetic stability of plants regenerated after cryopreservation was studied using AFLP markers. Polymorphic fragments were observed in non-cryopreserved and cryopreserved samples treated with PVS2 solution, the number of which increased with increasing durations of exposure to PVS2 solution.

Is there an androgen level threshold for aneuploidy risk in infertile women?

BackgroundLow functional ovarian reserve (LFOR) has been associated with hypoandrogenemia and increased embryo aneuploidy, while androgen supplementation has been reported to improve aneuploidy rates. We, therefore, assessed whether in infertile women undergoing in vitro fertilization (IVF) androgen concentrations are associated with aneuploidy rates.MethodsThis study was performed in 2 academically affiliated fertility centers in New York City and an academically affiliated steroid chemistry laboratory in Utah. Androgen concentrations were measured in blinded fashion from 84 infertile women (age 40.3 +/− 2.4 years) at New York University (NYU), using a validated LC-MS/MS method, in cryopreserved serum samples of patients who had undergone IVF with concomitant preimplantation genetic screening (PGS), utilizing a 24-chromosome platform. The Center for Human Reproduction (CHR) provided plasma samples of 100 historical controls (ages 38.6+/−5.0 years) undergoing IVF without PGS. Statistical comparisons were made of androgen concentrations, and of associations between androgen concentrations and embryo aneuploidy.ResultsWomen undergoing IVF + PGS at NYU revealed no association between embryo aneuploidy and androgen concentrations but demonstrated significantly lower androgen concentrations than the 100 control patients from CHR,ConclusionsThough this study revealed no association between androgen levels and embryo ploidy, the extremely low androgen levels in the NYU study group raise the possibility of a threshold effect below which testosterone no longer affects aneuploidy. Before an androgen effect on embryo ploidy can be completely ruled out, a patient population with more normal androgen levels has to be investigated.

P0515 : Biological stability and diagnostic accuracy of ELF® (enhanced liver fibrosis test) to identify liver fibrosis in patients with chronic hepatitis C using cryopreserved serum samples during 25 years

P0515 : Biological stability and diagnostic accuracy of ELF® (enhanced liver fibrosis test) to identify liver fibrosis in patients with chronic hepatitis C using cryopreserved serum samples during 25 years

Expression of a surface antigen (MA6) by peripheral blood CD34+ cells is correlated with improved platelet engraftment and may explain delayed platelet engraftment following cord blood transplantation.

CD34(+) cell dose provides a measure of hematopoietic tissue that predicts the rate of engraftment upon transplant. It is positively correlated with multiple measures of hematopoietic recovery, including platelet engraftment. Here we identify a subpopulation of CD34(+) cells that coexpress a surface antigen--MA6, which is more positively correlated with platelet engraftment in a clinical setting than CD34(+) alone. The specific identity and function of MA6 remain to be determined, however, it is expressed by primitive megakaryocyte (MK) progenitors, but is lost with differentiation and is not expressed by platelets. Commitment of CD34(+)MA6(+) cells to the MK lineage was confirmed by in vitro assays and their significance in hematopoietic transplantation explored by flow cytometric analysis of cryopreserved samples of granulocyte colony stimulating factor-mobilized peripheral blood progenitor cell (PBPC) products along with a retrospective analysis of platelet engraftment data. Platelet engraftment by day 21 was predicted by receipt of ≥ 6 × 10(6) CD34(+) cells/kg or ≥ 0.3 × 10(6) CD34(+)MA6(+) cells/kg. Subsequent analysis of cord blood (CB) CD34(+) cells revealed <0.2% coexpressed MA6(+), compared to 8% of PBPC CD34(+) cells. This low proportion of CD34(+)MA6(+) cells may be responsible, at least in part, for the delayed platelet engraftment associated with CB transplantation. However, platelet engraftment is markedly improved in recipients of ex vivo-expanded CB. This may be a consequence of an increased proportion of CD34(+)MA6(+) cells present in the ex vivo-expanded product and also suggests that optimizing ex vivo culture conditions to generate CD34(+)MA6(+) cells might further improve platelet engraftment in CB recipients.

Accuracy of five serologic tests for the follow up of Strongyloides stercoralis infection.

BackgroundTraditional faecal-based methods have poor sensitivity for the detection of S. stercoralis, therefore are inadequate for post-treatment evaluation of infected patients who should be carefully monitored to exclude the persistence of the infection. In a previous study, we demonstrated high accuracy of five serology tests for the screening and diagnosis of strongyloidiasis. Aim of this study is to evaluate the performance of the same five tests for the follow up of patients infected with S. stercoralis.MethodsRetrospective study on anonymized, cryo-preserved samples available at the Centre for Tropical Diseases (Negrar, Verona, Italy). Samples were collected before and from 3 to 12 months after treatment. The samples were tested with two commercially-available ELISA tests (IVD, Bordier), two techniques based on a recombinant antigen (NIE-ELISA and NIE-LIPS) and one in-house IFAT. The results of each test were evaluated both in relation to the results of fecal examination and to those of a composite reference standard (classifying as positive a sample with positive stools and/or at least three positive serology tests). The associations between the independent variables age and time and the dependent variable value of serological test (for all five tests), were analyzed by linear mixed-effects regression model.ResultsA high proportion of samples demonstrated for each test a seroreversion or a relevant decline (optical density/relative light units halved or decrease of at least two titers for IFAT) at follow up, results confirmed by the linear mixed effects model that showed a trend to seroreversion over time for all tests. In particular, IVD-ELISA (almost 90% samples demonstrated relevant decline) and IFAT (almost 87%) had the best performance. Considering only samples with a complete negativization, NIE-ELISA showed the best performance (72.5% seroreversion).ConclusionsSerology is useful for the follow up of patients infected with S. stercoralis and determining test of cure.

Stem cells, progenitors & regenerative medicine: A retrospection.

It is well evident that the embryonic stem cells (ESCs) are pluripotent, can differentiate into all the three germ layers namely ectoderm, mesoderm and endoderm and into 200 odd cell types present in the body, are immortal, can expand in large numbers in vitro, and are genetically stable over long periods in culture. Three groups in the country have successfully derived well characterized human ES (hES) cell lines including Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Bengaluru1, Reliance Life Sciences2 and National Institute for Research in Reproductive Health (NIRRH)3, Mumbai. However, hES cells have the associated issues of immune-rejection and risk of teratoma formation. On the other hand, the adult stem cells (ASCs) do not expand in culture but these can be isolated from the patients’ own bone marrow and thus are considered safe. Though studies/trials have been undertaken using autologus stem cells in India4, at the global level this approach has not shown the desired results5,6,7. The National Guidelines for Stem Cells Research and Therapy became available in 2007, and has been recently revised (http://icmr.nic.in/guidelines/NGSCR%202013.pdf) and are named National Guidelines for Stem Cells Research indicating that stem cells are not yet ready for therapy and more research is required before these are put to translational use. Simultaneously, Central Drugs Standard Control Organization (CDSCO), New Delhi has put up a draft guidance document for regulatory approval of Stem Cell and Cell Based Products (SCCPs) (http://www.cdsco.nic.in). Several companies/clinics have started culturing and using mesenchymal stem cells (MSCs) for treating various conditions in India. The situation is alarming as it is not clear whether these therapies would benefit the public. Are mesenchymal cells true stem cells or just stromal cells? Similar situation exists with cord blood banking to treat multiple diseases and claims that cord blood may serve as a future health insurance for the baby. It has become evident that (i) besides blood related disorders, cord blood stem cells may not regenerate other tissues as these do not possess trans-differentiation potential, and (ii) autologus cryopreserved cord blood sample will never suffice when the baby grows up as an adult8. Use of fresh unrelated donor cord blood sample may be a viable alternative as emerging studies suggest that mismatched allogeneic cord blood stem cell transplant is easily tolerated with 1-2 antigen mismatch and is associated with lower anticipated risk of graft versus host disease (GVHD)9,10. Moreover, the present article suggests that pluripotent stem cells exist in adult body organs and the need to bank cord blood as a source of stem cells may be a futile exercise. The concerns raised in a recently published editorial11 are timely. More research is required to understand how normal body stem cells interact with their niche, differentiate and get mobilized under disease conditions to bring about regeneration. Such information is crucial to exploit therapeutic potential of stem cells in the field of regenerative medicine. Here the author discusses the existing confusion as to what are the true stem cells in the body, how these are different from the tissue-specific progenitors and whether these may be implicated in initiating cancers. It also provides an interesting perspective to the stem cell field based on available literature and highlights the importance of microenvironment/niche in deciding the stem cell fate.

Ultrastructural features of human sperm cells cryopreserved by different methods

Cryopreservation of human spermatozoa has been recognized as a key strategy for management of male fertility. Nevertheless, current protocols of sperm freezing are neither optimal nor standardized between different labs (1). In this study we compare the ultrastructural features of human normospermic sperm samples (according to WHO parameters 2010) from 5 different freezing techniques in order to identify the best methods of cryopreservation. After informed con- sent, 21 normospermic patients (from the Medically Assisted Procreation PMA Center of the Fondazione IRCCS Policlinico San Matteo in Pavia) were recruited and both traditional and improved analysis of sperm quality were applied, in order to define critical steps of cryopreservation. Cryopreservation of human spermatozoa has been related to decreased motility associated with impaired velocity and viability of sperm pre-freeze and post-thaw. For all applied methods there was a significant reduction of progressive and total motility (P) as a result of freezing. To investigate ultrastructural details, 5 additional cryopreserved samples by the best two freezing methods were analyzed with elec- tron microscopy (TEM). Preliminary data showed the minimal differences between the protocols, with a large number of queues detached and large quantities of cyto- plasmic debris after of the first protocol. Spermatozoa appear to be better preserved in the second analyzed method, despite both procedures induced deteriorations at ultrastructural level (2). Other non-routine analysis will be performed to determine whether the cooling time to +4°C may affect the procedure; Comet Assay (to assess the degree of sperm DNA fragmentation) (3) and flow cytometry (to study light scat- ters patterns and membrane integrity) (4) will be applied.

Pre-Transplant C-Reactive Protein (CRP), Ferritin and Albumin As Biomarkers to Predict Transplant Related Mortality (TRM) after Allogeneic Hematopoietic Cell Transplant (HCT)

▪HCT entails substantial TRM justifying continued efforts to better risk stratify patients. Single institutional studies have suggested several clinically available biomarkers of CRP, ferritin and albumin influence TRM if not overall survival (OS). We sought to confirm the independent prognostic value of these biomarkers in a large multi-institutional cohort.The study population consisted of 784 adults with AML in remission (80%) or MDS (20%) undergoing unrelated donor HCT between 2008 and 2010 and available cryopreserved samples through the Center for International Blood and Marrow Transplant Research (CIBMTR) repository. CRP and ferritin were centrally quantified by ELISA from cryopreserved plasma whereas albumin levels obtained from center reported data. Correlation studies for biomarkers required log transformation of CRP and ferritin to produce a normal distribution. Multivariate models were fitted separately for each biomarker applying protocol specified thresholds generated from the literature of CRP > 10 mg/L, ferritin > 2500 ng/mL, albumin < 3.5 g/dL on TRM. Further analysis explored optimal cutpoints for this cohort for all significant clinical variables for TRM.HCT characteristics included a median age of 50 (range 18-78) years, HCT-CI (co-morbidity index) 3 or more in 35%, single allele/antigen mismatch (7/8) in 23%, PB as stem cell source in 83%, and myeloablative conditioning in 72%. Biomarker data were available in 783, 781 and 695 cases for CRP, ferritin and albumin respectively. The median values and ranges for each biomarker were as follows: 5.0 mg/L (0.3 - 316) for CRP, 1148 ng/mL (51 – 14,298) for ferritin and 3.6 g/dL (0.6-5.3) for albumin. Log transformed CRP and ferritin showed a modest correlation (r=0.35, P<0.001), and log CRP was marginally associated with albumin (r=-0.12, P=0.002). HCT-CI had no correlation to CRP and albumin. Higher ferritin was associated with HCT-CI (P=0.014) and disease (P<.001). In the entire population, TRM and overall survival (OS) at 2 years were 23% and 54%, respectively. The table shows the independent association of each biomarker with TRM and OS. Ferritin had no association with these outcomes but only 12% had levels above 2500 by ELISA. The optimal cutpoints for TRM were defined as follows: CRP > 3.67 (HR=1.75, P=0.001); albumin < 3.4 (HR 1.70, P <0.001); and ferritin as a continuous variable (HR 1.30, P = 0.008). A risk score was created modeling optimal biomarker thresholds to predict TRM based on the following formula: risk score = 0.44633*I(CRP>3.67)+0.18330*ln(Ferritin)+0.44730*I(albumin<3.4), where “I” is an indicator function and represent 1 or 0 depending on the presence of the abnormal biomarker level in parenthesis. The risk score included adjustment for HCT-CI. The biomarkers did not influence the incidence of relapse or graft-versus-host disease with any of these models.Elevated CRP and ferritin and decreased albumin prior to HCT were associated with impaired transplant survival, primarily through higher TRM. Integration of a biomarker panel in clinical practice once validated can enhance risk-stratification, improve adjustment for comparative studies and point towards the design of biomarker driven trials.Abstract 422. Table:Multivariate analysis of TRM and OS for biomarkers, and biomarker panel risk score*Abnormal,N (%)Transplant related mortalityOverall SurvivalBiomarkerHR95% CIPHR95% CIPProtocol definedCRP >10 mg/L184 (24)1.521.11 – 2.070.0081.220.98 – 1.530.072Ferritin >2500 ng/mL93 (12)1.260.84 – 1.880.271.150.86 – 1.540.35Albumin < 3.5 g/dL290 (42)1.481.10 – 2.00.0101.391.13 – 1.720.002Optimal threshold, combined model**CRP >3.67 mg/L497 (64)1.561.11 – 2.190.0101.241.00 – 1.550.054Log(Ferritin), linearn/a1.200.99 – 1.460.071.201.04 – 1.380.010<3.4 g/dL203 (29.5)1.581.16 – 2.150.0041.371.10 – 1.720.005Biomarker Score**NLow (<1.5)2001.0--1.0Intermediate (1.5-2.0)3311.661.10-2.490.0151.381.06-1.800.017High (>2.0)1592.721.77-4.19<0.0012.011.51-2.70<0.001*biomarkers adjusted for significant covariates of BMI, disease, mismatch, and CMV serostatus and stratified on conditioning regimen**combined model includes all biomarkers and HCT-CI DisclosuresNo relevant conflicts of interest to declare.

Genetics of Disease Progression in Diffuse Large B-Cell Lymphoma: Clonal Selection and Acquisition of Newly Acquired Somatic Mutations at Relapse

Introduction:Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoma with ~30% of cases failing treatment or relapsing after R-CHOP chemotherapy. Treatment options for refractory/ relapsed DLBCL are limited and often have a poor outcome. Tumour evolution and selection of a chemo resistant clone are likely explanations making it important to understand clonal heterogeneity at diagnosis and relapse. Green et al have noted a hierarchical acquisition of mutations with disease progression in follicular lymphoma (FL), with BCL2 and CREBBP mutations seen in earlier precursors and MLL2 and TNFRSF14 mutations proposed to be later events. Notably, 1 case analysed at diagnosis and at relapse had variable IgVH indicating origin in a more immature precursors. [Green, M.R., Blood, 2013]. More recent studies indicate that the dominant clone of FL and transformed FL arise by either convergent or divergent evolution from a common mutated precursor through the acquisition of additional genetic events [Pasqualucci, L., Cell Rep, 2014]. Defining the deregulated pathways and oncogenic mutations at diagnosis and later at relapse could help identify novel therapeutic targets for treatment at relapse. We aimed to map clonal evolution at relapse using immunophenotyping, sequencing for the Immunoglobulin variable heavy chain gene (IGHV) and genotyping for known somatic mutations in key lymphoma genes, within matched diagnostic and relapsed DLBCL pairs.Methods:Six matched diagnostic and relapsed DLBCL cryopreserved samples were subjected to comprehensive immunophenotyping using 10-colour B-cell flow cytometry panels based on normal ontogeny. PCR on initial and relapsed samples was performed to amplify clonally rearranged IgHV genes from fresh/paraffin embedded primary diagnostic samples. Sanger sequencing was done for IgHV mutation status by targeting the conserved framework regions (FR) FR1 [IGHA: VHFR1-JH] or FR3 [IGHC: VHFR3-JH] using the Invivoscribe kit based on the Biomed-2 protocols. A custom Haloplex library preparation kit designed and ordered from Agilent was used to capture the exons of ~40 genes commonly mutated in DLBCL. Targeted fragments were PCR amplified and target enriched samples sequenced on an Illumina MiSeq sequencerResults:Table 1. Immunophenotypic diversity was noted among cases but the phenotype did not change at relapse. IGHV sequencing showed that 3 cases with two distinct lymphoma clones at diagnosis evolved to a single detectable IGHV clonal population at relapse. Lymphoma-associated somatic mutations detected at diagnosis were preserved at relapse and in 4 cases, acquisition of additional mutations was observed.Table 1:Immunophenotype, IgHV sequence and somatic lymphoma mutations in paired DLBCL samples at diagnosis and relapsePatient numberDisease courseImmunophenotypeIgHV sequenceLymphoma mutationsDL157DiagnosisIgM+, IgD+++, IgG-, CD27-, CD21+++, CD38++, CD10-N/A (not available)EP300 I->V EZH2 D->H NOTCH2 L->H TRAF2 A->TDL157RelapseSame as diagnosisIgHV3-64 IgHV3-30EZH2 D->HFOXO1G->D NOTCH2L->HTRAF2 A->TDL214DiagnosisIgM+++,IgD++, IgG-, CD27-, CD21-, CD38+++, CD10+IgHV4-30 IgHV4-39N/ADL214RelapseSame as diagnosisIgHV4-39EP300 I->V MYD88 L->P NOTCH1 R->W PRDM1 C->Y SPEN D->E TP53 Y->NDL215DiagnosisIgM-,IgD- IgG++, CD27++,CD21++, CD38-, CD10-IGHV3-23 IGHV3-23DKMT2D R->C MYD88 L->P NOTCH1 E->KDL215RelapseSame as diagnosisIGHV3-23BTG1 H->Y BTG1 Q->Stop BTG2 S->N FOXO1 V->E GNA13 R->H KMT2D R->CMYD88 L->PNOTCH1 E->KPIM1 L->F PIM1 K->N PIM1 V->LDL245DiagnosisN/AIGHV3-11 IGHV3-33EP300,I->V,997 NOTCH2,L->V,1559DL245RelapseN/AIGHV3-11EP300,I->V,997NOTCH2,L->V,1559DL213DiagnosisIgM+,IgD-, IgG-, CD27-,CD21+++, CD38+++, CD10++IGHV3-15ETS1 A->T EZH2 D->H PRDM1 L->F TNFRSF14 C->SDL213RelapseSame as diagnosisIGHV3-15ETS1 A->TEZH2 D->HKMT2D R->Stop PRDM1 L->FSTAT3 R->WDL252DiagnosisIgM-,IgD++, IgG-, CD27-,CD21-, CD38+, CD10-IGHV1-2NOTCH2,P->A,2359 TNFRSF14,V->I,241DL252RelapseSame as diagnosisIGHV1-2N/AConclusions:While multiple IGHV sub-clones are present at diagnosis of DLBCL, evolution of a single clone is noted at relapse (convergent model) with acquisition of additional somatic mutations. Immunophenotyping using B-cell maturation flow cytometry panels demonstrates heterogeneity of differentiation between cases but no change in immunophenotype at relapse. DisclosuresNo relevant conflicts of interest to declare.

C-21: Clinical banking of amniotic membrane allografts

Amniotic membrane (AM) is currently collected from caesarean section placenta, processed into allograft pieces and cryopreserved by the Comprehensive Tissue Centre (CTC) in Edmonton, Alberta, Canada for regional and national ophthalmological and dermatological transplant applications. Anti-inflammatory, anti-immunogenic, and anti-fibrosis properties of AM allografts are important for post-transplant epithelialization. The purpose of this study was to evaluate quality of cryopreserved AM allografts. AM allograft sections were cultured for bacterial and fungal growth after recovery from donors and after antibiotic decontamination. Non-linear controlled-rate cooling was used to cryopreserve AM allografts (sizes: 3 cm2, 5 cm2, 10 cm2). Post-thaw AM viability was assessed by a fluorescent membrane integrity assay (ethidium bromide/Syto® 13) and confocal microscopy. Cell Tissue Organ Surveillance System (CTOSS) forms provided information on AM quality from surgical end-users. While 40.9% of AMs showed positive bacterial culture results upon recovery, with 55.6% of positive culture due to Propionibacterium sp, 100% of cultures were negative after the antibiotic decontamination (n = 22). CTOSS feedback indicated quality of 16.3% AM allografts was excellent, 61.2% satisfactory and 2.0% unsatisfactory (n = 49). Percent viabilities for cryopreserved AM allografts, including 3 cm2 (n = 8), 5 cm2 (n = 7) and 10 cm2 (n = 6) were 57.8 ± 11.0%, 42.7 ± 8.5%, and 47.7 ± 8.2%, respectively, with no statistically significant difference among the allograft sizes (p = 0.54). There was statistical significant difference between percent viability of “fresh” allografts (47.7 ± 13.6%) when compared to cryopreserved samples (p = 0.78). The results of this study provide important information on quality of AM allografts for transplant applications. The CTC takes great pride in providing safe allografts of high quality, through excellence in practice and leadership in research. Disclosure: Author was or is employed by University of Alberta.

Ultra performance liquid chromatography - mass spectrometry studies of formalin-induced alterations of human brain lipidome.

The development of 'omics' sciences offers new opportunities for the study of neurodegenerative diseases but increases at the same time the sample demand on brain banks that collect and store valuable human post-mortem tissue. Our study aims to evaluate in lipidomics the potential of formalin-fixed tissue compared with the cryopreservation method, considered as the gold standard for biochemical research. Two complementary liquid chromatography/mass spectrometry analytical platforms were used on the basis of hybrid quadrupole time-of-flight and triple quadrupole mass spectrometers. Untargeted fingerprinting, semitargeted profiling of specific lipid classes and targeted monitoring of lipid species were performed in formalin-fixed and cryopreserved samples to provide detailed information at the molecular level on the formalin-induced alterations of the brain tissue. In vitro incubations of lipid standards were also performed to further describe the degradation processes induced by formaldehyde. Phospholipid compounds were found to be extensively hydrolysed, whilst the sphingolipid ones were preserved. N-methylation and N-formylation of amine-containing phospholipids have also been evidenced. These findings show that the potential detrimental effect of formalin on the analytes of interest must be taken into account when analysing formalin-fixed samples.

Comprehensive study of hydrostatic pressure treated human umbilical cord blood cells via response surface method

Amelioration of the survival parameters of cryopreserved samples after thawing has already been addressed through several techniques including vitrification to avoid the formation of ice cores. However, this approach cannot be followed in the case of samples with higher volumes. Hydrostatic pressure (HP) treatment has been proven to increase some qualifying parameters (e.g., motility, insemination efficiency) of certain biological samples. Accordingly, the preparation of umbilical cord blood (UCB) samples through an active (mechanical) pre-stressing process to increase the survival rate of cryopreserved samples can be regarded as a novel strategy that calls for basic experimental studies. The goal of our study was to assess the effects of HP treatment on the qualifying parameters (DNA fragmentation by agarose gel electrophoresis and capillary electrophoresis, Total Nucleotide Cell (TNC) count, CD34+/CD45+ count, and superoxide dismutase activity (SOD) of human umbilical cord blood (UCB) derived cells). The experimental arrangement was set to provide data for response-surface analysis to take into account the common effects of the individual variables of pressure and time exposure. 3D visualization of experimental data revealed that 50-min long HP treatment at 12.5MPa can significantly (α=0.05) enhance white blood cell (WBC) and CD34+/CD45+ cell counts. However no DNA fragmentation was observed even at higher pressures, SOD activity was triggered over 15.0MPa. As a conclusion, HP treatment may contribute to the optimal cryopreservation of UCB cells by significantly increasing WBC and CD34+/CD45+ cell counts without adverse effects neither on DNA stability nor on triggering SOD activity.