Posting maternal blood samples for free beta-human chorionic gonadotrophin testing.
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Blood Specimen Collection↗
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Mass spectrometric analysis of formalin-fixed paraffin-embedded tissue: unlocking the proteome within.
The predominance of tissues stored worldwide in hospitals and clinical laboratories exist in formalin-fixed paraffin-embedded (FFPE) blocks that are generated by simple and well-established protocols. Although generation of FFPE tissues has facilitated their characterization by such techniques as histopathology, they have proven refractory to biomarker discovery investigations using state-of-the-art MS-based proteomic methodologies. Very recently new methods have been developed that enable proteins extracted from FFPE tissues to be analyzed by MS. This review will highlight and discuss those efforts that have led to this exciting recent progress. Although these developments are quite new, the ability to conduct MS-based proteomic analyses of FFPE tissues opens heretofore intractable clinical samples for discovery-based biomarker research.
Cryopreservation↗
Ultrastructure of tracheal surface liquid: low-temperature scanning electron microscopy.
A layer of liquid lines the airways in the lung. Previous microscopic studies have suggested that it is in two phases, with a mucous gel lying above a periciliary sol. However, shrinkage artifacts due to chemical fixation, dehydration, and drying have prevented reliable estimates of the depth of these layers. To avoid such problems, we have studied the surface liquid of bovine trachea by low-temperature scanning electron microscopy (LTSEM). A polished copper probe cooled to liquid nitrogen temperature was applied to the mucosal surface of sheets of excised tracheal epithelium to effect rapid freezing of surface liquid. Tissue sheets were then mounted in an LTSEM (AMRay 1000A with Biochamber) which maintains samples at -180 degrees C with a Joule-Thompson refrigerator built into the stage. Tissues were fractured at right angles to the epithelial surface, coated with gold, and viewed, all at 10(-5) to 10(-6) torr without transfer through air. The sample was stable under the electron beam at accelerating voltages up to 20 kV. Epithelial features (nuclei, cilia, microvilli, mucous granules) were well preserved. The mucosal surface of the cells was covered with material on the order of 8 microns in depth. The mucous gel and periciliary sol could be seen as distinct layers and could be distinguished by the size and pattern of ice crystal voids generated by radiant-etching of the fractured surface of the sample.
Animals↗
Collection and concentration of tear proteins studied by SDS gel electrophoresis. Presentation of a new method with special reference to dry eye patients.
An obvious obstacle in the analysis of tear film samples from patients with dry eyes is the drastically reduced tear volume. To overcome this problem we have developed a method by which the surface of the eye is flushed with saline, diluting the sparse tear fluid, followed by a concentration of the wash fluid. We compared undiluted tears, diluted tears, tear fluid collected by flushing, reconcentrated diluted tears and reconcentrated flush fluid. Gel electrophoresis of the tear samples obtained showed a representative collection of tear proteins, with bands ranging in molecular weight from about 10 kDa to about 90 kDa. The method may therefore provide a useful alternative to the analysis of undiluted tear fluid in such patients. After storage at -80 degrees C for 1 month, electrophoresis often showed some weakening of band intensities. However, no specific loss of bands was found, and by ultracentrifugation a band profile similar to that of fresh, undiluted tears was obtained. For most purposes, therefore, samples may be kept by this means for later analysis.
Cryopreservation↗
Stability of morphine and buprenorphine in whole blood.
Delays between time of sampling and time of toxicological analysis are common, therefore the length of time that postmortem blood can be stored at various temperatures (e.g. 4 degrees, 25 degrees, -20 degrees C) was evaluated for the effect on the stability of morphine and buprenorphine from day one up to one year. Solid phase extraction and GC-MS were used for the isolation and quantification of the drugs. Morphine and buprenorphine were found to be very stable for up to 6 months under these storage conditions, where at least 85% and 77% of morphine and buprenorphine respectively, were recovered. The study showed that a reasonable amount of the drugs (not less than 70%) was still detectable after one year of storage regardless of the temperature when blood samples were stored in silanized glass vessels.
Blood Preservation↗
Optimal use of fresh and frozen-thawed testicular sperm for intracytoplasmic sperm injection in azoospermic patients.
PURPOSE: To optimize the use of fresh and frozen-thawed testicular biopsy specimens from patients with azoospermia. METHODS: Fifty-one patients suffering from obstructive and non-obstructive azoospermia underwent testicular sperm extraction (TESE). The specimens were divided and used for either in vitro maturation or freezing for a future intracytoplasmic sperm injection (ICSI) cycle. RESULTS: At initial testicular sperm extraction, very few motile spermatozoa were seen. After 24 h of in vitro maturation, sperm motility increased remarkably, with a maximum motility rate seen between 48 and 72 h of culture. Motile spermatozoa were observed up to 120 h in culture. In the 22 fresh ICSI cycles, a total of 294 oocytes were injected using motile sperm and 212 oocytes demonstrated normal 2PN formation (fertilization rate, 72.1%). In 36 frozen-thawed ICSI cycles, a total of 454 oocytes were injected and 302 oocytes became 2PN (66.5%). On day 3, high quality embryos were observed in 54.2% of fresh cycles and 54.1% of frozen cycles (P > 0.05). The clinical pregnancy rate did not show a significant difference between using fresh (59%) and frozen (55.5%) testicular biopsy sperm for ICSI (P > 0.05), but the embryo implantation rates did differ significantly between fresh (29.5%) and frozen-thawed (22.2%) cycles (P < 0.05). A total of 33 healthy babies have been born from 22 women, giving birth after 58 embryo transfer attempts (38%). CONCLUSION: The freezing and in vitro culturing of testicular biopsy tissue is a very reliable approach for the management of testicular biopsy specimens from azoospermic patients, and offers the possibility of several treatments of IVF/ICSI from a single sample.
Adult↗
Particle size distribution in ground biological samples.
Modern trace and retrospective analysis of Environmental Specimen Bank (ESB) samples require surplus material prepared and characterized as reference materials. Before the biological samples could be analyzed and stored for long periods at cryogenic temperatures, the materials have to be pre-crushed. As a second step, a milling and homogenization procedure has to follow. For this preparation, a grinding device is cooled with liquid nitrogen to a temperature of -190 degrees C. It is a significant condition for homogeneous samples that at least 90% of the particles should be smaller than 200 microns. In the German ESB the particle size distribution of the processed material is determined by means of a laser particle sizer. The decrease of particle sizes of deer liver and bream muscles after different grinding procedures as well as the consequences of ultrasonic treatment of the sample before particle size measurements have been investigated.
Animals↗
Secretion of soluble HLA-G by day 3 human embryos associated with higher pregnancy and implantation rates: assay of culture media using a new ELISA kit.
Identification of chemical markers in human embryo culture media that relate to embryo quality and implantation potential could be an invaluable tool in embryo selection for transfer. Embryonic secretion of soluble human leukocyte antigen G (sHLA-G) has been postulated to be a marker for 'embryonic competence'. This study examines sHLA-G concentrations in day 3 culture media droplets of embryos that were selected for transfer, as well as those being cryopreserved. A total of 712 embryo culture supernatants from 83 patients were assayed. Soluble HLA-G was detected in 306 of the 712 samples tested. In the 58 transfers in which at least one embryo selected for embryo transfer was positive for sHLA-G, the pregnancy rate was 64% (37/58) and the implantation rate per embryo transferred was 38%. In contrast, patients receiving only embryos that were negative for sHLA-G had both a lower pregnancy rate of 36% (9/25; P < 0.05) and a decreased implantation rate (19%; P < 0.05). Expression of sHLA-G was also related to increasing cell stage. Concentration of sHLA-G in embryo culture media was variable and in the low range (3-10 ng/ml). These data suggest an association between implantation potential and embryonic secretion of sHLA-G. The commercial assay kit utilized allowed for same day assessment of sHLA-G secretion. Addition of sHLA-G status to traditional morphological criteria may be useful as a clinical tool for embryo selection.
Adult↗
Measurement of urinary glycosaminoglycans in dogs.
OBJECTIVES: To measure urine concentrations of sulfated glycosaminoglycans (GAGs), determine optimal storage conditions for urine samples, establish a reference range, and determine whether there is correlation between 24-hour total urine GAG excretion and the GAG-to-creatinine ratio (GCR). ANIMALS: 14 healthy adult dogs. PROCEDURE: Single urine sample GAG concentrations and GCRs were measured in samples collected from 14 healthy dogs at the start of the 24-hour collection period. Twenty-four-hour total urine GAG excretions were determined from urine collected during a 24-hour period in the same 14 dogs. Total sulfated GAG concentrations were also measured in urine from these dogs after the urine had been stored at 4 degrees C and -20 degrees C for 1, 7, and 30 days. RESULTS: Urine GAG concentrations were not significantly different from baseline values after urine was stored at 4 degrees C for up to 1 day and -20 degrees C for up to 30 days. Neither single urine sample GAG concentration (R2, 0.422) nor GCR (R2, 0.084) was an adequate predictor of 24-hour total urine GAG excretion. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study provide data that can be used to establish a reference range for 24-hour total urine GAG excretion in dogs and adequate conditions for sample storage. Contrary to findings in humans, there was no significant linear correlation between 24-hour total urine GAG excretion and single urine sample GCR in dogs, limiting clinical use of the single urine sample test.
Animals↗
[Flow cytometric DNA analysis of frozen samples].
Now that flow cytometric DNA analysis is becoming an established procedure, many samples that are being received from various hospitals cannot all be analyzed within a day. Therefore, samples must be stored for subsequent analysis in a flow system. To determine a proper temperature for storage by freezing, samples of the same material were stored for 2 weeks at 5 degrees C, -80 degrees C, and -195 degrees C. Measurement and analysis at the end of this period revealed no major differences among the subsequent histograms of these samples. Thus, it seems that a flow cytometric DNA analysis can be made of frozen samples for diagnostic and prognostic evaluations.
Cryopreservation↗
Further assessment of glasswool column filtration of semen.
Although not a common cause, increased viscosity may occasionally be associated with infertility. Specimens healthier than those with increased fertility potential should maintain their motility after reasonable intervals of time and after cryopreservation and freezing. Glasswool column filtration and alpha-amylase have been shown to improve the longevity and survival of progressive sperm in viscous samples compared to controls and the use of Alevaire and disruption methods. It appears that the use of Alevaire is detrimental to sperm.
Cell Separation↗
Hematopoietic potential of IL-2-cultured peripheral blood stem cells from breast cancer patients.
Patients receiving autologous transplants for various malignancies generally experience an increased incidence of relapse compared with patients receiving unmanipulated allogeneic transplants. We initiated a protocol for IL-2 activation of peripheral blood stem cells (PBSC) for induction of in vitro and in vivo autologous graft-versus-tumor (GVT) activity in patients with breast cancer. In this study we analyzed the effects of 24 h of IL-2 incubation on the hematopoietic potential of PBSC from these patients. Cells collected by leukapheresis were first cryopreserved and stored in liquid nitrogen, then thawed rapidly and incubated with IL-2 in a serum-free system for 24 h, with samples analyzed before and after incubation. Although there was a significant drop in mononuclear cells (MNC) (from 4.5 to 3.7 x 10(8)/kg) and CD34+ cells (from 12.3 to 7.5 x 10(6)/kg) after 24 h in culture, there was no significant change in colony-forming units (CFU) (from 12.5 to 11.5 x 10(5)/kg). Time to engraftment (neutrophils: < 0.5 x 10(9)/l; platelets: > 20 x 10(9)/l) was comparable to a cohort of similar patients receiving non-cultured PBSC transplants. These results indicate that mobilized frozen/thawed PBSC which have been cultured in IL-2 for 24 h retain adequate potential for hematopoietic reconsistution in this group of patients.
Breast Neoplasms↗
Prenatal T-cell reconstitution after in utero transplantation with fetal liver cells in a patient with X-linked severe combined immunodeficiency.
OBJECTIVE: Fetuses with severe combined immunodeficiency may be treated with intrauterine transplantation of fetal hematopoietic stem cells. In previous reports on intrauterine transplantation with T-cell-depleted bone marrow, repeated injections have led to partial immunoreconstitution at birth, with subnormal T-cell counts and a delayed response to mitogens. STUDY DESIGN: A male fetus with X-linked severe combined immunodeficiency because of a stop mutation in the gene encoding the common gamma chain of cytokine receptors was transplanted in week 14 of gestation with a single injection of 7 x 10(7) cryopreserved nucleated fetal liver cells (9 x 10(8) cells per estimated kilogram fetal weight) into the fetal abdomen. At 24 and 33 weeks of gestational age, fetal blood samples were taken to detect evidence of engraftment. Fetal mixed chimerism was determined using polymerase chain reaction amplification of a variable number of tandem repeats and was verified by genomic HLA class II typing and flow cytometry. RESULTS: The course of pregnancy, delivery, and the first 18 months of life have been uncomplicated. At week 24 of gestation, donor HLA class II alleles were detected at a low level in the background of the recipient's fetal HLA genotype. The chimeric proportion of donor cells was about 10% at 24 weeks of gestation, increasing to 50% at 33 weeks of gestation. Whereas the T-cell fraction was still markedly reduced in week 24, it increased thereafter and was in the normal range from week 33 of gestation. In vitro response to T-cell mitogens was normal from birth. CONCLUSION: In utero transplantation of cryopreserved fetal liver cells in week 14 of gestation with a single injection led to complete T- and NK-cell reconstitution at birth. Signs of engraftment were found already in week 24 of gestation. We consider intrauterine transplantation a valuable experimental method and a useful adjunct to postnatal transplantation and gene therapy in the treatment of severe combined immunodeficiency.
Female↗
Large scale purification of human blood CD34+ cells from cryopreserved peripheral blood stem cells, using a nylon-fiber syringe system and immunomagnetic microspheres.
Isolation of large numbers of human peripheral blood CD34+ cells could lead to therapeutic applications, including purging of malignant cells from blood cell transplantations, purging of T cells from allogeneic bone marrow, and even blood cell transplantation. This procedure has limitations if there are not sufficient numbers of progenitor cells in the leukapheresis concentrates available for selection after detection of tumor cells in apheresis products. Use of frozen/thawed peripheral blood mononuclear cell (PBMC) samples would make feasible pooling of two or even more stem cell harvests collected at different time points and the total number of CD34+ progenitor cells available would increase. We established an efficient method for purification of CD34+ cells from cryopreserved apheresis products, using a nylon-fiber syringe system and immunomagnetic microspheres. We compared purity, recovery rate and clonogenicity of CD34+ cells purified from fresh (n = 22) and cryopreserved apheresis products (n = 14), using a nylon-fiber syringe system and immunomagnetic microspheres. The purity of CD34+ cells from cryopreserved products was less than that from fresh products (85.9 +/- 14.4% vs 94.6 +/- 10.0%), but the recovery rate of CD34+ cells and colony-forming cells was comparable between fresh and cryopreserved products. One patient underwent grafting with peripheral blood CD34+ cells selected after freezing, with good success. Therefore, these cells are capable of rapidly reconstituting hematopoiesis after high-dose chemotherapy. Bone Marrow Transplantation (2000) 26, 787-793.
Antigens, CD34↗
Ultrastructural changes of articular cartilage chondrocytes associated with freeze-thawing.
In an attempt to justify the use of cryopreserved versus fresh articular cartilage (AC) allografts, we used transmission electron microscopy (TEM) to study the ultrastructure of fresh versus frozen-thawed AC with or without a dimethyl sulfoxide (DMSO) treatment. AC explants were cut aseptically from the femoral condyles of healthy, mature rabbits when they were killed. Half of all explants were incubated in Ham's F-12 medium, supplemented with antibiotics and with or without 7.5% DMSO, frozen to -80 degrees C, stored for 24 h, and thawed rapidly. These, and the control explants, were fixed with glutaraldehyde, paraformaldehyde, and acrolein in cacodylate buffer. Sections were stained for acid phosphatase (APase), postfixed with osmium, embedded, and examined under TEM. The typical organization of the matrix and the cells was noted in control sections. The chondrocytes contained intact nuclei, organelles, and discrete plasma membrane. Although some endoplasmic reticula and nuclear membrane appeared intact, distinct ultrastructural changes were observed in frozen-thawed samples treated with DMSO. These changes included condensation of chromatin, large lipid droplets, partly disrupted plasma membrane, and pericellular precipitation of APase-positive crystalites. In sections not treated with DMSO, the cytoplasm was extensively vacuolated and no distinct organelles could be detected in the chondrocytes. Little difference was noted between the matrix organization of fresh or frozen-thawed samples. Our results suggest that distinct ultrastructural changes occur in the chondrocytes following freeze-thawing of intact AC and that DMSO pretreatment may contribute to improvement in the cryopreservation of AC.
Acid Phosphatase↗
Computer-assisted sperm head morphometry analysis (ASMA) of cryopreserved ram spermatozoa.
Normal sperm morphology has been shown to be indicative of male fertility; however, subjective methods of assessing morphology are highly variable. Computer-assisted sperm morphometry analysis (ASMA) has been developed for the objective analysis of sperm head dimensions. Developing applicable protocols for sperm head morphometry analysis increases the efficiency of these systems. The objective of the current study was to develop accurate methods for employing ASMA of ram sperm heads. Staining methods, optimal sperm sample numbers microscopic magnification and sampling variation within and between technicians were assessed. Frozen semen from 10 fertile rams was thawed and prepared on slides for morphometric analysis. Staining spermatozoa with hematoxylin and rose bengal stains yielded the best results. Ram sperm head morphometry was accurately evaluated on at least 100 spermatozoa at x 40 objective magnification. Using these techniques, a sample could be analyzed in approximately 3 min. No significant differences in sperm head measurements were detected between 2 technicians. The system properly recognized and digitized ram spermatozoa 95% of the time. The morphometric measurements of sperm heads for all rams were as follows: length = 8.08 microns, width = 4.80 microns, width:length ratio = 0.59, area = 29.13 micron 2 and perimeter = 23.93 microns. The mean within analysis coefficients of variation for all individual analyses and parameters ranged from 4.8% for length to 6.0% for area. The variation between replicate analysis was 2.4% or less for both technicians. When applying proper sample preparation and analysis procedures no differences in measurements or variation were observed between the 2 system operators.
Animals↗
Determining viability of fresh or cryopreserved homograft valves at implantation.
Studies of homograft valves in the past two decades have suggested that "viable" valves, i.e., those maintaining chemical and structural integrity of their leaflet intercellular matrix, have a better long-term function than nonviable valves. The most effective qualitative methods of assessing leaflet viability involve destruction of the valve leaflets; thus, these methods have been limited to random use in selected valves. A study was conducted in swine in an attempt to establish a control tissue which could be tested in place of the homograft leaflets, thereby determining viability levels of every valve clinically implanted and correlating the results with long-term clinical function. Thirty samples each of the aortic and pulmonary artery wall and tricuspid leaflet were compared with aortic and pulmonary leaflets. Utilizing the technique of C14-proline uptake, viability was assessed at procurement, following sterilization, and following cryopreservation and short-term storage. The tricuspid leaflet was found to retain the same level of viability as the aortic and pulmonary leaflets before and after the cryopreservation period. It was concluded that the tricuspid leaflet could be utilized as the control tissue.
Animals↗
In vitro maintenance, cooling and cryopreservation of red wolf (Canis rufus) spermatozoa.
A current priority for the preservation of the endangered red wolf (Canis rufus) is the development of a sperm-based genome resource bank. The aims of this study were to examine the effects of (i) holding temperature on the motility of spermatozoa over time, and (ii) cooling methods on the characteristics of spermatozoa after cooling and cryopreservation. Electroejaculates (n = 11; fresh) were evaluated for the percentage of motile spermatozoa, cell and acrosome morphology (Spermac (Meditech 1st Canada Inc, Montreal, Ontario) and fluorescein isothiocyanate-labelled Pisum sativum agglutinin lectin (PSA/FITC; Sigma Diagnostics, Oakville, Ontario) staining), and zona penetration. Semen samples were then divided into two equal samples and centrifuged to remove seminal plasma. One half of the ejaculate sample was re-suspended in sperm-Tyrode's albumin lactate pyruvate (TALP), divided into three aliquots and maintained either at room temperature (approximately 21-23 degrees C), 0 degree C or 37 degrees C. Sperm motility was examined at 0.5 and 1.0 h, and subsequently every hour for 10 h. Motility of spermatozoa decreased after 2 h, but was consistently greater at room temperature than at 37 degrees C or 0 degree C. The other half of the ejaculate sample was re-suspended in an egg yolk-based extender and divided into two aliquots. One aliquot was cooled in a refrigerator (5 degrees C) for 30 min, whereas the second aliquot was put into a beaker containing water at 37 degrees C, which was then placed into an ice bath until the sample reached 0 degree C (approximately 120 min). Spermatozoa were evaluated after cooling and after freezing and thawing treatments. No differences were observed between cooling treatments either after cooling or freezing and thawing. However, marked decreases in intact acrosomes, post-thaw motility and normal morphology of spermatozoa after treatment demonstrate that further investigations are necessary to improve cryopreservation methods in this species.
Animals↗