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PubMed2006

Relationship between preexisting anti-varicella-zoster virus (VZV) antibody and clinical VZV reactivation in hematopoietic stem cell transplantation recipients.

Reactivation of latent varicella-zoster virus (VZV), presenting as localized zoster or as disseminated infection, is a common and potentially serious complication in hematopoietic stem cell transplantation (HSCT) recipients. We retrospectively studied anti-VZV immunoglobulin G titers by the immune adherence hemagglutination method after HSCT and also studied VZV DNA by real-time PCR during clinical VZV reactivation using cryopreserved serum samples. No significant difference was found between anti-VZV titers in 13 patients with VZV infection (localized zoster in 11 patients and disseminated zoster in 2 patients) and in 13 subjects without VZV infection at each time point after HSCT. Preexisting anti-VZV titers of disseminated zoster cases tended to be lower than those of localized zoster cases (P=0.10). Serum VZV DNA copy numbers at the onset of disseminated zoster cases tended to be higher than those of localized zoster cases (P=0.09). A strong inverse correlation was found between preexisting anti-VZV titer and serum VZV DNA at onset (r=-0.90, P=0.006). In HSCT recipients, preexisting antibody does not prevent the development of VZV reactivation but may contribute to decreased viral load at onset, resulting in a mild clinical course.

Adult↗
PubMed2003

Quality assessment program for genotypic antiretroviral testing improves detection of drug resistance mutations.

Genotypic antiretroviral testing is now widely used for the management of patients who are undergoing antiretroviral therapy for human immunodeficiency virus infection. The assays are complex, and there is considerable potential for variation between laboratories. Informative and ongoing quality assessment programs (QAPs) which address all aspects of testing are required. The panel distribution of clinical material is a critical component of QAPs. We report on the results and data from a recent panel. Four cryopreserved plasma samples from treated donors were distributed to nine laboratories. Three laboratories performed testing by commercial assays, and six laboratories used in-house assays, with one laboratory reporting results from two in-house assays. There was complete concordance between results for 95.9% of the nucleotide sequence and 94.5% of the amino acid sequence. Despite this overall high level of concordance, the degree of concordance at drug resistance mutation (DRM) sites when DRMs were present was considerably less (38% of DRM sites). Consequently, only 3 of the 10 methods reported 100% of DRMs as present. This elevated discrepancy rate is almost certainly a result of variability in the identification of mixtures of nucleotides (mixtures) at any site within the sequence. In addition, laboratories differed in the number of codons in the reverse transcriptase gene that were sequenced and their ability to amplify all samples. This panel distribution demonstrated a requirement for laboratory participation in ongoing QAPs and the optimization of assays with standards that contain mixtures.

Anti-HIV Agents↗
PubMed2006

Use of chemical mutagenesis in mouse embryonic stem cells.

The chemical mutagenesis of mouse embryonic stem (ES) cells is an approach complementary to chemical mutagenesis of spermatogonia in whole animals. It has great potential to contribute significantly to the generation of a comprehensive collection of multiple alleles for most mammalian genes and to facilitate the progression from gene sequence to gene functional analyses. The general strategy includes the treatment of ES cells with a chemical mutagen and the isolation of individual mutagenized clones that are then cultured in duplicate. Whereas one set of samples is cryopreserved for archiving and subsequent generation of germline chimeras by means of blastocyst injection, the second set is used for genotype- or phenotype-driven screens to identify mutant alleles.

Alleles↗
PubMed1997

Changes in plasma HIV RNA levels and CD4+ lymphocyte counts predict both response to antiretroviral therapy and therapeutic failure. VA Cooperative Study Group on AIDS.

BACKGROUND: Markers are needed for assessing response to antiretroviral therapy over time. The CD4+ lymphocyte count is one such surrogate, but it is relatively weak. OBJECTIVE: To assess the association of changes in plasma human immunodeficiency virus (HIV) RNA level and CD4+ lymphocyte count with progression to the acquired immunodeficiency syndrome (AIDS). DESIGN: Analysis of data from a subset of patients in a multicenter, randomized, clinical trial. SETTING: Six Veterans Affairs medical centers and one U.S. Army medical center. PATIENTS: 270 symptomatic HIV-infected patients from the Veterans Affairs Cooperative Study on AIDS. INTERVENTION: Patients were randomly assigned to receive zidovudine or placebo initially; a cross-over protocol was established for patients receiving placebo who had disease progression. MEASUREMENTS: Reverse transcriptase polymerase chain reaction on cryopreserved plasma samples, previously obtained CD4+ lymphocyte counts, and clinical events. RESULTS: For each decrease of 0.5 log10 copies/mL in plasma HIV RNA level, averaged over the 6 months after randomization, the relative risk (RR) for progression to AIDS was 0.67 (P < 0.001). In a subset of 70 treated patients with long-term follow-up, a return to baseline plasma HIV RNA levels within 6 months of randomization was associated with progression to AIDS (RR, 4.28; P = 0.004). Plasma HIV RNA levels or CD4+ lymphocyte counts over time were more strongly associated with progression to AIDS than were baseline levels or counts. CONCLUSIONS: An adequate virologic response after initiation of antiretroviral therapy seems to require a decrease in plasma HIV RNA level of at least 0.5 log10 copies/mL that is sustained for at least 6 months. The independent relation between plasma HIV RNA level and CD4+ lymphocyte count over time and clinical outcome suggests that the measurement of plasma HIV RNA level, in addition to the CD4+ lymphocyte count, has a role in guiding the management of antiretroviral therapy.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Field preservation of marine invertebrate tissue for DNA analyses.

Successful preservation of tissue samples is a prerequisite for long field studies in remote areas. However, there is little published information concerning field preservation of marine invertebrate tissues for DNA analyses. This omission is significant because marine biodiversity is centered in the Indo-Pacific, where immediate DNA analysis is often impossible. Consequently, we used an assay based on polymerase chain reaction (PCR) to examine the effect of five storage solutions and three temperature regimens on the degradation of DNA from four common classes of marine invertebrates (Anthozoa, Gastropoda, Polychaeta, and Scyphozoa). Control samples were cryopreserved. Storage solution and the type of tissue preserved were the best predictors of preservation success. The length of time in storage and the storage temperature also affected the preservation of DNA. A field test demonstrates that a solution of dimethylsulfoxide and sodium chloride (DMSO-NaCl) preserves a wide range of tissues for DNA analyses and is very simple to use in remote field locations.

Animals↗
PubMed2004

Parental exposure to medications and hydrocarbons and ras mutations in children with acute lymphoblastic leukemia: a report from the Children's Oncology Group.

Ras proto-oncogene mutations have been implicated in the pathogenesis of many malignancies, including leukemia. While both human and animal studies have linked several chemical carcinogens to specific ras mutations, little data exist regarding the association of ras mutations with parental exposures and risk of childhood leukemia. Using data from a large case-control study of childhood acute lymphoblastic leukemia (ALL; age <15 years) conducted by the Children's Cancer Group, we used a case-case comparison approach to examine whether reported parental exposure to hydrocarbons at work or use of specific medications are related to ras gene mutations in the leukemia cells of children with ALL. DNA was extracted from archived bone marrow slides or cryopreserved marrow samples for 837 ALL cases. We examined mutations in K-ras and N-ras genes at codons 12, 13, and 61 by PCR and allele-specific oligonucleotide hybridization and confirmed them by DNA sequencing. We interviewed mothers and, if available, fathers by telephone to collect exposure information. Odds ratios (ORs) and 95% confidence intervals (CIs) were derived from logistic regression to examine the association of parental exposures with ras mutations. A total of 127 (15.2%) cases had ras mutations (K-ras 4.7% and N-ras 10.68%). Both maternal (OR 3.2, 95% CI 1.7-6.1) and paternal (OR 2.0, 95% CI 1.1-3.7) reported use of mind-altering drugs were associated with N-ras mutations. Paternal use of amphetamines or diet pills was associated with N-ras mutations (OR 4.1, 95% CI 1.1-15.0); no association was observed with maternal use. Maternal exposure to solvents (OR 3.1, 95% CI 1.0-9.7) and plastic materials (OR 6.9, 95% CI 1.2-39.7) during pregnancy and plastic materials after pregnancy (OR 8.3, 95% CI 1.4-48.8) were related to K-ras mutation. Maternal ever exposure to oil and coal products before case diagnosis (OR 2.3, 95% CI 1.1-4.8) and during the postnatal period (OR 2.2, 95% CI 1.0-5.5) and paternal exposure to plastic materials before index pregnancy (OR 2.4, 95% CI 1.1-5.1) and other hydrocarbons during the postnatal period (OR 1.8, 95% CI 1.0-1.3) were associated with N-ras mutations. This study suggests that parental exposure to specific chemicals may be associated with distinct ras mutations in children who develop ALL.

Adolescent↗
PubMed1990

External quality control in the andrology laboratory: an experimental multicenter trial.

An external quality control study for semen analysis was performed involving 10 andrology laboratories in geographically separate locations. For the evaluation of sperm concentration, eight samples with different concentrations, and for the assessment of sperm morphology three slides prepared from different semen samples were distributed. Sperm motility was evaluated in five samples delivered cryopreserved to the participants. The coefficients of variation (CVs) for sperm counts varied with the sperm concentrations showing highest variability for low and lowest for high concentrations (range 23% to 73%). The CV for sperm morphology ranged from 25% for normal heads to 87% for abnormal midpieces. The CV for motility of sperm was 21%. For comparison the mean CVs for internal quality control were as follows: 10% for concentration, 8% for morphology (normal heads), and 8% for motility.

Clinical Laboratory Techniques↗
PubMed1995

[Early relapse after peripheral blood stem cell transplantation in acute myelogenous leukemia with t(8;21)].

We report a 3-year-old boy with acute myelogenous leukemia, who relapsed very early after peripheral blood stem cell transplantation (PBSCT). He was admitted with a tumor in maxillar sinus and hemorrhagic diathesis and was diagnosed as having acute myelogenous leukemia with t(8;21). He achieved complete remission with etoposide, cytosine arabinoside and mitoxantrone. After 8 courses of consolidation therapy and marrow ablative chemotherapy, he received PBSCT. G-CSF was given from day 0 because of severe infection. WBC and platelete counts rapidly increased, however, from day 20 platelet count spontaneously decreased. Concomitantly bone marrow examination revealed the presence of blastic cells. RT-PCR showed that the presence of AML 1/MTG 8 chimera mRNA in the cryopreserved PBSC samples. In vitro analysis also revealed that leukemic cells had G-CSF receptors and increased 3H-thymidine uptake in the presence of G-CSF. These findings strongly suggest that the reinfusion of leukemic cells in PBSC and the administration of G-CSF after PBSCT might be relevant to early relapse in this patient.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1996

Determination of activable proacrosin/acrosin in bovine sperm using an irreversible isocoumarin serine protease inhibitor.

The activable proacrosin/acrosin levels in bovine sperm were examined using fluorescent staining and flow cytometry. The proportion of sperm with active acrosin were determined using the biotinylated isocoumarin serine protease inhibitor, Bi-Aca-Aca-OMe-IC (BIC). The presence of bound inhibitor on sperm was then determined by secondary labeling with avidin fluorescein conjugate. The proportion of sperm with activable proacrosin/acrosin was assessed by using detergent treatment to expose the active acrosin in intact sperm. The difference between untreated and detergent-treated aliquots was used to estimate the proportion of sperm with activable proacrosin/acrosin. In the 24-h stored samples from six bulls, the mean proportion of sperm with activable proacrosin/acrosin was 78.8 +/- 2.8%, whereas the mean proportion with exposed acrosin after cryopreservation of these samples was 55.8 +/- 4.1%. Significant differences (p < 0.05) were found among bulls in the proportion of sperm with activable proacrosin/acrosin both before and after cryopreservation. Activable proacrosin/acrosin levels in samples of cryopreserved sperm from five bulls were not correlated with fertility. These results do indicate, however, that the irreversible isocoumarin serine protease inhibitor BIC can be used to determine the proportion of sperm cells that retain activable proacrosin/acrosin after cryopreservation and thawing.

Acrosin↗
PubMed1999

Cell cultures from cryopreserved renal biopsies and other tissue samples.

Answering questions regarding research or clinical aspects, histological and histochemical examinations of tissue specimens are playing an increasing role. The same is true for cell cultures obtained from organ specimens. In most cases, tissue samples are obtained only once and have to be examined immediately. This is often impracticable and therefore, it is necessary to store tissue samples or cells from established cell cultures so as to be able to continue examinations at a later time. Very rare reports exist on the preservation of tissue for performing cell culture examinations and they exclusively refer to tumour tissue and bone marrow, but not to normal organ tissue and biopsy samples. Therefore, in this study cell cultures from several organs have been prepared immediately after obtaining the tissue and compared with those established after cryopreservation several months later. The tissue specimens were obtained from rats (kidney, skin, heart) and from humans (kidney, placenta) or were biopsy specimens from the kidney and skin. From all these cryopreparations, typical cells were cultured. There was no significant difference in the mean population doubling time (MPD) and regarding morphological cell criteria between cell cultures obtained from fresh tissue samples after biopsy and those prepared several months after cryopreservation. There was nearly the same ratio between most cell types present in the tissue. From these results we can conclude that in the examined organs cells from cryopreserved tissue can be cultured even months or more than one year later. At least, these results make it possible to answer new questions and repeat different experiments at any time.

Actins↗
PubMed2005

The viability of cryopreserved PBPC depends on the DMSO concentration and the concentration of nucleated cells in the graft.

BACKGROUND: DMSO is widely used as a cryoprotectant for PBPC. It is desirable to reduce the amount of DMSO without jeopardizing the quality of the stem cell product. The present study was undertaken to investigate whether recovery and survival of CD34+ cells would be significantly altered when PBPC used for autologous transplantations were cryopreserved with four different DMSO concentrations. METHODS: Apheresis samples of PBPC from 20 consecutive patients were mixed in parallel with 2%, 4%, 5% and 10% DMSO, frozen with identical cell concentrations at a controlled rate, and stored in liquid nitrogen for 6-8 weeks. PBPC samples from 11 consecutive patients were also cryopreserved with two different cell concentrations (150 and 300 x 10(6) nucleated cells/mL) to investigate the effect of increasing the cell concentrations while decreasing the DMSO concentration. The flow cytometric absolute count method, based on ISHAGE guidelines, was used to measure the absolute count of total and viable CD34+ cells in the post-thaw samples. RESULTS: PBPC cryopreserved at 150 x 10(6) cells/mL with 2% DMSO yielded significantly inferior CD34+ cell recovery (P < 0.001) and survival (P < 0.001) compared with cryopreservation with 4% and 5% DMSO. This was also observed when comparing higher cell concentrations. However, a reduced cell survival (P = 0.02) was observed when the nucleated cell concentration was increased from 150 to 300 x 10(6) cells/mL in samples cryopreserved with 5% DMSO. DISCUSSION: We conclude that 5% DMSO may be the optimal dose for cryopreserving PBPC as long as the cells have not been concentrated at much more than 200 x 10(6) nucleated cells/mL.

Antigens, CD34↗
PubMed2001

Testicular sperm extraction in a patient with metachronous bilateral testicular cancer.

A new indication for testicular tissue cryopreservation is demonstrated in a patient with metachronous bilateral testicular tumours and azoospermia. At the age of 18 (1982) the patient underwent left orchidectomy and radical retroperitoneal lymphadenectomy for a testicular teratoma (pT1N0M0). Semen samples were not cryopreserved because of absence of motile spermatozoa after thawing. Seventeen years after the primary testicular cancer, a seminoma of the contralateral right testis was diagnosed (pT1N0M0). Since the patient was azoospermic, no semen samples could be cryopreserved. However, spermatozoa were detected in testicular biopsy material of the right testis and were cryopreserved for ICSI. Since all spermatozoa were dead after thawing, testicular sperm extraction (TESE) was performed in the remaining tissue samples at the time of ICSI treatment. Only spermatids could be extracted from frozen-thawed samples due to the inhomogeneous distribution of spermatogenic activity in the testicular tissue. Although one oocyte was fertilized with these spermatids, a clinical pregnancy was not achieved. Despite the disappointing results of ICSI in the couple presented here, this case report demonstrates that cryopreservation of testicular tissue and TESE should be considered in patients with bilateral testicular tumours and azoospermia, if frozen semen samples are not available.

Adolescent↗
PubMed2005

Optimizing cryopreservation of human testicular tissue: comparison of protocols with glycerol, propanediol and dimethylsulphoxide as cryoprotectants.

BACKGROUND: Cryopreservation of testicular tissue is an option in fertility preservation for pre-pubertal boys who will lose spermatogenic cells as a result of chemotherapy. We compared three different protocols and cryoprotectants in cryopreservation of testicular tissue. METHODS: Testicular tissue obtained from 16 infertile men was evaluated by light microscopy(LM), immunostaining against MAGE-A4, transmission electron microscopy (TEM) and organ culture. Seminiferous tubules (1312) from non-frozen (n = 16) and frozen-thawed samples (n = 34) were studied following cryopreservation using protocols with either 1,2-propanediol (PrOH), glycerol or dimethylsulphoxide (DMSO) as cryoprotectants. RESULTS: Normal structure was seen in 86 +/- 6% (mean +/- SD) of the fresh tissue. After freezing with DMSO, 70 +/- 6% and after PrOH, 37+/-3% of the tubules were judged to be good. When glycerol was used, the structure of the basal compartment of the tubules was severely damaged. The ultrastructure of the cryopreserved samples as revealed by TEM and MAGE-positive spermatogonia confirmed the findings. Cryopreserved Leydig cells maintained their morphology and ability to release testosterone in culture. CONCLUSION: DMSO as a cryoprotectant (at a 0.7 mol/l concentration) proved to maintain the structure of testicular tissue, especially spermatogonia, after cryopreservation better than PrOH or glycerol.

Adult↗
PubMed2002

Flow cytometric measurement of apoptosis and necrosis in cryopreserved PBPC concentrates from patients with malignant diseases.

The number of viable precursor cells actually reinfused into patients after high-dose chemotherapy is one of the most clinically important variables determining graft success or failure. A modified, previously described flow cytometric method based on annexin V staining was therefore applied to assess the degree of apoptosis and necrosis in cryopreserved PBPC concentrates from patients with malignant diseases. Twenty-two samples of unmanipulated cryopreserved PBPC concentrates were analyzed by flow cytometry. The samples were triple-stained with anti-CD34 PE, annexin V-FITC and actinomycin D, which enabled the separation of viable, early apoptotic and late apoptotic/necrotic CD34(+) precursor cells. Apotosis and necrosis were also measured in the total cell population of the concentrates. Eighty-one percent (range 49-97) of the CD34(+) cells were viable, while 7% (range 1-15) were early apoptotic and 12% (range 2-36) were late apoptotic/necrotic after freeze/thaw. There was no difference in apoptosis and necrosis in CD34(+) cells harvested from mildly pretreated patients with multiple myeloma and heavily pre-treated patients with non-Hodgkin's lymphoma. Apoptosis and necrosis were higher in the total mature cell population of the concentrates. Thirty-two percent (range 7-69) of the cells were apoptotic and 33% (range 12-60) were necrotic. We conclude that flow cytometric analysis of annexinV/actinomycin D binding in PBPC concentrates is a simple technique that can give additional information of the viability status of the cells post thaw. The present study confirms the relative robustness of human CD34(+) precursor cells concerning the freeze/thaw procedure, which are carried out in daily clinical practice.

Adult↗
PubMed2006

The effect of cryopreservation on sperm head morphometry in Florida male goat related to sperm freezability.

The Sperm Class Analyzer was used to investigate the effect of freeze-thawing procedure on Florida buck sperm head morphometry, and to relate possible changes in sperm head dimensions to cryopreservation success. Semen samples (n=76) were frozen with tris and milk-based extenders and thawed. Sperm quality samples (motility, morphology, acrosome), and sperm head morphometric values (length, width, area, perimeter, ellipticity) were compared between fresh and frozen-thawed samples. Sperm freezability was judged according to the sperm quality parameters assessed. Fertility data was obtained after artificial insemination with cryopreserved semen. Cryopreservation success was different between freezing methods. Sperm head dimensions were significantly (p<0.05) smaller in cryopreserved tris and milk spermatozoa respectively than in those of the fresh samples. The sperm head morphometric parameters that had changed after cryopreservation were lower in suitable semen samples after thawing and with successful pregnancies after artificial insemination. These data suggest that changes in sperm head morphometry might reflect spermatozoa injury occurred during cryopreservation.

Animals↗
PubMed1997

Fluorometric assessments of acrosomal integrity and viability in cryopreserved bovine spermatozoa.

The combination of specific fluorometric staining and flow cytometry provides a rapid and precise means of assessing the functional status of cells. We sought to utilize this approach to quantify two important seminal characteristics, acrosomal integrity and sperm viability, and to compare these with classical microscopic measurements of acrosomal integrity and sperm motility. Samples of thawed, cryopreserved sperm packaged in 0.5-ml French straws were obtained from 12 Holstein bulls. Classical acrosomal assessments and sperm motility estimates were made using differential interference contrast microscopy. Fluorescent acrosomal probes included LysoTracker Green DND-26 (LYSO-G), fluorescein-labeled peanut agglutinin, the biotinylated isocoumarin serine protease inhibitor Bi-Aca-Aca-OMe-IC that was secondarily labeled with fluorescein-avidin, and rabbit antibodies to bovine acrosin that were secondarily labeled with fluoresceinated anti-rabbit immunoglobulin. The fluorescent probes for sperm viability were a combination of SYBR-14 and propidium iodide (PI) or of SYTO-17 and PI. Significant differences were found among methods and among bulls, but not among straws (n = 3). All four fluorescent measures of acrosomal integrity showed highly significant correlations with both classical measurements. These data indicated that the quality of cryopreserved bovine sperm samples could be readily quantified using a variety of organelle-specific fluorescent staining techniques.

Acrosin↗
PubMed2002

[Thermometry of intracellular ice crystal formation in cryopreserved platelets].

The temperature of platelet intracellular ice crystal formation (IIF) is one of the most important physical parameters to instruct platelet cryopreservation. In this study, the range of temperatures for platelet IIF was measured by means of biological and physical methods. All platelet samples were graded cooling, and two samples of per 5 degrees C decrease were thawed by 2 different ways: 37 degrees C directly (T 37 degrees C) and 37 degrees C after keeping in liquid nitrogen (LN) for 2 hours. The phosphatidylserine (PS) positive rate, plasma lactate dehydrogenase (LDH) concentration and platelet aggregate rate were measured in all samples. The heat release graphs of platelets cryopreserved with or without 5% DMSO were also measured by differential scanning calorimeter (DSC). The results showed that the PS positive rates and aggregate rates in platelets and plasma LDH concentrations gradually increased in T 37 degrees C group and decreased in LN group until the arrival of -35 degrees C, and then there were no further changes of the 3 parameters. A small second heat release peak was detected at about -35 degrees C in the platelet samples cryopreserved without DMSO. It is concluded that the temperature of intracellular ice crystal formation in platelet is from -30 to -40 degrees C (-35 degrees ).

Blood Platelets↗