Measurement of yield and quality of DNA in human buffy coat is extraction method dependent.

During the last few years, an important element in the improvement of the molecular biology techniques has been the necessity for availability of high quality and functionality DNA. Several DNA extraction procedures with different results in both performance and quality, have been proposed. In this study our objective was to determine the most reliable extraction method that balances DNA quantity, and to assess the sample quantification of the fluorometric DNA quantification methods. For this, blood extracted by venopunction from 20 healthy volunteers was used to obtain DNA from buffy coat, and 4 commercial DNA extraction kits were assessed as well as two fluorometric DNA quantification methods with protocols of different complexity. Results suggest that manual methods achieve higher quality and larger yields of DNA. DNA purity obtained with the 4 extraction kits evaluated through the 260/280 and 260/230 ratio showed that the Qiacube kit fulfilled the criteria established in this work, followed very close by the Flexigene kit. On the other hand, the fluorometric DNA methods used in the samples quantification showed a higher variability when using QuantiFlour method, obtaining better results probably due to the simplicity of this protocol.

Search for asymptomatic carriers of SARS-CoV-2 in healthcare workers during the pandemic: a Spanish experience.

BACKGROUND: Asymptomatic carriers of SARS-CoV-2 can be a vehicle for transmission of the infection. This fact is of particular importance in the case of healthcare workers (HCWs). DESIGN: Cross-sectional study in HCWs in a medium size hospital in the South of Spain. METHODS: between April 15 and 25, 2020, naso and oropharyngeal PCR determination was performed together with IgG and IgM antibody determination by immunochromatography to the HCWs of the Costa del Sol Hospital in Marbella of the units involved in patient care with CoVID-19: Emergencies, Intensive Care and Anesthesia, Internal Medicine and Pneumology. Other units not directly involved in the care of these patients were offered to participate. On the day of sampling, a health questionnaire was answered, reporting symptoms on the same day and in the previous fourteen days. RESULTS: 498 HCWs were studied. Two individuals were detected with PCR for SARS-CoV-2 positive. Both were asymptomatic on the day of sampling, but one of them had had a CoVID-19 compatible picture in the previous two weeks and had positive IgG and IgM; therefore, only one subject was truly asymptomatic carrier (0.2%). 9 workers with positive IgG (1.8%) were detected. CONCLUSIONS: the prevalence of asymptomatic carriers among health workers of the services directly involved in the care of patients with CoVID-19 was very low in our center. This type of strategy can be one more tool in controlling the pandemic.

Detection methods predict differences in biology and survival in breast cancer patients.

BACKGROUND: The aim of this study was to measure the biological characteristics involved in tumorigenesis and the progression of breast cancer in symptomatic and screen-detected carcinomas to identify possible differences. METHODS: For this purpose, we evaluated clinical-pathological parameters and proliferative and apoptotic activities in a series of 130 symptomatic and 161 screen-detected tumors. RESULTS: After adjustment for the smaller size of the screen-detected carcinomas compared with symptomatic cancers, those detected in the screening program presented longer disease-free survival (RR = 0.43, CI = 0.19-0.96) and had high estrogen and progesterone receptor concentrations more often than did symptomatic cancers (OR = 3.38, CI = 1.72-6.63 and OR = 3.44, CI = 1.94-6.10, respectively). Furthermore, the expression of bcl-2, a marker of good prognosis in breast cancer, was higher and HER2/neu expression was lower in screen-detected cancers than in symptomatic cancers (OR = 1.77, CI = 1.01-3.23 and OR = 0.64, CI = 0.40-0.98, respectively). However, when comparing prevalent vs incident screen-detected carcinomas, prevalent tumors were larger (OR = 2.84, CI = 1.05-7.69), were less likely to be HER2/neu positive (OR = 0.22, CI = 0.08-0.61) and presented lower Ki67 expression (OR = 0.36, CI = 0.17-0.77). In addition, incident tumors presented a shorter survival time than did prevalent ones (RR = 4.88, CI = 1.12-21.19). CONCLUSIONS: Incident carcinomas include a variety of screen-detected carcinomas that exhibit differences in biology and prognosis relative to prevalent carcinomas. The detection method is important and should be taken into account when making therapy decisions.

Alterations in sperm protein phosphorylation in male infertility.

Protein phosphorylation is involved in sperm capacitation, so the effect of protein phosphatase inhibitors on the capacitation of spermatozoa of males with unexplained infertility was investigated. d-mannose ligand specific receptor expression in fresh, living spermatozoa, capacitated or treated with calyculin A (an inhibitor of protein phosphatases 1 and 2A), was studied in three groups of men: pre-vasectomy (fertile) males, males in couples with male infertility, and males in couples with infertility of unknown aetiology. Flow cytometry showed significant differences between infertile couples with a male factor and fertile couples (P < 0.05), both after capacitation and after treatment with calyculin A. In the group of couples with infertility of unknown aetiology (n = 15), d-mannose receptor expression was diminished in six cases after classical capacitation. However, when the spermatozoa of these six men were treated with calyculin A, five showed an increased specific d-mannose receptor expression. From these results it is suggested that in vitro treatment of spermatozoa with inhibitors of protein phosphatases may be of great value in some cases of unexplained infertility.

Quality assurance of point-of-care testing in the Costa del Sol Healthcare Area (Marbella, Spain).

Traditionally, point-of-care testing (POCT) has been used throughout the healthcare system without the involvement of the central laboratory. After an exhaustive study of the situation of these laboratories in the Costa del Sol Healthcare Area, we designed a quality control program for the POCT. This program targeted the tests done at the points of care throughout the hospital and the Primary Healthcare Area (PHA), using the Joint Commission on Accreditation of Healthcare Organisations (JCAHO) standards for waived testing. We developed two programs: hospital-based tests and ambulatory POCT for outpatients in PHA. The hospital-based POCT apparatus was used for gases, glucose, qualitative urinalysis, Helicobacter pylori detection in gastrointestinal biopsies and coagulation tests. Ambulatory POCT was used to detect glucose, qualitative urinalysis and pregnancy tests. The personnel responsible are nursing staff with no continuing training program. There was no explicit quality control program and most of the results were used as screening except for glucose in the neonatal department. Criteria for selection of kits and devices were basically based on ergonomic and economic evaluation. Therefore, we performed an evaluation of precision and accuracy of two glucose meter devices. We implemented the internal and external quality programs (IQC and EQC) for glucose testing. We elaborated a guide of standard proceedings for quantitative and qualitative POCT and created an annual course for nursing staff. The annual evaluation of the indicators showed 96% for degree of compliance with IQC; 54% of nursing staff participated in the training program; 98% of the glucometers were operating; and 88% agreement between central laboratory and POCT. As there is no previous experience in our healthcare system, this represents a promising new area of working with nurses, who show great interest in participating in these new programs.

Decreased sperm function of patients with myotonic muscular dystrophy.

Myotonic muscular dystrophy (MMD) is a genetic disease caused by a defective enzyme, myotoninkinase. Male patients with MMD are reported to have reduced fertility. The purpose of this work was to study sperm capacitation and acrosome reaction in the ejaculates of sterile males with MMD and of healthy males (control group). The expression of the specific D-mannose receptors was explored by microscopic examination and by flow cytometry analysis. In addition, the binding patterns of Pisum sativum (PSA) lectin to acrosome content and outer acrosomal membrane in the spermatozoa of each group were analysed. Both the capacitation and the acrosome reaction in the spermatozoa of the MMD group were deficient and these findings strongly suggest that these anomalies may account for the sterility of these patients.

Increase of large granular lymphocytes in human ejaculate containing antisperm antibodies.

Using flow cytometry, we studied the expression of the CD16 antigen by lymphocytes present in human semen samples from three groups of patients: 60 fertile men attending for vasectomy, 60 sterile patients without antisperm antibodies (ASA) and 18 immunological sterile patients with ASA in their ejaculate. No significant difference was found in the concentration of leukocytes or subpopulations of these cells (monocytes, lymphocytes and granulocytes) between fertile, sterile without ASA and immunological sterile groups. However, we detected a predominance of macrophages/monocytes within the population of seminal leukocytes. No statistically significant difference was found in the absolute number of T and B lymphocytes between the three groups studied. However, a significant increase in the number of CD16+ lymphocytes was observed in the ejaculate of sterile patients with ASA as compared to the other groups. This finding might establish an important parameter in the follow-up and prognosis of patients with immunological sterility.

Thyroid function in acute pancreatitis.

OBJECTIVES: To analyze changes in the thyroid function in patients with acute pancreatitis. METHODS: Admission serum levels of triiodothyronine (T3), reverse triiodothyronine (rT3), thyroxine (T4) and thyrotropin (TSH) were determined in 20 patients with pancreatitis and 20 healthy control patients. Another group of 20 patients with upper digestive haemorrhage was included to study possible changes in the pattern of thyroid function in hemodynamic alterations. In addition, laboratory indicators of liver, renal and pancreatic functions were measured in all groups. RESULTS: Our results demonstrated low levels of T3 in 20% of patients with pancreatitis and increased rT3 levels in 75% of them. Thyrotropin was always among reference ranges and only one case presented a low level of T4. No significant alterations were detected in patients with upper digestive haemorrhage. CONCLUSIONS: These results suggest that pancreatitis may play a role in the genesis of these changes, since other factors such as diet and cellular hepatic alteration appear to have had no effect on the levels of thyroid hormones in these patients. In other studies those changes in the thyroid function can be relationed with the prognosis in acute pancreatitis.

Differential expression of MHC class II genes in lung tumour cell lines.

Molecular characterization of HLA class II expression was investigated in five lung tumour cell lines at the protein and mRNA levels. The cell lines exhibited a differential expression of HLA-DR, HLA-DP and HLA-DQ products and also showed differences in the inducibility of HLA class II genes by gamma-IFN. Gamma-IFN stimulation induced only HLA-DR expression to varying degrees in three cell lines, while only one cell line showed stimulation for HLA-DP and none for HLA-DQ antigens. These results suggest locus-specific regulation for the three loci. The presence of DR protein on the cell-surface membrane was always positively correlated with the presence of HLA-DR mRNA in the cells. After treatment with 5-azacytidine in the A549 cell line, which expressed the lowest values, there was no effect on HLA class II levels. This suggested that methylation does not play an important role in the lack of MHC class II antigen expression. In addition to studying mRNA levels of HLA class II antigens, we analysed mRNA of the proto-oncogene c-myc and observed a positive correlation of two mRNA: the increments in HLA-DR expression were associated with increments in c-myc expression. This suggests a relationship between the regulatory and HLA-DR antigens control the expression of c-myc and HLA-DR antigens in lung tumour cell lines.

Lack of correlation between codon 12 K-ras mutations and major histocompatibility complex antigens in bronchogenic carcinomas.

In experimental systems, an association between K-ras activity and expression of major histocompatibility complex (MHC) molecules has been reported. In this study, 52 surgically resected bronchogenic carcinomas were studied for human leukocyte antigen (HLA) class I and II expression, and for the presence of point mutations in codon 12 of the K-ras gene. HLA class I loss was detected in 18 carcinomas, and most of the tumors (43 cases) were found negative for HLA class II antigen expression by the APAAP technique with specific monoclonal antibodies. Analysis using the polymerase chain reaction (PCR), together with selective hybridization using mutation-specific synthetic oligonucleotides, demonstrated K-ras mutations in five cases, all of them corresponding to the adenocarcinoma subtype (31.2% of the adenocarcinomas included in our study) with a poor degree of differentiation. We did not find any correlation between K-ras mutations and HLA class I and II expression in bronchogenic carcinomas. Therefore, it would appear that downregulation of MHC antigens by point mutations of K-ras does not take place in vivo.

CD4+ cells in human ejaculates.

Using flow cytometry, we studied the expression of the CD4 antigen within the different cells present in human ejaculate, both in spermatozoa and round cells. In all, 20 samples of semen were obtained from fertile males; in 11 of these, we detected the presence of leukocytes, using the peroxidase test. Swim-up was performed for the analysis of the spermatozoa. From our results it may be concluded that there is no expression of the CD4 antigen on the surface of human spermatozoa or on CD45- ejaculate cells (epithelial and germinal cells). However, we did detect the presence of the CD4 antigen on the surface of the leukocyte cells (CD45+). A better characterization of these CD45+ cells made it apparent that the CD4+ cells of ejaculate are composed of T lymphocytes (helper/inducer T lymphocytes) and monocytes. Thus we may conclude that human spermatozoa do not express the CD4 antigen, the cell surface receptor for human immunodeficiency virus. However, we did detect CD4+ T lymphocytes and CD4+ monocytes in semen.

Undetectable expression of genomic progesterone receptor in human spermatozoa.

The expression of genomic progesterone receptor in human ejaculated spermatozoa was investigated. Spermatozoa from 10 fertile donors who exhibited normal semen parameters were analysed. Indirect immunofluorescence and an enzyme immunoassay using monoclonal antibodies against genomic progesterone receptor were used. Different types of spermatozoa were studied: fresh, post-swim-up (migrated), capacitated and post-artificial induction of the acrosome reaction by calcium ionophore A23187. Progestin receptor-rich T47D human breast cancer cells were used as a positive control, and progestin receptor-poor MDA-MB-231 human breast carcinoma cells were used as a negative control. Genomic progesterone receptor was not detected in fresh, migrated, capacitated and post-acrosome reaction induction human spermatozoa and MDA-MB-231 cells by either indirect immunofluorescence or enzyme immunoassay. However, in T47D cells a mean concentration of 1043.2 +/- 125.2 fmol genomic progesterone receptor/mg protein was observed by enzyme immunoassay, and indirect immunofluorescence results were positive using both flow cytometry and fluorescence microscopy. These findings suggest that the effect of progesterone on human spermatozoa is not mediated by genomic progesterone receptor.

Influence of incubation on the chromatin condensation and nuclear stability of human spermatozoa by flow cytometry.

Flow cytometry analysis was used for the accurate and objective evaluation of sperm chromatin condensation and chromatin stability of sperm nuclei. It was also possible to determine the influence of incubation on sperm chromatin. Different types of spermatozoa were studied: unprocessed spermatozoa at 1 and 45 min after ejaculation, after swim-up (migrated), spermatozoa incubated for 6 h in non-capacitating conditions (aged), or in B2 medium (capacitated) or B2 medium followed 1 h later with A23187 (reacted). All types of spermatozoa were analysed before and after treatment with various decondensation agents: sodium dodecyl sulphate (SDS), SDS plus EDTA and SDS plus disulphide-reducing agent [dithiotreitol (DTT)]. Sperm nuclei were enzymatically isolated and stained with propidium iodide. Three flow cytometric parameters were then measured: forward light scatter (cellular size), side light scatter (cellular complexity) and fluorescence (uptake of propidium iodide). Fluorescence was the most suitable parameter to study the degree of condensation and resistance to decondensation of DNA in the spermatozoa. Unprocessed spermatozoa 1 min after ejaculation underwent decondensation by all assessed treatments (anionic detergent, chelating or disulphide-reducing agents). Unprocessed spermatozoa 45 min after ejaculation and migrated spermatozoa did not undergo decondensation with SDS treatment, but decondensation occurred after treatment with SDS+EDTA or SDS+DTT. Spermatozoa incubated for 6 h under both non-capacitating (aged spermatozoa) and capacitating conditions (capacitated spermatozoa) and reacted spermatozoa were decondensed only after treatment with SDS+DTT.(ABSTRACT TRUNCATED AT 250 WORDS)