Sterile EDTA Research Articles

EFFECTS OF METFORMIN ON TNF-α RELEASE IN LIPOPOLYSACCHARIDE-INDUCED MONOCYTES IN RATS

Inflammation which is a response of immune system was demonstrated in many disorders such as atherosclerosis, hypertension, diabetes, cancer and rheumatoid arthritis. Metformin, an oral antidiabetic drug, has anti-inflammatory effect apart from blood glucose regulatory effect. However, the mechanism of its anti-inflammatory effect is not clearly understood. In this study, the effect of metformin on the release of cytokines (TNF-α and IL-6) from LPS stimulated rat mononuclear blood cells was investigated. Blood samples (5ml) were taken from healthy, male, 8-12 weeks old rats (n=5, 200-250g) through cardiac puncture under general anesthesia into sterile EDTA containing tubes. Monocytes were separated by centrifugation and were resuspended in RPMI 1640 media (3.3±0.2x105 /ml). Cells were then incubated with metformin (2.5 µM, 25 µM, 250 µM) for 2,5 hours followed by addition of LPS (100 ng/ml, 1µg/ml) for further 5 hours. After centrifugation, the supernatant was taken and TNF-α level was measured by ELISA. There was no statistically significant change in the amounts of TNF-α in the LPS + metformin groups compared to the 100 ng/ml LPS group (p>0.05). In LPS+metformin groups, compared to 1 µg/ml LPS, 2.5 µM and 250 µM metformin significantly increased TNF-α levels (p0.05). The amount of IL-6 was not within measurable range in this study. In summary, metformin increased the amount of released TNF-α rather than decreased in our study.

Serological Evidence of HIV/HBV Co-infection among HIV-infected patients in Onitsha, Anambra State, Nigeria

Research suggests that HIV-infected individuals who have also been co-infected with HBV face a greater risk of HIV progression. Hepatitis B and HIV infections pose serious public health challenges in sub-Saharan Africa. This study examined the possible HBV co-infection with the socio-demographic traits of HIV-infected individuals attending the HIV clinic at a referral specialist mission hospital in Onitsha, Anambra State, Nigeria. Two hundred and twenty (220) HIV-infected individuals gave consent to participate in the study. Between March 2022 and October 2022, blood samples (about 5ml) were aseptically collected during routine investigations into sterile EDTA bottles, and plasma samples were obtained by centrifugation. These samples were tested for HBsAg using a Monolisa HBsAg ULTRA kit (Bio-Rad, USA). CD4 counts were measured using the Partec CyFlow. Plasma viral loads (PVL) were also determined using the Abbott Real-Time HIV-1 Assay US protocol. HIV/HBV co-infection was 40.9%. The results showed that most (39.7%) study participants co-infected with HBV had a CD4 cell count of 350 cells/µl. The virological assay revealed that the highest HBsAg seroprevalence was detected in the study participants whose viral loads were between 40 and 1000µl (61.9%). The majority of the study participants were female HIV patients (74.5%), while those co-infected with HBV were mainly male patients (48.2%). HIV-positive individuals aged 18-30 (53.3%) were mostly affected. None of the socio-demographic variables tested was significantly associated with HBV in HIV-infected patients in Onitsha, Anambra State, Nigeria. This study showed a high HIV/HBV co-infection in Onitsha, Anambra State, Nigeria.

Co-infection of Hepatitis C among HIV-infected patients: A cross-sectional study from A University Teaching hospital in Anambra State, Nigeria

Hepatitis C virus co-infection (HCV) with HIV is regarded as a significant public health risk despite being less frequent than hepatitis B co-infection with HIV. This observation results from HIV's impact on the HCV life cycle and, ultimately, the hepatic system. In this study, potential HCV co-infection was examined in connection to the socio-demographic characteristics of HIV-positive patients attending an HIV clinic at a University Teaching Hospital in Awka, Anambra State, Nigeria. Between September 2021 and June 2022, 255 HIV patients who consented to participate in the study had their HCV levels checked using the anti-HCV antibody ELISA kit (DIA.PRO, Italy). Using the Partec CyFlow, CD4 counts were calculated. During routine investigations, blood samples (approximately 5ml) were aseptically collected into sterile EDTA vials, and plasma samples were obtained by centrifugation. With the aid of Abbott Real-Time HIV-1 Assay US methodology, plasma viral loads (PVL) were also calculated. A total of 11 (4.3%) HIV-positive people were also confirmed to have HCV infection, with women making up the majority (4.8%). Most of those affected were between the ages of 31 and 40 (6.8%). With marital status, HCV co-infection was found more amongst married patients, with a rate of 4.8%. The prevalence of HIV/HCV co-infection was higher (4.8%) in those with CD4 cell counts under >350 cells per ml than others. Most patients (3.1%) did not have their viral RNA detected (TND). Self-employed infected persons and those with secondary school educational backgrounds had the highest HIV/HCV prevalence rate of 6.6% and 7.6%, respectively. The study's findings show no statistically significant relationships between the patients' socio-demographic traits and HCV. Also, HCV co-infection prevalence was significantly low in the study participants.

Are VEGF and SCUBE1 gene expressions increased in diabetic retinopathy?

Abstract Objectives In the current study, we synergistically evaluated vascular endothelial growth factor (VEGF) gene expression levels and signal peptide-CUB-EGF domain-containing protein 1 (SCUBE1) gene expression levels in diabetic patients without retinopathy, non-proliferative diabetic retinopathy (NPDR), and proliferative diabetic retinopathy (PDR). Methods 94 blood samples from 26 healthy controls, 29 non-DR, 22 NPDR, and 17 PDR patients were collected in sterile EDTA tubes. Total RNA was obtained from these samples without waiting and then converted to cDNA. The expression levels of the VEGF and SCUBE1 genes were determined by quantitative real-time polymerase chain reaction (qPCR). Results SCUBE1 gene expression levels were 2.15 (p=0.015), 1.75 (p=0.799), 2.37 (p=0.037) times higher, and VEGF gene expression levels were 1.71 (p=0.023), 1.75 (p=0.012), 1.85 (p=0.031) times higher in the non-DR, NPDR, and PDR groups compared to the control group, respectively. VEGF gene expression levels were significantly higher in participants with HbA1c levels ≥5.7% compared to those with <5.7. SCUBE1 and VEGF gene expression levels were significantly higher in participants with fasting plasma glucose (FPG) levels ≥126 mg/dL than those with <126 mg/dL. Conclusions As a result, SCUBE1 gene expression levels are higher than VEGF gene expression levels, especially in the PDR group. Therefore, SCUBE1 may contribute to the pathology of DR just like VEGF by generating angiogenesis. However, we believe there is a need for experimental animal model studies with DR examining SCUBE1 gene expression levels in tissue samples.

Evaluation of Clinical , Laboratory and Genetic Parameters in Patients with Primary Myelofibrosis in Nineveh province

Background :Primary myelofibrosis is a Philadelphia-negative myeloproliferative neoplasm(MPN) with predominant proliferation of megakaryocytes and granulocytes in the bone marrow characterized by initial proliferative phase, followed by a reactive deposition of fibrous connective tissue in the bone marrow. Objectives : To evaluate the clinical and laboratory data in primary myellofibrosis and to correlate the genetic disorders with clinical and other laboratory parameters. Setting : This case series study conducted at Ibn-Sina Teaching Hospital/Outpatients Hematology Department from November 2019 to April 2020. Patients and method : Total fifteen patients with primary myelofibrosis included in this case series (retrospective and prospective) study. The records of old cases were reviewed from patients for clinical history, clinical examination, previous blood counts, bone marrow study and genetic study. For new cases clinical history and physical examinations were noted, ten mls of venous blood were aspirated from patients (new cases) by clean venipuncture and delivered into sterile EDTA and gel tubes for doing complete blood pictures, genetic study (JAK2) and biochemical tests. Data were collected and analyzed by using SPSS software version 24. Result : The males were (60%) of patients and females were (40%) with male to female ratio was 1.5:1, their age ranged from 43-75 years with a mean of 59.3 years. The most common complains were fatigue (86.7%), fullness in the abdomen (73.3%) and constitutional symptoms (66.7-80%). All patients(100%) had splenomegaly at presentation. The total mean Hb was 8.2 g/dl with a range of 4.7-12.5 g/dl. Leukocytosis had been observed in (40%) of patients, while thrombocytosis was found in (26.6%) of patients. Compared in hematological parameters in PMF patients with JAK2 Positive and JAK2 Negative, it was found that MPV significantly increased in JAK2 positive patients with p-value 0.03 . Conclusion : old age groups (55-64 years) were commonly affected by primary myelofibrosis and males were predominance over females. The most common complains were fatigue and constitutional symptoms, while splenomegaly was the most common clinical sign. The mean platelets volume (MPV) significantly increased in JAK2 positive patients with p-value 0.03.

Assessment of the Haematological Alterations in Cervical Cancer Patients Attending Sabratha

Background: Cervix cancer is one of the most common cancers in women worldwide, and is the third most common malignant disease in women. It is one of the main health problems in Libyan women. Blood act as a pathological reflector of the status of exposed patient to infections and other conditions. Laboratory tests on the blood are vital tools that help detect any deviation from normal in the human body As the disease progresses, changes appear in haematological parameters which have been of relevant consideration in context of cancer patients. Objectives: The present study aimed to evaluate the alterations in haematological parameters among cervical cancer patients in Sabratha National Cancer Institute. Subjects and Methods: The present study was conducted on 150 cervical cancer patients, attending the National Cancer Institute of Sabratha from the 11th February, 2006 to the 3rd February, 2020. This study was approved by the Research and Ethical Committee of Sabratha University and Sabratha National Cancer Institute. Age was extracted from patient files. Also, 60 healthy individuals without any chronic disease were recruited for the control group. Blood samples were collected by vein puncture, 3 ml of venous blood withdrawn from each participant in the study by using disposable syringes under aseptic technique; they then transferred to a sterile EDTA tube, for complete blood count. The statistical significance of differences between groups was evaluated with the Mann Whitney U test. Results: The results showed that the mean age of the cervix cancer patients was 53.37±11.6 years.RBCs count, hemoglobin concentration, Hct value, MCH, MCHC, and lymphocytes % were significantly (P<0.01) decreased compared with the healthy individuals. On the other hand, leukocytes and platelets count, mixed %, neutrophils %, PLTs/Lymph, and Neutrophils/Lymphocytes ratios were significantly increased as compared with the healthy individuals. Conclusion: It can be concluded that a significant increase in leukocytes and platelets count, mixed %, neutrophils %, and the studied inflammation related haematological parameters and a significant decreased in lymphocytes %, RBCs count and most its indices. Further haematological studies are needed to confirm these results. Also, there is need to routinely monitor the haematological parameters and among cervical cancer patients.

Assessment of the haematological alterations in cervical cancer patients attending sabratha national cancer institute, Western Libya

Background: Cervix cancer is one of the most common cancers in women worldwide, and is the third most common malignant disease in women. It is one of the main health problems in Libyan women. Blood act as a pathological reflector of the status of exposed patient to infections and other conditions. Laboratory tests on the blood are vital tools that help detect any deviation from normal in the human body As the disease progresses, changes appear in haematological parameters which have been of relevant consideration in context of cancer patients. Objectives: The present study aimed to evaluate the alterations in haematological parameters among cervical cancer patients in Sabratha National Cancer Institute. Subjects and methods: The present study was conducted on 150 cervical cancer patients, attending the National Cancer Institute of Sabratha from the 11th February, 2006 to the 3rd February, 2020. This study was approved by the Research and Ethical Committee of Sabratha University and Sabratha National Cancer Institute. Age was extracted from patient files. Also, 60 healthy individuals without any chronic disease were recruited for the control group. Blood samples were collected by vein puncture, 3 ml of venous blood withdrawn from each participant in the study by using disposable syringes under aseptic technique; they then transferred to a sterile EDTA tube, for complete blood count. The statistical significance of differences between groups was evaluated with the Mann Whitney U test. Results: The results showed that the mean age of the cervix cancer patients was 53.37±11.6 years. RBCs count, hemoglobin concentration, Hct value, MCH, MCHC, and lymphocytes % were significantly (P<0.01) decreased compared with the healthy individuals. On the other hand, leukocytes and platelets count, mixed %, neutrophils %, PLTs/Lymph, and Neutrophils/Lymphocytes ratios were significantly increased as compared with the healthy individuals. Conclusion: It can be concluded that a significant increase in leukocytes and platelets count, mixed %, neutrophils %, and the studied inflammation related haematological parameters and a significant decreased in lymphocytes %, RBCs count and most its indices. Further haematological studies are needed to confirm these results. Also, there is need to routinely monitor the haematological parameters and among cervical cancer patients.

Establishment of a miRNA profile in paediatric HIV-1 patients and its potential as a biomarker for effectiveness of the combined antiretroviral therapy

miRNAs have been extensively studied in pathological conditions, including viral infections, such as those provoked by HIV-1. Several cellular and circulating miRNAs are altered during HIV-1 infection, with either beneficial effects on host defenses or enhanced virus infectivity. Blood samples were collected in sterile EDTA tubes and plasma was separated and stored, as were PBMCs. RNA was isolated and reverse-transcribed. Finally, the miRNA gene expression profile was assessed using TaqMan Array Human microRNA Card A v2.0. A comprehensive statistical analysis was performed on the results obtained. This is the first study on miRNAs in HIV-1 paediatric patients, and a miRNA profile differentiating patients starting combination antiretroviral therapy (cART) at different times after HIV-1 diagnosis was established. Thirty-four miRNAs were observed to have different expression levels between the control group and the cART group. The data indicates the need to start cART as soon as possible after the establishment of HIV-1 infection to assure the best outcome possible. Finally, the selected 34 miRNAs may be used as biomarkers for prognosis and assessing therapy effectiveness. However, more research must be conducted to establish adequate quantitative correlations.

P0739 / #1900: CD64, CD11A, AND CD18 LEUKOCYTES EXPRESSION IN A CASE SERIES OF CHILDREN WITH SARS-COV-2 MULTISYSTEM INFLAMMATORY SYNDROME AND KAWASAKI DISEASE: SIMILAR BUT NOT THE SAME

Aims & Objectives: A new paediatric inflammatory syndrome named as “pediatric multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS)” has been described. It has been compared to Kawasaki Disease. The aim of this report is adding to the PIMS-TS clinical and analytical description the application of immunophenotyping by flow cytometry (FC). We describe CD64, CD18, and CD11a leukocytes expression in three children with SARS-CoV2 infection and compare it with three cases of Kawasaki Disease. Methods: Three children were studied after informed consent obtained from their parents or legal guardians. Their blood samples were collected in sterile EDTA at room temperature or refrigerated at 4°C and analyzed within 24 hours. Expressions were measured in monocytes, neutrophils, and lymphocytes. At least 10 000 events were recorded for each sample. The intensity of CD64, CD18, and CD11a surface expression were measured as mean fluorescence intensity in arbitrary units (MFI). They were compared with three previous cases of KD. Results: The median CD64, CD18 and CD11a MFI expression in PIMS-TS versus KD cases are described in Figure 1. The CD64 and the CD11a expression on neutrophils and monocytes are higher. The CD11a in CD8 Lymphocytes is higher too.Conclusions: We compare for the first time the immunophenotype of children with PIMS-TS infection versus KD. We observed significant but higher upregulation of CD64, CD18, and CD11a expression. This response appears to be similar but different than in KD. Prospective studies with a higher number of cases should be conducted to confirm this observation.

Impact of Insecticide Treated Nets and Intermittent Preventive Treatment in Reducing Malaria Morbidity among Pregnant Women in Gombe, Nigeria

Malaria in pregnancy is a major public health problem affecting women fetuses and new borns. Many studies highlight the critical importance of continuing the use of Insecticide Treated Nets (ITN) and Intermittent Preventive Treatment In Pregnancy (IPTp) among pregnant women to reduce the adverse consequences of malaria in pregnancy. This study was conducted in order to determine malaria prevalence in relation to the use ofITN and IPTp among the pregnant women in the study area. Five (5) ml of blood was obtained from each participant by the use of a sterile syringe and placed in a sterile EDTA container for laboratory analysis. The malaria parasite was detected by microscopic examination of Giemsa-stained thick blood films. Information on the use of ITN and IPTP was collected using administered questionnaire. A high prevalence of 78.4% was observed among the studied population. Although 74.4% of those that use ITN were positive for malaria parasite as against the 83.6% of those that reported not using the ITN, the difference was statistically not significant (p<0.05). 70.0% of those reported using IPTp were positive however, higher percentage was observed for those reported not using IPTp (83.7.0%). The difference was statistically significant in this case. This study has shown the influence of malaria prevention method during pregnancy on malaria infection and the need for targeted preventive starategies when designing and implementing policies aimed at improving uptake of these measures during pregnancy in Gombe.
 Keywords: malaria, pregnant women, ITN, Gombe, IPTp

Polyomaviruses BK and JC DNA infection in peripheral blood cells from blood donors

ObjectivesTo investigate the prevalence of human polyomavirus (BK and JC viruses) infection in peripheral blood mononuclear cells of healthy blood donors. MethodsThe study included 250 healthy blood donors. Five-milliliter blood was drawn into sterile EDTA tubes and PBMCs were isolated from whole blood. The isolated PBMCs were counted and stored at −70°C for future investigation. DNA was extracted and subjected to simple, sensitive and specific semi-nested PCR as well as QPCR using both general and specific primers for different assays. ResultsOf 250 blood samples, 66 (26.4%) were positive for BKV DNA (146–34,514 copies/106 cells). JC DNA was found in 45 (18%) blood samples (65–21,250 copies/106 cells). Co-infection with these viruses were found in 11 (4.4%) out of 250 blood samples. DiscussionOur study provides important data on polyomavirus infection in peripheral blood mononuclear leukocytes in immunocompetent individuals. These data indicate significant differences between the prevalence of BKV and JCV infection in healthy blood donors. The prevalence of BK and JC virus infection is higher in the age range 30–39 years compared to other age ranges.

Raised PDW Common Hematological Parameters as a Prognostic & Recovery Survival Index of Plasmodium Vivex Malaria for Acute Disease

Author Dr Akshay Surana Curewell Hospital Pvt. Ltd 19\1-C New Palasia Indore MP Email: asurana06@yahoo.com 7741906945 Abstract Aims & Objectives: Improve the survival rate of patients. Reduced the morbidity and mortality of malaria due to plasmodium vivex Reduced the incidence of plasmodium vivex induced acute illness and prevent the complication. Material & Methods: Blood was collected in a sterile EDTA containing tube and processed following our established laboratory protocol .A complete blood counting including HB%, PCV, Red cell indices, platelet count with platelets indices PDW and total white cell count and differential was done by Automated blood cell counter and peripheral blood smear examination by thin film and thick film. The all cell count indices including RBC, WBC count with differential along with platelets count was further confirmed by manual oil immersion smear study method. Peripheral smears study was done with field A and B stain and leishman stain. Conclusion: Thrombocytopenia is one of the common complications of acute illness of the P. vivax malaria. After the Thrombocytopenia, raised platelets distribution width is a as prognostic tool and recovery survival index after acute illness p.vivex malaria. Keywordplatelets distribution width, Thrombocytopenia.

Raised Red Cell Distribution Width as a Prognostic Tool for Acute Aluminium Phosphide Poisoning (AALPP) Patients

Objectives & Aims: Identify the acute aluminium phosphide poisoning. Identify the acute aluminium phosphide poisoning induced changes in hematological parameters .Raised red cell distribution used as a prognostic tool for acute aluminium phosphide poisoning . The changed hematological parameter used as a survival index of acute aluminium phosphide poisoning patients. Material & Methods: Blood was collected in a sterile EDTA containing tube and processed following our established ISO certified hospital laboratory protocol .A complete blood counting including HB%,PCV, Red cell indices ,platelet count and total white cell count and differential was done by Automated blood cell counter with peripheral blood smear examination and further confirmed by manual oil immersion smear study method. Peripheral smears study was done with field A and B stain and leishman stain. Conclusion: Aluminium phosphide poisoning caused changes in size of peripheral red blood cell due to lipid per oxidation and oxidative stress of cell membrane. These oxidative stress and lipid per oxidations lead to damage the cell membrane of RBC. These damage directly related with severity of exposer of the Aluminium phosphide. This type of damages decided the outcome survival of patients. So variable size of RBC occurs in peripheral blood used as a prognostic tool for severity of toxicity, monitoring of patients ,follow-up and outcome survival in the form of RDW .The RDW is a hematological parameter is easily generated by all type of hematological cell counters used as a good prognostic tool for acute aluminium phosphide poisoning Keyword: aluminium phosphide poisoning, Red cell distribution width. Material & Methods Study area and designThis present study was conducted at the Department of Pathology curewell hospital Pvt Ltd Indore MP. The study was designed as a observational hospital based study over a period of time from 2011 to 2016 years. Ethical ConsiderationBlood was collected in a sterile EDTA containing tube and processed following our established laboratory protocol then generate the report of each patient. Take informed consent was obtained from all study participant for use of your blood sample for medical research after doing physician request investigating and generate the report. Patient’s Selection CriteriaThe study target all the acute aluminium phosphide poisoning toxicity patients on the basis of clinical signs, www.jmscr.igmpublication.org Impact Factor 5.244 Index Copernicus Value: 83.27 ISSN (e)-2347-176x ISSN (p) 2455-0450 DOI: http://dx.doi.org/10.18535/jmscr/v4i10.28

The Role of p53 Gene in Cervical Carcinogenesis.

To investigate the role of p53 gene in cervical carcinogenesis. A total 50 cases and controls were taken after setting exclusion criteria. Venous blood (3ml) samples were collected in sterile EDTA sterile vials. Both punch biopsy of cervical growth in cases and biopsy from cervix after hysterectomy in controls were performed. Genomic DNA was extracted from tissue and blood using standard protocol of Miller et al. 1994 using chloroform-phenol method. Gene was amplified using specific forward and reverse primers and p53 gene expressions were studied. The present study of p53 gene regulation analyzed the expression of 279-bp bands on 1.5 agarose gel. Out of the total 50 samples of cases and controls, we were able to isolate DNA from 38 cases and 28 controls in blood and in 22 cases and 22 controls in tissue. In cases of carcinoma cervix, p53 expression is either downregulated or absent in 71.06% of cases compared to 50% of controls in blood and 72.73% of cases compared to 59.09% of controls in tissue, but these figures were not statistically significant (p=0.67 and p=0.167, respectively). p53 positivity rate was only in 27.78% of squamous cell cancer and 50% of adenocarcinoma. Three out of nine patients (33.3%) with L.N. positive status have p53 gene positivity, whereas 23% (3 out of 13) with L.N. negative status have p53 gene positivity, which is not significantly associated. In our study, p53 overexpression increases with the various stages of cervical cancer. In our study, we found that there is the increased frequency of upregulation or overexpression of p53 gene in control in both blood (50%) and tissue (40.9%), but this association is statistically nonsignificant. In the present study, there is a lack of relationship between p53 overexpression and prognosis in the cervical cancer patients. However, our study lacked larger sample size which otherwise would have been able to lend support to truly significant findings through much larger combined and comparative datasets.

Raised Red cell distribution width is common hematological finding of HIV infection

Background: Discuss relevant laboratory findings raised RDW with age and sex distribution. Establish care guidelines for HIV infected person and altered haematopoiesis resulting in with raised red cell distribution width. Aims & Objectives: To Review the pathogenesis of the haematological manifestations of human immunodeficiency virus (HIV). Materials and Methods: Blood was collected in a sterile EDTA containing tube and processed following our established laboratory protocol and by universal precaution as per the guideline of National aids control organization (NACO, India). A complete blood counting including HB%, PCV, Red cell indices, platelet count, Red cell distribution width and total white cell count and differential was done by Automated blood cell counter analyzer of all the patient on antiretroviral therapy. The all cell count indices including WBC count with differential and platelet count, was further confirmed by manual oil immersion smear study method. Peripheral smears study was done with field A and B Stain and Leishman stain. Results: In our study out of 300 HIV cases 189 cases (63%, n=300) shows increased RDW in which male cases are 125 cases (66.48%, n=188) and female cases are 64 (57.14%, N=112). Conclusion: RDW is commonly affected haematological parameter in HIV infective cases. Higher red blood cell distribution width is associated with a worse virologic and clinical situation in HIV infected.

EFFECTS OF THE COMBINATION ANTIBIOTIC—EDTA–TRIS IN THE TREATMENT OF CHRONIC BOVINE ENDOMETRITIS CAUSED BY ANTIMICROBIAL-RESISTANT BACTERIA

The combined effects of the uterine infusion of EDTA–Tris solution and antibiotics have been evaluated in 75 cases of slight, moderate or severe bovine endometritis which did not respond to local routine antimicrobial therapy. Antibiotic-resistant bacteria were isolated from uterine swabs. The cows were divided into three groups on the basis of the severity of endometritis and treated with 100 ml of sterile EDTA–Tris solution (250 mMEDTA and 50 mMtris, pH 8) and the same antibiotic used in the first unsuccessful treatment (oxytetracycline, enrofloxacin, lincomycin–spectinomycin or amikacin). Control groups consisting of six animals treated with antibiotic alone were used. Clinical evaluations performed 2, 15, 21, 42 and 63 days after treatment revealed good therapeutic results, as 53 cows showed a complete recovery with renormalization of the subsequent oestrus cycle. Artificial insemination was followed by pregnancy in about 90% of treated cows. In control animals the second treatment performed using only the antibiotic gave variable and unsatisfactory results, particularly in animals affected by severe endometritis.