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  • br Blood samples and CRP assay br Serum isolation

    2020-08-14


    Blood samples and CRP assay
    Serum isolation
    Blood samples were collected from patients in clot activator vacutainers after the disease was confirmed. The blood was stored in clot activator vials at 37°C until the serum was separated. The upper most layer from the clot activator vial was transferred into an eppendorf. Then the tube was centrifuged for 10 min at 3000 rpm so that Trichostatin A the remaining unseen RBCs get settled down. The tube was taken out and supernatant was carefully transferred into previously labeled cryogenic vial. The tubes were discarded in the biohazard bin. The serum was stored at −80 °C till estimation.
    Estimation of CRP levels
    CRP levels in the serum samples were detected by fully automatic Bioanalyzer Erba XL 200 (Mannheim, Germany) using commercially available kit CLT20009 procured from ERBA Mannheim (Germany) as per the manufacturer’s instructions. The reagents provided with the kit included phosphate buffered saline, polyethylene glycol, and sodium azide. Antiserum and calibrator was also provided with the kit. The principle is based on measurement of antigen-antibody reaction by end point method. The patients were divided into 3 groups based on CRP levels that is normal levels (0-1mg/dl), moderate levels (1-2mg/dl), and high levels (more than 2 mg/dl). The 3 groups were categorized in accordance with the previous studies.12,13 The mod-erate levels and high levels were not merged with a purpose to monitor the association of mod-erate as well as elevated levels of CRP with disease outcome.
    Demographic profile of the patients.
    Demographic features
    Patients Controls P value (patient vs controls)
    Median age
    4 8 N/A Family history of breast cancer 11 Nil N/A Family history of other cancers 19 Nil N/A Chemotherapy
    242 Nil N/A Surgery
    Follow-up
    The follow-up of the patients was conducted out wth the help of a clinician during the follow-up visit of the patients to the hospital. However, some of the patients who did not turn up for follow-up where contacted telephonically (either the patient or a first degree relative) to confirm if the patient was dead. Follow-up was carried out at an interval of 3, 6, 12, 15, 18, 21, 24, and 27 months after hospital discharge. Median follow-up was 13.5 months. Interviews with the patient and clinician were conducted to check metastasis, recurrence and death.
    Statistical analysis
    The data were expressed as mean ± SD. The data was not normal. It was positively skewed. So non-parametric Mann-Whitney U test was performed for comparison of mean values of CRP among cases and controls. It was also performed to determine association of CRP levels with age and triple negative breast cancer. Association of high, normal, and moderate levels of CRP among controls and patients was estimated by odds ratio with 95% CI and χ 2 analysis using Open Epi software (Open Epi version 2.3.1 from Department of Epidemiology, Rollins school of public health, Emory University, Atlanta, GA 30322, USA). Mann-Whitney test was used to es-timate association of CRP levels with histo-pathologic subtypes and body mass index among patients and controls. The association of CRP levels with outcome including death, recurrence and metastasis was estimated by Mann-Whitney U test.
    Results
    Two hundred forty-two female breast cancer patients and equal number of age and sex-matched controls were included in the study. All the cases were recruited from Malwa region of Punjab. Mean age of patients at the time of disease diagnosis was 53.62 ± 12.31 years. Median age of the patients was 53 years. Youngest patient with breast cancer was an unmarried female of 23 years. More than half of the patients (55%) were residing in the urban area. None of the patients was pregnant at the time of diagnosis. Family history of cancer including breast and other cancers was seen in 12.3% of patients (Table 1).
    Carcinoma of left breast was observed in 55% of patients. Modified radical mastectomy and lumpectomy was performed in 89% and 11% of patients respectively. All the patients under-went adjuvant chemotherapy (Cyclophosphamide and doxorubicin). Most prevalent histopatho-logic subtype of breast cancer was infiltrating ductal carcinoma followed by infiltrating lobular carcinoma. Data indicating estrogen receptor/progesterone receptor (ER/PR) and human epider-mal growth factor receptor 2 (HER2) status was available for all patients. Among these 39.2%, 37.6%, and 15% were positive for ER, progesterone receptor, and HER2 receptors, respectively,
    Table 2
    Mean CRP levels in patients and controls.
    Group
    Table 3
    Normal, moderate, and high CRP levels in patients and controls.
    Study group Normal levels Moderate levels High levels P value and odds ratio
    and around 17.5% of cases were suffering from triple negative breast cancer which is the most aggressive type of breast cancer.