Hematology Department Lab SOPs

Introduction to ELISA Enzyme-Linked Immunosorbent Assay (ELISA) is a powerful biochemical technique used to detect the presence and quantify the concentration of antigens (proteins, peptides, hormones, etc.) or antibodies in a sample. ELISA is widely utilized in clinical diagnostics, research, and quality control due to its high sensitivity, specificity, and ease of use. Principles of ELISA The fundamental principle of ELISA involves the specific binding of an antibody to its corresponding antigen, which is then detected by an enzyme-linked antibody capable of producing a measurable signal, usually a color change, upon addition of a substrate. The intensity of the signal is directly proportional to the amount of antigen or antibody present in the sample. Types of ELISA ELISA can be broadly categorized into four main types based on the format and the way the antigen or antibody is detected: 1. Direct ELISA:    - Principle: In direct ELISA, the antigen is immobilized on the micr...

Clinical Significance The Typhidot test is a rapid serological assay used to detect antibodies (IgM and IgG) against Salmonella typhi, the causative agent of typhoid fever. This test is significant in diagnosing typhoid fever, particularly in regions where the disease is endemic. Early and accurate diagnosis helps in the prompt initiation of appropriate antibiotic therapy, reducing morbidity and preventing complications. Principle The Typhidot test is based on the detection of specific IgM and IgG antibodies against the 50 kDa outer membrane protein (OMP) antigen of Salmonella typhi using a dot enzyme immunoassay (EIA) format. The presence of these antibodies indicates recent or current infection with Salmonella typhi. Requirements - Test kit components: Typhidot test strips, buffer solution, conjugate, substrate, and washing solution. - Patient sample: Serum or plasma. - Laboratory equipment: Micropipettes, pipette tips, test tubes, timer, incubator, and absorbent paper. - Personal...

Polyacrylamide Gel Electrophoresis (PAGE) is a widely used technique for separating proteins based on their size and charge.  Clinical Significance 1. Protein Analysis: PAGE is used to analyze protein mixtures, including detecting abnormal protein patterns in diseases such as multiple myeloma, autoimmune disorders, and certain cancers. 2. Genetic Research: Identifies genetic variations and mutations by analyzing proteins or nucleic acids. 3. Diagnostics: Helps in diagnosing conditions by analyzing serum proteins, hemoglobin variants, and enzyme deficiencies. 4. Biomarker Discovery: Essential in the discovery and validation of biomarkers for various diseases. Principle PAGE separates proteins based on their electrophoretic mobility, which is a function of the length, conformation, and charge of the molecule. The gel matrix acts as a sieve, with smaller molecules moving faster than larger ones. Requirements - Equipment:    - PAGE apparatus (gel casting unit, electropho...

The Montoux Test, also known as the Tuberculin Skin Test (TST), is a diagnostic tool used to determine whether a person has been infected with Mycobacterium tuberculosis, the bacteria that cause tuberculosis (TB). Clinical Significance Diagnosis of Latent TB Infection (LTBI): Helps identify individuals who are infected with TB bacteria but do not have active TB disease. Screening Tool: Used for screening high-risk populations such as healthcare workers, people with weakened immune systems, and individuals who have been in contact with TB patients. Public Health Surveillance: Assists in controlling and preventing TB by identifying and treating latent infections before they progress to active disease. Principle The test involves the intradermal injection of a purified protein derivative (PPD) of the TB bacterium. If the person has been exposed to TB bacteria, their immune system will recognize the PPD and mount a localized reaction at the site of injection. This reaction is measured 4...

 Several weak antigen D forms are recognized, including what was formerly called antigen Du. The term weak antigen D is used to describe those forms of antigen D where the number of red cell D receptors is reduced. Such weak D cells react less strongly than red cells with normal numbers of D receptors. Monoclonal IgM anti-D serum will detect weak antigen D. In some African and other populations, weak antigen D has been found in up to 10% of people. Requirements         Purple top vacutainer blood whole blood         Anti-D antisera         Coombs reagent         Test tubes         Centrifuge         Microscope         Slides Procedure Prepare 5% cell suspension of patient. Place one drop of anti-D serum in a test tube. Add  to  it  one  drop  of  patient’s  cell  suspension. Incubate the t...

Platelet count is a crucial parameter in assessing the coagulation status and overall health of an individual. It measures the number of platelets present in a specified volume of blood and helps in diagnosing various disorders, such as thrombocytopenia or thrombocytosis. One of the commonly used methods to determine platelet count is the Neubauer chamber method, which utilizes a specialized counting chamber and a microscope. Requirements: To perform platelet count using the Neubauer chamber method, the following materials are required: Neubauer counting chamber: A specially designed slide with a grid pattern and a known depth to enable accurate counting. Microscope: A compound microscope with appropriate magnification (typically 10x or 40x objective) to visualize platelets. Hemocytometer cover slip: A cover slip that is placed over the counting chamber to create a defined volume for counting. Phosphate-buffered saline (PBS): A solution used to dilute the blood sample for better visual...