Hematology Department Lab SOPs

 1. Clinical Significance To estimate the hemoglobin concentration in a blood sample using the cyanmethemoglobin method and a spectrophotometer. 2. Principle Hemoglobin in the blood reacts with Drabkin's reagent. Potassium ferricyanide oxidizes hemoglobin to methemoglobin, which then reacts with potassium cyanide to form cyanmethemoglobin (hemiglobincyanide). The resulting colored compound is measured spectrophotometrically at 540 nm . Its absorbance is proportional to the hemoglobin concentration in the sample. 3. Requirements Spectrophotometer Clean test tubes Micropipette (20 µL) Pipette for 5 mL reagent Drabkin's reagent EDTA-anticoagulated blood sample Appropriate cuvettes Gloves and laboratory protective equipment 4. Procedure Switch on the spectrophotometer and allow it to stabilize for 20 mins. Label a clean test tube with the sample identification. Add 5.0 mL of Drabkin's reagent to the test tube. Using a micropipette, add 20 µL of well-mixed EDTA-anticoagulated b...

1. Introduction Spectrophotometry is an analytical technique used to measure the amount of light absorbed or transmitted by a substance at a selected wavelength. In a spectrophotometer, light of a selected wavelength passes through a sample. Some of the light is absorbed by the substance, while the remaining light passes through the sample and reaches a detector. The instrument measures this light and displays the result as absorbance or percentage transmittance. 2. Principle The principle of spectrophotometry is based on the absorption of light by molecules at specific wavelengths. According to the Beer–Lambert law, the absorbance of a solution is directly proportional to the concentration of the absorbing substance and the path length of light through the solution, provided the measurement is made under suitable conditions. Where: T  = Transmittance I0  = Incident light intensity I  = Transmitted light intensity Percentage transmittance is calculated as: Important: High...

 Practical 1: Capillary Blood Collection 1. Purpose To obtain a small volume of blood for laboratory investigations when capillary sampling is appropriate, including: Peripheral blood smear preparation. Hemoglobin estimation and selected hematological tests. Point-of-care glucose testing. Selected microcollection tests, according to the laboratory's validated procedure. 2. Principle Capillary blood is obtained by puncturing the skin with a sterile, single-use lancet. The specimen contains blood from small vessels and may include variable amounts of interstitial fluid if excessive squeezing is used. Therefore, appropriate puncture depth, site selection, and gentle collection are essential for specimen quality. 3. Requirements and materials Examination gloves and other PPE as indicated. Hand-hygiene supplies. Sterile, single-use safety lancet. 70% isopropyl alcohol swab, where appropriate. Sterile, dry gauze or cotton. Microcollection tubes or approved capillary collection devices. M...

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...