Fall 24-25 Practical Session 2-Lab1 Physiology BMS201 PDF

# Determination of Hemoglobin Content, Hematocrit Value, and Calculation of Blood Indices ## Practical session 2-Lab 1-BMS201-Physiology **Prof. Sahar El Agaty | Professor of Physiology** **GALALA UNIVERSITY** **Powered by Arizona State University** ## Intended Learning Outcomes - Interpret th...

# Determination of Hemoglobin Content, Hematocrit Value, and Calculation of Blood Indices ## Practical session 2-Lab 1-BMS201-Physiology **Prof. Sahar El Agaty | Professor of Physiology** **GALALA UNIVERSITY** **Powered by Arizona State University** ## Intended Learning Outcomes - Interpret the complete blood count - Assess the Hb concentration by cyanmethemoglobin method - Determine the hematocrit value (Hct). - Calculate red blood cell indices. - Recognize causes of leucopenia, leucocytosis, thrombocytosis and thrombocytopenia. ## Complete Blood Picture (Complete Blood Count, CBC) A complete blood count (CBC) is a blood test that helps to monitor or diagnose health conditions. It can detect blood cancers, anemia, and infections. Also, it is used to follow the progress of certain medical conditions and to diagnose certain diseases that affect the number of the WBCs and platelets. It is performed by a variety of automated instruments including coulter. ### Complete Blood Picture | TEST NAME | RESULT | UNIT | BIOLOGICAL REFERENCE | |---|---|---|---| | Haemoglobin | L 11.4 | g/dl | 11.5-15.5 | | Haematocrit (PCV) | 36.3 | % | 36-45 | | RBCs Count | 4.47 | millions/cmm | 4.0-5.2 | | MCV | 81.2 | fl | 80-100 | | MCH | L 25.5 | pg | 27-33 | | MCHC | 31.4 | g/dl | 31-37 | | RDW-CV | 13.0 | % | 11.5-15 | | Platelet Count | 302 | thousands/cmm | 150-450 | | Total Leucocytic Count | 5.8 | thousands/cmm | 4-11 | | Differential Leucocytic Count | Percent Values | | Absolute Values | | Neutrophils | 43.0 | % | 2.50 x10⁹/L | 2-7 | | Lymphocytes | 39.3 | % | 2.29 x10⁹/L | 1-4.8 | | Monocytes | 14.6 | % | 0.85 x10⁹/L | 0.2-1 | | Eosinophils | 2.4 | % | 0.14 x10⁹/L | 0.1-0.45 | | Basophils | 0.7 | % | 0.04 x10⁹/L | 0-0.1 | | Other Cells | | | | | **Comment:** | MILD HYPOCHROMIC ANEMIA.RELATIVE MONOCYTOSIS SERUM IRON, TIBC, FERRITIN & FOLLOW UP ARE RECOMMENDED | | | ## Red Blood Count ### Normal Value: - **Adult males:** 5.0-5.5 million/mm³ - **Adult females:** 4.5-5.0 million/mm³ - **Newly born infants:** 4.8-7.1 million/mm³ - **Children:** 4-5.5 million/mm³ - **N.B. mm³ = µl= 10^-6 L** ### Increased RBC count (Polycythemia) ### Decreased RBC count (anemia) ### Recognize causes of polycythemia and anemia. ## Hemoglobin Concentration **Normal value:** - **Adult Male:** 16 ± 2 gm /dl - **Adult Female:** 14 ± 2 gm /dl - **Infants at birth:** 18 gm/dl - **Children 6-12y:** 13.5 gm/dl. ### Increased Hb concentration (Polycythemia) ### Decreased Hb concentration (anemia) ### List types of hemoglobin ## Measurement of Hemoglobin Concentration by Using Cyanmethemoglobin Method It is the internationally recommended method for determining the hemoglobin concentration of blood. ### Principle: - Blood is diluted 1:201 in a solution containing potassium ferricyanide and potassium cyanide. Potassium ferricyanide oxidizes hemoglobin in the sample to methemoglobin. The methemoglobin further reacts with potassium cyanide to form a stable-colored cyanmethemoglobin (hemiglobincyanide) complex. The intensity of the colored complex is measured at 540 nm which is directly proportional to the amount of hemoglobin present in the specimen. ### *Diagram of Hemoglobin reaction* ``` Potassium ferricyanide Hemoglobin (Fe2+) ---> Methemoglobin ---> Cyanmethemoglobin (Stable red colored complex) Potassium cyanide ``` ### Specimen: Capillary or venous blood. Venous blood should be anticoagulated with EDTA ### Reagents: 1. Reagent 1: ferricyanide and cyanide reagents 2. Hemoglobin standard ### Method: - Label three clean, dry test tubes as Blank, Standard, and Test. - Pipette as follows: | | Blank | Standard | Test | |---|---|---|---| | Reagent 1 | 5 ml | 5 ml | 5 ml | | Hemoglobin standard | - | 20 µl | - | | Sample | - | - | 20 µl | - Mix well and allow to stand at room temperature (25°C) for 5 minutes. - Measure the absorbance of the standard and test sample at 540 nm (green filter) against blank in a spectrophotometer (colorimeter). The color will be stable for up to several hours. ### Calculation: **Conc. of Hemoglobin in the specimen (g/dl) = (Absorbance of Test / Absorbance of Standard) x Conc. of Standard** ### Calculate the Hb concentration by using the following parameters: The standard blood sample concentration is 20 and absorbance of standard is 2, and the absorbance of the test sample is 1.5. **Hb concentration (gm/dl) = (1.5 / 2) x 20 = 15 gm/dl** ## Hematocrit value (Hct) or packed cell volume (PCV) ### Definition: The percentage ratio of red blood cells in the total blood **Hct = (Volume of RBCs / Volume of Blood) x 100** ### Normal values: - **Adult males:** 45% - **Adult Females:** 42% - **Infant at birth:** 60% - **Children 6-12 y:** 40% ### Measurement: It can be measured by: 1. Coulter 2. Microhematocrit method 3. Wintrobe's method ### Uses: Determination of blood volume and help to determine the type of anemia ## Measurement of Hct by microhematocrit method 1. It is carried out on blood contained in capillary tubes 75 mm in length and having an internal diameter of about 1 mm. 2. The tube used may: - contain anticoagulant heparin for direct collection of capillary blood (from skin puncture or venous blood) - be plain in case of using anticoagulated blood sample. 3. At least 15 mm of the tube is left unfilled. 4. The tube was sealed from one end by plasticine. 5. Then, the tube is centrifuged (centrifugal force of 12 000) for 5 min. 6. The Hct was determined by using hematocrit reader. ## Measurement of Hct by Wintrobe's method 1. Venous blood is collected in EDTA and withdrawn in a Wintrobe tube (110 mm in length, 3 mm internal diameter, and has 100 markings). 2. The tube was centrifuged at 2300 g for 30 min. 3. Measure the height of RBC layer. ## Enumerate physiological and pathological factors affecting Hct. ## Red cell indices - They are measurements that describe the size and Hb content of red blood cells. - These indices can provide a basis for classifying anemias. They include: 1. **Mean corpuscular volume (MCV)** 2. **Mean corpuscular hemoglobin (MCH)** 3. **Mean corpuscular hemoglobin concentration (MCHC)** 4. **Red cell distribution width (RDW)** - It is an index of the variation in cell volume within the red blood cell population and is expressed as a percentage. ### Normal value: **RDW = 11.5-14.5%** - A high RDW indicates that the red blood cells are more variable in volume than normal. ### Causes of increased RDW: 1. Anemias caused by deficiencies of iron, vitamin B12, or folic acid. 2. Abnormal hemoglobins, such as in sickle cell anemia, can change the shape of red blood cells as well as cause them to hemolyze (cell fragments) 3. Conditions that cause more immature large cells to be released into the bloodstream, such as severe blood loss. ## Mean corpuscular volume (MCV) - **It measures the average volume of a red blood cell.** - **The MCV categorizes red blood cells by size.** ### Calculation: **MCV= (Hct x 10 / RBCs count in millions/mm³) = (45 x 10 / 5) = 90 μ³ = 90 fl** ### Normal value: **MCV= 80-95 fl (femtoliters)** **Femtoliter = 10^-15 Liter** ## Morphologic Classification of Anemia - **MCV** - **Low:** Microcytic- Iron deficiency anemia - **Normal:** Normocytic- Chronic disease, Renal failure - **High:** Macrocytic- Vitamin B12 and folic acid deficiency anemia ## Mean corpuscular hemoglobin (MCH) - **It is the average weight of hemoglobin content in a red cell.** ### Calculation: **MCH = (Hb content (g/100 ml) x 10 / RBCs count in millions/mm³) = (15 x 10 / 5) = 30 pg** ### Normal value: **MCH = 27-31 pg (picograms)** **Pg = 10^-12 g** ## Mean corpuscular hemoglobin concentration (MCHC) - **It measures the average concentration of hemoglobin in 100 ml of red blood cell.** - **The MCHC categorizes red blood cells according to their concentration of hemoglobin.** ### Calculation: **MCHC = (Hb content (g/100 ml) x 100 / Hct) = (15 x 100 / 45) = 33 g/100 ml** ### Normal value: **MCHC = 30-35 g/100 ml of RBCs.** ## Total leukocytic count **Normal value: 4,000–11,000 / mm³** - **Leukocytosis:** Increased WBCs count > 11,000 / mm³ in adult. - **Leukopenia:** Decreased WBCs count < 4,000 / mm³ in adult. ## Differential leukocytic count: 1. **Neutrophils (60-70%)** 2. **Eosinophils (1-4%)** 3. **Basophils (0.25-0.5%)** 4. **Lymphocytes (25-33%)** 5. **Monocytes (2-6%)** ## Identify causes of abnormal leukocytic count. ## Platelet count **Normal value:** 150-450 x10³/mm³ of blood - **High platelet count** is called **Thrombocytosis.** - **Low platelet count** is called **Thrombocytopenia.** ## Causes of thrombocytosis: 1. **Reactive thrombocytosis:** blood loss, cancer, infection. 2. **Bone marrow abnormally produces too many platelets** ## Causes of thrombocytopenia: 1. **Bone marrow depression** 2. **Autoimmune disease** 3. **Splenomegaly** ## Questions **Q1 Hct reading is 45 per cent and the red cell count is 5 million per cu mm of blood, Calculate the MCV.** **- The answer:** **MCV = (Hct x 10 / RBCs count in millions/mm³) = (45 x 10 / 5) = 90 fl** **Q2 The Hb content is 14 g/dl and the red cell count is 5 million per cu mm Calculate the MCH.** **- The answer:** **MCH = (Hb content (g/100 ml) x 10 / RBCs count in millions/mm³) = (14 x 10 / 5) = 28 pg ** ## Thank You This document describes the process of determining hemoglobin content, hematocrit values, and calculating blood indices. The content is an overview of the process, and not instructions to be executed by a layperson.