Lab 5: Practical Physiology Second class Hemoglobin Estimation Hemoglobin: is the major constituent of the red blood cell cytoplasm, accounting for approximately 90% of the dry weight of the mature cell. It is comprised of heme and globin, 2. Carriage of carbon dioxide from tissues to the lungs as carbaminohaemoglobin. 3. Hb as buffer: Hb helps to maintain its acid base balance. Sahli's (acid hematin) Method Principle: Blood is mixed with 0.1 N of HCl resulting in the conversion of all types of Hb to acid hematin which is brown in color. The solution is diluted till its color matches with the brown colored glass of the comparator box. The concentration of Hb is read directly. quipment required Haemometer which consists of: “Comparator box which has brown colored glass on either side + Hb pipette which is marked up to 20 mm*(0.02ml blood) + Tube with markings of Hb on one side Glass rod “* Dropper Reagents and instrument required 0.1 concentration from HCI % Distilled water *% Alcohol 70% “+ Whole blood LancetNormal Level of Hb in Human Body Normal Hb level in Males 14-16 (gms per 100 ml) Normal Hb level in Females 12-15 (gms per 100 ml) Age, sex, diurnal variation, altitude, exercise, excitement and adrenaline effect on the Hb level. Acid haematin method using a haemoglobinometer (Sahli’s method) 1. Add HCl into the graduated tube up to mark 10 2. Measure 0.02 ml (2011) of well-mixed blood, with the provided micropipette (Sahli’s pipette) and transfer it to the HCI in the tube. 3. With the pipette beneath the surface of the acid, gently blow the blood. 4. Rinse the pipette by sucking up and blowing out diluent 2-3 times. 5. Thoroughly mix blood and acid using a fine glass rod (HCL will react with the hemoglobin and convert it into acid-haematin, which has a brown color). 6. Wait up to 10 minutes after adding the blood to allow the color to develop sufficiently to achieve an accurate comparison. 7. Add distilled water gradually to the mixture and mix the solution with glass rode. 8. Place the tube in the haemoglobinometer and compare it with the standard. 9. Continue to add distilled water until the sample firstly appears to be detectable 10.Note the level of the liquid in the tube.Comparator box Tube with markings of Hb Glass rod DropperrN Lab 6: Practical Physiology Second class Blood groups The ABO typing is the most important test performed in transfusion practice. The most common cause of transfusion-related fatal factor is due to a patient being transfused with ABO-incompatible blood. Aim: The standard test is to determine ABO and Rh blood type. Principle: Blood typing involves the two types of molecules called antigens and antibodies. Agglutination of the test cells indicates the presence of the related antigen, while no agglutination indicates its absence. Types of blood group ‘There are four major blood groups determined by the presence or absence of two antigens — A and B—on the surface of red blood cells: + Group A—has gust the A antigen on red cells (and B antibody in the plasma) « Group B— has just the B antigen on red cells (and A antibody in the plasma) « Group AB~has both A and B antigens on red cells (but neither A nor B antibody in the plasma). It can take blood from all blood group types. 80 it called universal recipient, Put only give blood to AB blood group. «= Group O —has neither A nor B antigens on red cells (but both and B antibody are in the plasma). Itcan give blood to all blood group types. So it called universal donor. Put only take blood from O blood group. Rh blood group ‘The Rh blood groups have Rh antigens (antigen D), Most people are Rh+ (Rh positive), meaning that their RBCs carry the Rh antigen. If a Rh- person receives mismatched blood (that is, Rh+) hemolysis (rapture of RBCs) will occur, Note: The ABO blood type is indicated by using letters, and the Rh blood type is indicated by using the symbols (+) and (-).Material required 1, Glass slides 2. Antisera A, Antisera B and Anti D i 3. Wooden stick rod for mixing 4. Marker pen 5. Laneet 6. Whole finger blood 7. Cotton & alcohol 70% Procedure 1. Obtain a microscope slide. The slide must be very clean, so it does not interfere with the reaction. 2, With a marker pen, draw two lines on one surface of the slide to divide the surface into thirds. 3, Obtain three wooden stick rods. One wooden stick rod for each drop will be used. 4. Place one drop of the appropriate antiserum (anti A, anti B, anti D) on the slide. 5. Clean the finger with alcohol let it air dry. Lance the finger by lancet. 6. Put one drop of blood on each drop of antiserum. 7. Mix the blood and antiserum with wooden stick rod, and rock the slide gently for about 1-2 minutes. 8. Examine for agglutination with naked eye, if it present indicates a positive result. ‘The reaction patterns of the most common ABO phenotypes are shown in the following table: ABO GroupingGroup A Group B Group AB Group O Red blood cell type Mate Antibodies | ~ ~y ay ye in Plasma Anti-A and Anti- Antigens in Red Blood lutinogen saciinatee wi with ant-A) B (contains | agglutinogen B; agglutinates with ant-6) Type 0 (contains no Dicedgenes does not eaghutinate will with etther | sani}Lab 7: Practical Physiology Second class Bleeding time and Clotting time Bleeding time: is the time interval from oozing of blood after a cut or injury till arrest of bleeding. Aim: To determine the bleeding time of a patient to assess platelet function and the body’s ability for complete stopping of blood flow. Principle ‘The test involves making a puncture wound in a superficial area of the skin and monitoring the time needed for bleeding to stop. The bleeding Time test is us used of 1, Patients who have a history of prolonged bleeding after cuts. 2. Patients who have a family history of bleeding disorders. 3. The test is sometimes performed as a preoperative test to determine a patient's likely bleeding response during and after surgery. 4. The test helps identify people who have defects in their platelet function. Duke's Method 1. Clean the lobe of the ear or tip of a finger with alcohol and let dry. 2. Pierce the lower portion of the ear lobe (or tip of a finger) with the lancet making the incision 3-4 mm deep start the stopwatch. 3. Wipes the blood every 30 seconds witha filter paper without squeezing. 4, Atthe time when blood fails to appear on the filter paper, stop stop watch. 5. Count the spot of blood on the filter paper. & Record the result and calculate the bleeding time (each 2 spots = 1 min.). «The usual time is about 2-6 minutes. 4 Prolonged bleeding times are generally found when : 1-The platelet count is below 50,000/n1. 2-When there is platelet dysfunction.Material and instrument for bleeding time test: l- Lancet. 2- Filter paper. 3- Stop watch. 4- Cotton and Alcohol 70%. Clotting time: is the time interval from oozing of blood after a cut or injury till formation of clot. ‘Aim: To determine the clotting time ofa subject. Principle: A measure of the time required for blood to solidify (coagulate) after it has been removed from the body. Material and instrument for clotting time test: 1- Fine capillary glass tubes of about 10 mm length 2- Lancet. | 3. Stop watch. | 4- Cotton and Alcohol 70%. Procedure 1. Clean the finger with aleohol 70% and allow itso ary. 2. Prick the finger by lancet. 3, Draw blood up in the capil 4, Start the stop wateh. 5 After one minute start breaking small pieces of the capillary tube every 30 second until a fibrin thread is seen between the two broken ends. 6. Calculating the clotting time by = lary glass tube. (The waiting time after the glass tube is filled + no. of capillary tubes breaks x 30 second) % Normal duration : 3-8 minutesoe Lab 8: Practical Physiology ‘Second clas Measurement of Body Temperature Measurement of body temperature is a routine procedure used in all clinical work, and it is one of the main vital signs that must be monitored to ensure safe and effective care. It varies under many physiological and pathological conditions. Indications for measurement of body temperature Ly To obtain the baseline temperature that enable comparisons to be made with future recordings. observation in resolving hypothermia / hyperthermia. 2» To enable close fection. 3» To observe and monitor patients for changes indicating an in! 4 To erapy for infection. 5» Before and during a blood transfusion to m Factors increasing heat production: 1. Muscular activity. monitor the effect of treatment for antimicrobial th conitor for signs of a reaction. 2. Ingestion of food 3, Time of day: Bod y temperature tends to be at its highest in the late aftemoon or carly evening. 4, Emotional stress 5. Hormones 6. Infections 7, Increased temperature of the environment 8, Menstruation and pregnancy Body temperature is recorded either in degree centigrade (C) or degree Fahrenheit (F). *To convert centigrade to = (9/5 x C) + 32 «To Change Fahrenheit to Centigrade subtract 32 and multiply by 5/9. C= (F-32) x 5/9 Fahrenheit, multiply by 9/5 and add 32.Direct Method: 1, Oral 2. Rectal Indirect Method: 1. Axillary or groin 2. Aural Direct Method: 1- Oral temperature measurement: Most common, convenient and comfortable method: Procedure: 1. Clean the thermometer by rubbing its blub with cotton soaked in alcohol, and rotates it forward and backward until the column appears clearly. 2. If the mercury level is 45°C, lower its level to below this mark; make sure it reads less than 96°F (35.6°C). 3, Insert the bulb of the thermometer inside the mouth under the tongue and tell the patient to close his mouth and breathe from his nose in order not to let cold air comes inside his mouth and gives false reading. 4. Leave the thermometer for 2-5 minutes. 5. Remove the thermometer without touching the tip; take the reading of the thermometer. Apparatus used: | a. An accurate Clinical thermometer of good quality. b. Cotton and alcohol 70% to sterile thermometer. Contraindication of oral temperature: + Infants and children. + Unconscious patients. + Inflammation or surgery of mouth. + Persistent frequent coughing. + Mouth breathing patients.2-_Rectal Method (in the rectum): Most accurate because it is an intemal measurement, Clinical thermometer left in place for 2-3 to 5 minutes (thermometer is inside the body). The rectal temperature is often half to one degree higher than that recorded in the mouth. Indication of rectal temperature: 1. Used for children 2. Un conscious patient 3. The patients with epilepsy Indirect Method: 1. Axilla or Groin Because of the presence of skin and connective tissues, the temperature recorded half to one degree lower than that in the mouth. Less accurate because they are external temperatures This method used for babies and old ages patients, and if there is injuries in the face and the mouth. Procedure: 1. Clean the thermometers by rubbing its blub with cotton soaked in alcohol, and rotate it forward and backward until the column appears clearly. 2. If the mercury level is 45°C, lower its level to below this mark. 3. The area should be dry from sweat and the arm or leg must be steady and near the body. 4, Let the thermometer in the axillary flexure while upper arm is held close to body and or flex the thigh on abdomen. 5. Wait for 4 minutes and then take the reading. Clinical thermometer left in place for 2,3 minutes2. Aural ‘aken with a special thermometer that is placed in the ear or auditory canal, Thermometer detects and measures the thermal, infrared energy radiating from blood vessels in the tympanic membrane. Since this provides a measurements of body core temp, there is no normal range for aural Hours of ectivity and week, Hora of rest and step. ope ae lia tae ieee ei assaet : Tice wee am ASH EEE weal rAsS Pet RE i Oe ee & Ge Cr tN Cee SEECEEHEEEEEHEEE Ee es v0] | - = TT oo 4 ne = AH = a fe tH pes im COCA Important notes about body temperature ies between about 36.1 and 37.8 °C + Normal internal body temperature (which vari (97.0 and 100.0 °F). * An early moming temperature higher than 37.2 temperature higher than 37.8 °C (99.9 °F) is norma ¢F); there may be serious brain damage and °C (99.0 °F) or a late afternoon lly considered a fever. * Death normally occurs at 43°C (109.4 shock. Death usually occurs at 24-26 °C (75-79 °F) or less, due to irregular heart beat or respiratory arrest.