FHB101 & MSK101-2 Practical Physiology PDF

FHB101  ESR (ERYTHROCYTE SEDIMENTATION RATE)  Definition: It’s the rate of physical sedimentation of RBCs when suspended in a column of blood.  Apparatus: Westergreen tube and Westergren method.  The idea of the experiment: There are different electrostatic forces that...

FHB101  ESR (ERYTHROCYTE SEDIMENTATION RATE)  Definition: It’s the rate of physical sedimentation of RBCs when suspended in a column of blood.  Apparatus: Westergreen tube and Westergren method.  The idea of the experiment: There are different electrostatic forces that interact between the molecules of plasma proteins (specially fibrinogen and globulin) at their normal concentrations and the different cellular elements of blood, especially RBCs. This force favor the clumping of RBCs and the rate of their sedimentation. In some chronic inflammatory disorders, certain disease marker proteins and inflammation induced proteins are synthesized. These proteins disturb the electrostatic forces and affect the rate of RBCs sedimentation. It can be assessed by the vertical (perpendicular) distance or the height of plasma column above sedimented RBCs of an anti-coagulated blood sample in the calibrated Westgreen tube for an  Anti-coagulant used: In Westergreen method: sodium or potassium citrate at a concentration of 3.4%. At a ratio of 1amount of the anti-coagulant to 4 amounts of blood (1:4). In Wintrope method: EDTA (ethylene diamine tetra acetic acid). Same blood sample used in this method can be used to determine hematocrit as EDTA doesn’t disturb the shape or size of RBCs.  Normal values: male: 4-6 mm/hr. Female: 6-10 mm/hr.  Mechanism: RBCs in the blood aggregate together & form rouleaux …….. RBCs sediment leaving clear plasma.  Factors affecting  Increased ESR:  Physiological factors: 1. after meals. 2. pregnancy. 3. menstruation. 4. high atmospheric temp. 5. exercise.  Pathological factors: 1. Trauma. 2. Tonsilitis. 3. tumors. 4. Tuberculosis (TB). 5. Anemia: by decrease repealing forces in between RBCs. 6. Rheumatic fever. 7. Tissue destruction and inflammation due to increased fibrinogen and globulins. 8. Albumin as in liver disease.  Decreased ESR: 1. polycythemia (physiological in infants or due to hypoxia as in people in high altitude).  Significance of ESR: Non specific test. It is not a diagnostic test, but it is prognostic test. It’s used in follow up of the disease and effect of treatment. ESR is a non specific marker of inflammation and is affected by other factors, ESR results must be used along with other clinical findings. ESTIMATION OF Hb CONTENT (SAHLI’S METHOD)  SAHLI METHOSAHLI METHODD  Apparatus used: Sahli apparatus (Sahli hemoglobinometer or hemometer). Which is a manual device.  Materials: 1. A Comparator: with a brown glass standard. Opaque white glass in the back for uniform (even) illumination. 2. A Sahli’s pipette or Hb pipette: marked at 20µL or 0.02ml. 3. 0.1 N hydrochloric acid (0.01N HCl) mini bottle. 4. Sahli’s graduated hemoglobin tube: Marked in grams percent g% (2-24) and percentage% (10-140). 5. A Stirrer: Thin glass rod for mixing blood samples with diluting solution.  Experiment procedure: 1) Fill the graduated mini tube is Sahli apparatus with 0.1N HCl solution till the mark 5g% on the yellowish graduation. 2) Blow air into the rubber end of the micropipette to ensure it’s patency, by receiving air in your palm. 3) Sterilize the finger with alcohol and leave it to dry. 4) Prick the tip of the thumb with lancet. 5) Draw 20µL (0.02ml) blood sample by putting the tip of micropipette to the undersurface of blood globule (avoid air bubbles). 6) If extra volume of blood sample is taken by capillarity, just draw the extra volume by a cotton swab. 7) Blow off the collected 20µL blood sample over the 0.1N HCL in the mini tube (without delay). 8) Mix the blood sample with the 0.1N HCL till homogenous brown solution is formed. Hemoglobin + HCl acid hematin (brown color). 9) Place the standard mini tube in its track beside the standard and leave it for 5 minutes. 10)Dilute half gram by half gram % (3drops of the diluting solution) then mix each time to compare. 11)Compare the acid hematin solution with the bilateral colored control after each dilution, until the brown color of the movable mini tube matches the color of the standard two tubes when seen in good light. 12)Read the result at the surface of acid hematin diluted solution at the yellowish g % side.  The principle of Sahli’s method: Hydrochloric acid converts hemoglobin to acid hematin, which is then diluted until the color of the solution matches that of the comparator.  Anti coagulant used: NO anticoagulant is used. As the clotting factors are destroyed by HCl.  Normal values:  In neonates: up to 20 gm/dl.  In male: 14-17 gm/dl.  In female: 12-15 gm/dl. It decrease in anemia and increase with polycythemia.  Significance: Used to calculate MCH (mean corpuscular hemoglobin). Used to calculate MCHC (mean corpuscular hemoglobin concentration). H.V (hematocrit value), PCV (packed cell volume) & blood indices.  H.V (HEMATOCRIT VALUE), PCV (PACKED CELL VOLUME) & BLOOD INDICES  Definition: The percentage ratio (%) of the volume of RBCs to the total blood volume.  Experiment: - Heparinized capillary tube is filled with blood (Wintrobe capillary). - One end of the tube is closed by plastin. - The tube is centrifuged at a rate of 3000 rpm (revolutions per minute) for 5 minutes. - Calculate the % of RBCs by reading the scale.  Why is the tube HERPARINIZED? to prevent clotting of blood.  Why heparin not Na citrate? Na citrate will change plasma volume.  Normal value: - Males: 40-50% (45%) - Females: 38-44% (42%) - Infants: 50-60% (55% due to physiological polycythemia of newly born).  Physiological variants: 1. Age: Higher in infants due to physiological polycythemia. 2. sex: Males have higher HV than females because of higher Hb concentration and higher number of RBCs. 3. Vascular source of the blood sample (arterial, venous, capillary): Venous blood have higher HV than arterial blood.  Pathological variants:  Increased H.V: 1. RBCs … polycythemia. 2. Plasma volume (dehydration) …. Vomiting, diarrhea, burn.  Decreased H.V: 1. RBCs … anemia. 2. plasma volume (overhydration).  Importance of H.V determination: 1. Blood indices. 2. Blood volume.  Significance: 1. diagnosis and treatment of anemia. 2. diagnosis and treatment of polycythemia. 3. Determination of the extent of dehydration. 4. Decision of blood transfusion.  Blood indices Blood indices have important diagnostic value in diagnosis of the type and cause of anemia. 1) Hematocrit Value (Packed Cell Volume or Erythrocyte volume fraction): It’s the percent volume of RBCs to total blood volume. 2) Mean Corpuscular Volume (MCV): It’s the average volume of a single RBC (in cubic micron =fL (femtoliter) =80-90 Cuµ (µm³) or fL). = PCV in 1000 ml OR in 100ml X10 = 45 X 10 = 90 Cuµ. RBCs count (in million /mm³) 5  Normal value: 80 – 90 Cuµ.  Value less than 80 Cuµ indicate microcyte.  Value greater than 95 Cuµ indicate macrocyte. 3) Mean Corpuscular Hb (MCH): It’s the amount of Hb present in one RBC (in micro microgram= picogram) = Hb in gm/100 ml OR Hb con. In 100 m X10 = 15X10 = 30 Pg. RBCs count (in million/mm³) 5  Normal value : 25-32 Pg.  Value less than 25 Pg is called hypochromia.  Value more than 33 Pg is called hyperchromia. 4) Mean Corpuscular Hb concentration (MCHC) : It’s the amount of Hb present in 100 ml of packed RBCs. = Hb in gm/100ml blood X100 = 15 X100 =33g/dL. PCV in 100 ml of blood 45 Normal values: 32-38 g/dL. 5) Colour Index (CI): It’s the ratio between the percentage of Hb and the percentage of RBCs in blood relative to normal. = Hb% of normal = 0.9 -1.1 RBCs% of normal Normal value: 0.9-1.1 BLOOD GROUPING  AGLLUTINOGENS (ANTIGENS)  Cell membrane of RBCs contain certain antigens called Agglutinogens.  Agllutinogens are glycolipids, present in other tissues as salivary glands, pancreas, kidneys, liver, lungs and testes.  2 types of antigens are present on RBCs: antigen A and antigen B. Accordingly 4 blood groups are present: a. Group A: 42% of population, have agglutinogen (antigen) A only. b. Group B: 9% of population, have agglutinogen B only. c. Group AB: 3% of population, have both agglutinogens A & B. (least common group) d. Group O: 46% of population, have no agglutinogens (neither A nor B). (most common group)  Aggllutinogen A has 2 subtypes (A1 and A2), thus we have 6 blood groups: A1, A2, B, A1B, A2B, O.  AGGLUTININS (ANTI-BODIES) Plasma contain gamma globulins (immunoglobulin M) called Agglutinins. Agglutinins are antibodies for agglutinogens. Immunoglobulin M ( ABO antibodies) are large and can’t cross the placenta.  At birth the level of Agglutinins is zero, it start to appear few months after birth reaching maximum at 8-10 years, then it gradually decrease. They develop against agglutinogens not present in the own RBCS. Agglutinogens and agglutinins never present in the same blood otherwise Antigen- antibody reaction will occur leading to hemolysis. There are 2 types of agglutinins (antibodies): a. α (Anti A) antibodies. b. β (Anti B) antibodies. So: c. Group A: have anti B agglutinins (anti bodies). d. Group B: have anti A agglutinins. e. Group AB: have no agglutinins (neither anti A nor anti B). f. Group O: have both anti A and anti B agglutinins.  DETERMINATION OF BLOOD GROUPS (SLIDE TECHNIQUE)  Steps: 1) Sterilize and prick the finger to obtain 2 drops of blood on a slide. 2) Add a drop of saline to each blood drop to dilute it. 3) Add anti A serum to the 1st drop and anti B serum to the 2nd drop. 4) Mix and examine after 2 min.s for agglutination (clumping) (Antigen- antibody reaction) and record the results as the following: a. If agglutination (clumping) with Anti-A (Blue in color) only RBCs contain agglutinogen A only: Group A. b. If agglutination with Anti-B (yellow in color) only RBCs contain agglutinogen B only: Group B. c. If agglutination with both Anti-A and anti B RBCs contain both A and B agglutinogens : Group AB. d. If no agglutination occurs RBCs contain neither A nor B agglutinogens : Group O.  RH FACTOR (RHESUS FACTOR)  It’s an agglutinogen (Antigen) normally present in the RBCs of Rhesus monkey.  There are 6 variations which are: C, D, E, c, d,e. It’s found in human especially D-antigen which is the strongest.  It’s inherited from both parents. With the dominance of D, while d is recessive. So DD & Dd are +ve Rh, while dd is –ve Rh.  Rh +ve forms 85% of population, while Rh –ve are 15%.  Rh antibodies are not normally present in the plasma and formed only by sensitizations (blood transfusion from Rh+ve individua to Rh –ve individual).  In addition to ABO and D agglutinogens, there are up to 500 billion other agglutinogens on RBCs membrane. However the most important are Rh, M and N antigens.  Steps to examination: 1) Drop of Anti-D is placed on the glass slide. 2) One drop of blood is placed on the anti- D drop. 3) Mix well and observe: - Agglutination occur with anti D Rh +ve blood group. - No agglutination occur with anti D Rh –ve blood group.  Difference between Rh antigen and ABO antigen: 1. ABO antibodies are formed spontaneously. 2. ABO incompatibility is associated with immediate reaction, while Rh incompatibility occurs after previous exposure (sensitization). 3. Rh antibodies are small sized, so they cross the placenta. (Immunoglobulin G)  EYTHROBLASTOSIS FETALIS (HEMOLYTIC DISEASE OF THE NEWBORN)  Complications of incompatible Rh transfusion: 1. transfusion reaction: when Rh –ve person receive Rh +ve blood, antibody formation will occur hemolysis of the transfused blood on 2nd exposure. 2. Erythroblastosis fetalis: o It’s a serious disease that affect Rh +ve fetus whose mother is Rh –ve and was previously sensitized by Rh +ve blood. o The formed antibodies are small and can cross the placenta (immunoglobulin G) and cause hemolysis to the fetal RBCs leading to: a. Die in utero. b. Delivered with sever anemia and jaundice. c. The immature erythroblasts are rapidly delivered from the bone marrow (site of there formation) to blood stream as a compensatory mechanism of the sever anemia, hence the name erythroblastosis. d. Liver maybe severely damaged. e. Brain damage due to deposition of bilirubin specially in the basal ganglia leading to kernicterus which is a fatal condition.  Sensitization of Rh –ve female by Rh +ve blood: 1) Previous receiving of Rh +ve blood (even during childhood) so the 1st baby will be affected. 2) If Rh-ve female is married to Rh+ve male, and got pregnant with Rh +ve 1st child (which will be born normal) , there was escape of fetal blood to the mother by any mean leading to sensitization (formation of Rh antibodies) a 2nd baby will be affected.  Causes of escape of fetal blood into the maternal blood: 1) Commonly during labor. 2) External trauma.  The fetus of the 1st pregnancy is usually born normal (no sensitization yet), serious effects occur in 3% of the 2nd pregnancy fetus and 10% of the 3rd pregnancy fetus.  Fetal RBCs doesn’t normally cross the placenta , so the maternal Rh antibodies are not always produced.  55% of the Rh +ve fathers are heterozygous (Dd), so the fetus maybe (dd) genotype Rh –ve.  Avoidance and treatment of erythroblastosis fetalis: 1. Avoid transfusion of Rh +ve blood to Rh –ve females. 2. Injection of anti Rh antibodies (Anti anti-D) to any Rh –ve female who delivered Rh +ve child within 48 hours of delivery to prevent formation of Rh agglutinins in her plasma. (with each delivery) 3. Erythroblastosis fetalis fetus is treated by repeated exchange transfusion with Rh –ve blood during the 1st weeks after delivery. 4. 400ml of ABO compatible blood are transfused slowly (over 2 hours) with removal of the baby’s blood at the same time leading to removal of the Rh+ve RBCs as well as agglutinins from the fetal blood.  BLOOD TRANSFUSION  What is agglutination: Antigen- antibody reactions leads to clumping followed by hemolysis of RBCs (irreversible).  Importance of blood grouping: - Blood transfusion - Disputed parenthood (strong –ve test)  Universal donor and recipient: - Universal donor: Group-O (no antigens). - Universal recipient: Group- AB (no antibodies).  Types of blood transfusion: 1) Heterologous transfusion: transfusion of foreign blood, from another person. 2) Autologous transfusion: transfusion of own blood. - It could be done before surgery: blood is withdrawn from the patient before operation and transfused to him again during or after surgery. - It’s a safe procedure as it avoid transmission of infections as AIDS, also it eliminate the risks of Heterologous transfusion.  the plasma agglutinins of the donor’s usually has no ill effects on the recipient R.B.Cs, as it is diluted by the large volume of plasma of the recipient however, it is not a rule because the level of agglutinins may be high in the donor’s plasma which may agglutinate the recipient R.B.Cs so that, it is preferable to perform inter-group transfusion and if it not possible a double cross matching  Indications of blood transfusion: 1. Hemorrhage. 2. Sever anemia. 3. Purpura. 4. hemophilia and para hemophilia. 5. Leukopenia. 6. Erythroblastosis Fetalis. 7. Sever infection (to supply γ globulins). 8. hypo proteinaemia. 9. Chronic leukemia.  Precautions before blood transfusion: 1. The transfused blood should be compatible (ABO & Rh). 2. Hb content should be 90% or more. 3. The transfused blood should be free from diseases (AIDS, malaria, hepatitis …). 4. The transfused blood should be fresh not frozen (stored at 4°C not exceeding 21days). 5. Direct cross matching test should be done.  Cross matching test: o Pre-transfusion test. It’s a safe guard against complications of transfusion. o It’s done by matching the donor’s RBCs against the recipient plasma. o It’s also advisable to match the recipients' RBCs against the donor’s plasma (Double cross  Complications of blood transfusion: A) General complications: 1. Excess blood transfusion circulatory overload. 2. Hypothermia if cold blood. 3. Transmission of infections. 4. Transmission of diseases (AIDS, malaria, hepatitis, syphilis..). 5. Excess blood citrate toxicity plasma Ca++ Tetany (Hypocalcaemia). 6. stored blood for a long time Hyperkalemia. B) Complications of incompatible transfusion reaction: 7. Severe chest pain: the clumping of cells closure of blood vessels in chest causing ischemia and pain. 8. Sever hypotension: due to release of histamine from the hemolysed cells VD and shock. 9. Jaundice: RBCs hemolysis Hb Bilirubin. 10. Acute Renal failure, due to: - Hypotension: decreased renal blood flow. - Release of Hb: filtered by the kidney and block the renal tubules. 5. Hyperkalemia arrhythmia.  BLOOD SAMPLING 1) Small blood sample (in microliters): By pricking the thumb or ear pinna by disposable lancet needle or sterilized surgical needle.  Precautions:  Good sterilization with 70% ethyl alcohol.  The area must be dry …. To avoid dilution of the blood sample.  The sample is taken when the blood form a globule.  Wipe off the first drop of blood and use the second drop …. As the first drop maybe mixed with tissue fluids.  Uses: 1. RBCs counting. 2. WBCs counting. 3. Hemoglobin contet. 4. Blood indices. 5. Hematocrit value. 6. Blood grouping. 7. Bleeding time. 2) Large blood sample (in milliliters):  Procedure (Venesection or phlebotomy): 1. Select a palpable, superficial vein (dorsum of the hand). 2. Clean the skin over the selected vein with alcohol. 3. Apply a tourniquet firmly to upper arm to cause engorgement of the vein. 4. Use a sterile, dry, proper gouge needle at 30º angle inside the selected vein (the bevel should be facing upwards). 5. As blood appears under the proximal plastic end of the needle, decrease the angle. 6. Proceed inside the vein for half the length of the needle. 7. Aspirate the required amount of blood. 8. Release the tourniquet before removing the needle to prevent bruises. 9. Apply a piece of sterile cotton, pressing it against the venipuncture.  Uses: 1. Erythrocyte sedimentation rate ESR. 2. Erythrocyte osmotic fragility. 3. Clotting time.  HEMOSTASIS 1) BLEEDING TIME  Definition: Time from injury of a small blood vessel till bleeding stop spontaneously without formation of blood clot. It’s caused by Vasoconstriction(VC) of blood vessels and platelets reaction. It’s a measurement of platelets number and function.  Normal platelets count: 150-400 X10³ /mm³ (150.000-400.000/mm³).  Decreased platelets count:  Below 50.000 /mm³ : bleeding if traumatized.  Below 20.000 /mm³ : petechiae (less than 2mm) & ecchymosis (more than 2mm).  Thrombocytopenia VS. Thromocutopenia:  Thrombocutopenia : decreased platelets number.  Thrombocytopathia (Thromboashthenia) : normal number but abnormal platelets function.  Mechanism of hemostasis: 1) V.C : 1- myogenic in origin 2- neurogenic : pain cause VC. 3- activation of sympathetic system 4- chemical: release of serotonin, adrenalin, thromboxane A2 from platelets. 2) Platelets plug formation.  Steps: (Duke method) 1. Sterilize the finger and leave it to dry. 2. Puncture it with a sterile needle and start a stopwatch immediately. 3. Use a blotting or filter paper or a piece of cotton to wipe blood coming from the prick every 15 sec (without squeezing or touching the skin).  Normal bleeding time: 3-5 minutes.  Causes of prolonged bleeding time: 1. Defect in blood vessel wall : old age, scurvy (vit. C deficiency). 2. platelets number less than 50.000 /mm³. 3. Platelets adhesion due to vWF. 4. Platelets aggregation. 5. Disorder in platelets function or structure … thrombasthenia.  2) CLOTTING TIME (C.T) (COAGULATION TIME)  Definition: The time from injury of blood vessels till formation of fibrin clot (fibrin threads). It is a measure of the clotting factors of the intrinsic pathway.  Steps: (capillary method) 1. Sterilize the finger and leave it to dry. 2. prick the finger with sterile needle and start a stopwatch immediately. 3. Put the end of long glass capillary tube (NON heparinized) at the base of the blood drop, blood will fill the tube by capillarity. 4. Break a small piece of the tube every 30 seconds till fibrin threads appear.  Normal clotting time: 4-8 minutes.  Mechanism: By the intrinsic pathway.  Causes of prolonged clotting time: 1) Hemophilia (decreace in clotting factors):  Hemophilia A: factor VIII.  Hemophilia B: factor IX. (Christmas disease)  Hemophilia C: factor XI. 2) Parahemophilia : factor V. 3) Vit K deficiency : factors II, VII, IX and X. 4) Liver disease : decrease all the clotting factors. 5) Dicumarol : anti vitamin K effect. 6) Heparin: prevents conversion of prothrombin to thrombin & facilitate action of AT (antithrombin) III (inhibition of IX, X, XI, XII). 7) Citrate : Ca++.  VITAMIN K DEFICIENCY Occur in cases as: 1) Obstructive jaundice:  Here bile can’t reach the intestine decreased absorption of fat and fat soluble vitamins including vitamin K. 2) Newly born infants:  Intestinal bacterial flora is the main source of vitamin K.  Newel born infants don’t have intestinal flora yet so decrease vitamin K.  This is why surgical operations in infants are better to be postponed for one month till bacterial flora is formed. 3) Prolonged use of antibiotics: affect intestinal flora. 4) Liver disease. 5) Patients receiving heparin or dicumarol.  VON WILLEBRAND DISEASE  It’s a bleeding disorder characterized by excess bleeding even in mild truma.  Is due to deficiency in Von Willebrand factor, which is a protein secreted by endothelium of damages blood vessels and platelets.  This protein is responsible for: 1. adherence of platelets to the endothelium of blood vessels during hemostasis after injury. 2. Survival and maintenance of factor VIII in plasma.  Deficiency in VWF suppress platelets adhesion, and cause deficiency in factor VIII, so the result will be excess bleeding that resemble what happen in cases of platelets dysfunction or hemophilia.  HEMOPHILIA VS. PURPURA Purpura Hemophilia Cause Due to platelets count than Due to factor VIII, IX or 40.000/mm³. XI. Types 2 types: 3 types: 1. Thromobcytopenia 1. A 2. Thrombocytopathia 2. B 3. C Inheretance NOT inherited Inhereted on X chromosome (sex linked) Sex affected Both sexes are affected Males are affected Females are carriers Bleeding Spontaneous Excessive bleeding due to minor truma Bleeding time Prolonged Normal Clotting time Normal prolonged PT (PROTHROMBIN TIME)& PPT (PARTIAL PROTHROMBIN TIME)  PT (PROTHROMBIN TIME)  Definition:  Time needed for blood to clot after adding tissue thromboplastin (factor III).  Time needed for clot formation by the extrinsic and common pathways.  It indicates the total quantity of prothrombin present in blood.  It measure clotting factors I, II, V, VII, X (extrinsic factor VII. Common factors I, II, V, X)  Steps: 1- collect a blood sample and add oxalate (v.imp) to it calcium is precipitated (Ca oxalate) j so prothrombin (factor II) in not converted to thrombin. 2- Add a large quantity of tissue thromboplastin (factor III) and Ca+² to the blood sample calcium stop the effect of oxalate & tissue thromboplastin activates prothrombin (factor II) ; so blood clotting occurs.  Normal value: 10 -12 seconds.  Prolonged PT in: 1. Prothrombin deficiency (factor II). 2. Deficiency in clotting factors : I, V, VII, X. 3. Vitamin K deficiency. 4. Use of warfarin and heparin. PT is normal in hemophilia.  Importance of PT: 1. In patients how aren’t taking anticoagulants and has signs and symptoms of a bleeding disorder. 2. Patients who will undergo an invasive medical procedure such as surgery, to ensure normal clotting ability. 3. To monitor oral anticoagulant therapy (Warfarin /Coumadin).  PPT (PARTIAL PTOTHROMBIN TIME) ACTIVATED PROTHROMBIN TIME OR ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT)  Definition:  Time needed for blood to clot after adding an activator such as phospholipid, along with calcium to the blood sample.  Time needed for clotting by intrinsic and common pathways.  Phospholipid serve as platelet substitute.  Commonly used activators: phospholipid , Kaolin.  Normal value: 30 – 45 seconds.  Prolonged PPT in: 1. Deficiency in clotting factors: I, II, V, VIII, IX, X, XI & XII. (intrinsic factors XII, XI, IX, VIII. And common factors I, II, V, X) 2. Use of warfarin or heparin. 3. Vitamin K deficiency. 4. Hemophilia. (V.imp)  THROMBIN TIME (TT)  definition:  Time needed for blood to clot after adding thrombin (active factor II) to it.  It’s done to detect the presence of heparin in plasma or to detect fibrinogen (factor I) abnormalities.  Normal value: 12 – 20 seconds.  Prolonged TT: 1. Heparin therapy. 2. Dysfibrinogenemia (abnormal function of fibrinogen with normal fibrinogen level).  INTERNATIONAL NORMALIZED RATIO (INR)  Definition:  It’s rating of a patient’s prothrombin time (PT) when compared to an average.  It measures the extrinsic clotting pathway.  Indications: 1. Test of liver synthetic function. 2. Before starting anticoagulant therapy. 3. Monitoring of anticoagulant drug therapy as warfarin (oral anticoagulant) and adjusting the dosage of anticoagulants: Patients with AF (atrial fibrillation) are usually treated by warfarin to protect against blood clots which can cause stroke. These patients must have regular blood INR tests to adjust the warfarin dosage. 4. Bleeding diathesis in patients with coagulating factors deficiency: fibrinogen, factors II, V, VII or X or a combined deficiency in the extrinsic pathway. 5. DIC Disseminated intravascular coagulation.  Normal INR: About 1.  Interpretation of INR results:  Blood takes longer time to clot if INR is higher.  Patients taking anticoagulant therapy for AF INR should be between 2 and 3.  Patients with heart valve disorders INR should be between 3 and 4.  INR greater than 4 indicates that blood is clotting too slowly and there is a risk of uncontrolled blood clotting (increased risk for bleeding). OSMOSIS   Osmosis: The flow of a solvent from less concentrated solution into a more concentrated solution through a semipermeable membrane.  Osmotic pressure: - The force by which a solution drags it’s solvent across the semipermeable membrane. - It’s measured by the applied pressure that can stop the movement of the solvent across the semipermeable membrane.  Tonicity: The osmolarity of a solution relative to the osmolarity of plasma. - An isotonic solution: has the same osmolarity as the plasma (280 mosmol/L). - A hypertonic solution: has higher osmolarity than the plasma. - A hypotonic solution: has lower osmolarity than the plasma.  What happen to RBCs in different tonicities : - In isotonic solution.. NO change - In hypotonic solution.. RBCs swell. - In hypertonic solution.. RBCs shrink.  Application : Examples of isotonic solutions that can be used clinically and experamintally :  0.9% NaCl: - It expand the ECF and remain mainly in the ECF (extracellular fluid). - Useful in cases of salt depletion. - Might increase the arterial blood pressure due to expansion of plasma volume. - It’s not indicated in cases of dehydration due to primary water depletion (lack of water intake as in prolonged coma or dysphagia).  5% Glucose solution: - Glucose is metabolized by cells and water remains to rehydrate the body, so excessive infusion with this solution may lead to overhydration. - It’s dangerous in cases of adrenocortical insufficiency (Addison’s disease) where there is sodium depletion. So after infusion with this solution, glucose is used and water remains in the body >>> hypotonicity of plasma and ECF >>> swelling of the cells of the hypothalamic thermostat >>> rise in body temperature (Glucose fever). - If the infusion was excessive >>> swelling of brain cells >>> confusion >>> coma >>> death.  1.8% urea solution: - Isotonic, but RBCs membrane is permeable to urea. So if RBCs is suspended in this solution, urea diffuses into the cells down it’s concentration gradient (from high urea concentration in the solution to low urea concentration inside RBCs). - Water follows urea, so >>> RBCs will swell >>> hemolysis. MSK101-2 Eclectic stimulator  EFFECT OF SINGLE STIMULATION TO THE MUSCLE  Types of stimuli according to intensity: 1- Subthreshold (subminimal) stimulus: is ineffective causing no response. 2- Threshold (minimal) stimulus: leads to weak contraction through response of most excitable fibers. 3- Supra minimal stimulus: leads to strong contraction through response of greater numbers of fibers. 4- Maximal stimulus: maximal contraction through response of all muscle fibers. 5- Supramaximal stimulus: same as maximal as each muscle fiber obeys all or non law. * Strength of the muscle contraction depends on: 1- the number of active motor units 8 2- the frequency of nerve impulses.  MECHANICAL CHANGES IN MUSCLES (MUSCLE CONTRACTION)  Types of muscle contraction: 1- Isotonic contraction: means muscle contraction without changes in its tension. 2- Isometric contraction: means muscle contraction without changes in its length. Isotonic Isometric Muscle length Decrease (Muscle shortens) Constant (No change) Muscle tension Constant (no change) Increase Duration of contraction Long Short Sliding of actin and Occurs greater Less than isotonic myosin Energy needed Greater Small O2 and nutrient Greater Lesser required Work done Work done (Carry weight) No work done (Waste heat) Mechanical efficiency 25% (lifting weight) Zero  SIMPLE MUSCLE TWITCH (SMT)  Definition: It is a brief muscle contraction followed by relaxation, produced by single maximal stimulus.  Phases and durations of SMT 1- Latent Period: It the time passes between application of stimulus and beginning of mechanical response (contraction). Its duration is 0.01 second (10 ms) in isotonic and 2 ms (0.002 sec) in isometric contraction. 2- Contraction Period (phase): the muscle shortens and reaches its maximal point. Its duration is 0.04 second. 3- Relaxation Period (phase): the muscle returns to its original length. Its duration is 0.05 second. The total time of contraction in the frog’s muscle is 0.1 second while in the mammalian muscle the time is much shorter 0.05 sec.  Factors Affecting S.M.T (4 factors) 1) Type Of The Muscle: In the skeletal muscles of the mammals, there are 2 types of muscle fibers. a- Red muscle fibers (type I) b- White muscle fibers (type II): it is subdivided into type IIA (rare in humans) and type IIB.  Red muscle fibers (type I), slow Type IIB white (pale) muscle fibers, fibers fast fibers Color Darker than other fibers Lighter Diameter small diameter Larger diameter Innervation Innervated by small slowly Innervated by large rapidly conducting conducting motor neurons motor neurons Myoglobin High Low content Oxidative Large amount of oxidative enzymes Large amounts of glycolytic enzymes enzymes (many mitochondria) ATPase activity Low ATPase activity Hight ATPase activity Capillary Many capillary : more blood supply Fewer capillaries: less blood supply supply Rate of Slow Rapid contraction and relaxation Fatigue Not easily fatigued (more resistant Easily fatigued to fatigue) Latent period Long Short Adaptation Adapted for long posture Adapted for skills movement e.g. extra maintaining contraction e.g. ocular muscles and some of the hand 2) Effects of Temperature on SMT:  Warming of the muscle leads to fast and strong contraction than normal due to: a- acceleration of metabolic (chemical) reactions so, all phases of the SMT increase in amplitude and decrease in duration. b- decrease muscle viscosity leading to decreased frictional forces in the muscle so, enhances the sliding movements of actin filaments leading to powerful contraction.  Cooling of the muscle has the opposite effect i.e. decrease the amplitude and increase the duration of all phases of SMT.  Excessive heating (over 45o): causes no contraction due to heat coagulation.  Excessive cooling: causes no contraction due to stoppage of the metabolic reactions. 3) Effects of Fatigue on SMT: DEFINITION: - It is the temporary decrease in muscle force of contraction due to previous contractile activity. It produce similar effects as cooling i.e. decrease the amplitude (strength of muscle -.contraction) and increase the duration of all phases of SMT - If the muscle is allowed to rest, it can recover its ability to contract upon re-stimulation THE ONSET OF FATIGUE DEPENDS ON: 1) Duration & intensity of contractile activity; High-intensity LEADS TO more rapid fatigue 2) Type of muscle fiber, some muscle fibers resists fatigue. SITES OF FATIGUE:  1- MEP (motor end plate).  2- muscle fibers.  3- Central psychological fatigue Causes of fatigue in isolated muscle: A) Muscular fatigue:  Accumulation of lactic acid with increase in the intracellular acidity leads to inhibition of the key enzymes in energy pathways.  Depletion of energy stores inside the muscle e.g. glycogen. B) Neuromuscular fatigue: It is due to depletion of A.ch stores at the motor end plate as the rate of A.ch release is more than its rate of synthesis. This type of fatigue occurs during fast-powerful activities. C) Central psychological fatigue: Occurs when the “central nervous system” no longer adequately activates the motor neurons supplying the working muscles. Athlete’s performance is not only dependent on the physical state of his muscles, but also, upon the well to win “ability to overcome psychological fatigue”. The nerve does not fatigue: WHY?? FATIGUE MAY occurs by indirect stimulation of the muscle OR Direct stimulation leads to contraction. Causes of delayed muscle fatigue inside the body: a) Cardiovascular system: supplies the muscles with O2 and nutrients and removes the metabolites from the muscle. b) CNS: regulates the muscle contraction so that not all the muscle fibres are contracted at the same time, but instead, there is alternation between contracted and relaxed muscle fibres. c) Hormones: e.g. adrenaline, noradrenalin, cortisol, thyroxin and insulin. 4) Effect of two successive stimuli: Three different effects according to the relation between the first and the second one: 1) Normal muscle twitch caused by the first stimulus. 2) Beneficial effect: Timing: When the second stimuli falls after the relaxation period of the first curve. Result: Two separate curves are obtained, and the force of the second contraction is greater than the first one. Cause: During the first contraction, the temp increases which lead to decrease in viscosity of muscle. So the force of the 2nd contraction is greater than the 1st one. 3) Superposition effect OR superimposition OR incomplete summation: Timing: When the second stimuli falls during the relaxation period 4) Summation OR complete summation : Timing: when the second stimuli is applied during contraction period or during the 2nd half of the latent period. Result: the two contractions are summed up and a single curve is obtained. * Summation curve is different from the simple muscle curve: Summation curve is greater than that of simple muscle curve; due to the summation of two contractions to give rise to one single curve. Base of the summation curve is broader than that of the simple muscle curve.  ELECTROMYOGRAPHY  Definition: Recording the electrical activity which occurs in skeletal muscles during its activity is called electromyogram.  The apparatus used: is called electromyograph.  The record is called: electromyogram (EMG). - It could be done by using microelectrodes which are either: 1- metal discs electrodes placed over the skin 2- or needle electrodes inserted beneath the skin (hypodermic electrodes) inserted in the muscle itself. With needle electrodes, it is possible to pick up the activity of a single muscle fiber. - During rest: a small potential are recorded (muscle tone). - During activity: an action potential reaching up 50 mV is recorded.  Clinical importance: 1. Investigation of muscle activity in health and disease. 2. Detection of the extent of paralysis in poliomyelitis. 3. In experimental animals to study the electrical activity of motor units and its relation to intensity of stimulus. Electromyogram during alternate contraction and relaxation of biceps muscle  MYASTHENIA GRAVIS  Definition:  it’s a disease characterized by sever weakness in skeletal muscles and easily fatiguability.  It’s more common in females.  Etiology : 1. Autoimmune disease; in which antibodies are formed against acetyl choline receptors of skeletal muscles. 2. Decreases sec of Acetyl Choline 3. Increased activity of acetyl cholinesterase enzyme.  Clinical picture: If the disease is sever, paralysis of skeletal m. particularly respiratory muscles may lead to death.  Treatment: Anti cholinesterase drugs as neostigmine or prostegmine + atropine (parasympatholytic). Thank You