Chemical Pathologist Lecture Notes PDF

‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ Chapter II Collection and Handling of blood samples Blood sampling Blood composition The blood as a tissue is composed of: • Plasma : The liquid part of blood, makes up about 60% of the blood's...

‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ Chapter II Collection and Handling of blood samples Blood sampling Blood composition The blood as a tissue is composed of: • Plasma : The liquid part of blood, makes up about 60% of the blood's volume. Plasma is mainly made from water, but contains many different proteins and other chemicals such as hormones, antibodies, enzymes, glucose, fat particles, salts, etc. • Blood cells : which can be seen under a microscope, make up about 40% of the blood's volume. Blood cells are made in the bone marrow by blood 'stem' cells. Blood cells are divided into three main types. ❖ Red cells (erythrocytes). These make blood a red color. Red cells contain hemoglobin. This binds to oxygen, and takes oxygen from the lungs to all parts of the body. ❖ White cells (leucocytes). There are different types of white cells such as neutrophils, lymphocytes, eosinophils, monocytes, basophils. They are a part of the immune system and are mainly involved in combating infection. ❖ Platelets. These are tiny and help the blood to clotting Types of blood used in laboratory Serum refers to the clear liquid that separates from blood when it clots Serum do not contain prothrombin or fibrinogen but it contain thrombin that is formed during blood clotting so it cannot be used in blood transfusion Plasma 1 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ Plasma is the pale yellow liquid obtained from centrifugation of blood with anticoagulant It contains clotting factors and has no thrombin Whole Blood Whole blood refers to plasma and cellular components of blood (used in hematology) Difference in composition between serum and plasma: Plasma > serum Ca, LDH and TP Serum > Plasma ALB, ALP, GOT, GPT, CK, glucose, Na, K, P, urea and uric acid Anticoagulants It is the substance that prevent coagulation of blood Mechanism of blood clotting EDTA ( Ethylene Diamine Tetra acetic Acid) Preparation: Found as disodium salt or dipotassium salt (K salt is more soluble so it is more preferable) Dissolve 1gm of powder in 100 ml DW then use 50 ul for each 1m1 blood Mechanism of action It acts by binding blood calcium ion to form insoluble salt by chelating calcium 2 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ Uses : Must be used in tests that need whole blood such as CBC, blood group, Rh, Osmotic fragility Advantage • Cellular elements are preserved well (but for limited time about 6 h) • Leukocyte stain ability is excellent • EDTA prevent platelets clumping Disadvantage • Excess EDTA will cause shrinkage of the blood cells (More than 1mg/m1 blood) • Excess EDTA cause platelets swelling and disintegration so high false platelets count can be obtained • EDTA is not suitable for coagulation tests as PT • EDTA inhibit the action of some enzymes as GOT, GPT, ALK Sodium citrate Preparation 38 gm in 100 ml DW (it is used as 3.8% solution) Mechanism of use It converts ionized Ca into unionized soluble complex Uses The most popular tests use citrate is: ESR test (400 ul citrate + 1.6 ml blood) PT test (200 ul citrate + 1.8 ml blood) Advantage Not poisonous and sheep Disadvantage • When used in dry form cause cell shrinkage • It cause dilution of blood when used as solution 3 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ • It decrease the color yield in phosphate measurement • Inhibit the activity of enzymes as ALK, GOT, GPT Sodium fluoride Mechanism of use Form a weakly dissociated Ca component It has anti-glycolytic effect (normally glucose decrease by about 10 mg/dl per 1h due to glycolysis) Note : the rate of glucose decrease is faster in newborn due to increased metabolic activity of their RBCs and in leukemia patient due to high metabolic activity of their WBCs Uses Used as a powder 5 mg/ml blood Only used for glucose samples Advantage Acts as good preservative for blood glucose as it interfere with enzyme system causing glycolysis Disadvantage • Very poisonous • It interfere with urease enzyme so cannot be used in urea determinations • It is poorly soluble so blood must be mixed well Heparin Preparation It is available as Li, Na, and k salts Mechanism of use It facilitates the action of anti-thrombin III which is a protease that inhibit active factors (IX, X, XI, XII) so inhibit conversion of fibrinogen to fibrin Uses 4 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ It is used in concentration of 10-50 iu/ml (0.1 ml 15m1 blood) There samples must be collected on heparin such as Blood gases, karyotyping , D-dimer , troponine Advantage Cause no changes in RBCs so it is suitable in PCV Disadvantage ► More expensive ► Blood should be examined with in 8 h (because it prevent coagulation for only limited period) ► Inhibit acid phosphatease activity ► Affect binding of T3 T 4 to their carriers proteins so increase the concentration of free T 3, T4 Acid citrate dextrose (ACD) Preparation It is a mixture of trisodium citrate, anhydrous citric acid, dextrose and DW It is used a ratio of one part to 4 parts of blood Uses It is used in blood transfusion (glucose provide some nourishment for the cell) Disadvantage Used as solution and this dilute blood Equipment's for blood sampling • Disinfection swabs Isopropyl alcohol, 70 %. • Tourniquet • Sterile Disposable syringes • Evacuated Collection Tubes • Gloves • Butterfly needle are very small needles used in phlebotomy of very young or elderly patients or patients with small veins and arteries. • Cotton 5 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ Vacutainer System The Vacutainer System consists of a double-pointed needle, a plastic holder or adapter and a series of vacuum tubes with rubber stoppers of various colors. The Vacutainer needle The Vacutainer needle has a sharp point at both ends, and usually is covered by a rubber sheath, with one end being shorter than the other. The long end of the needle is used for penetrating the vein, the shorter end is used to pierce the rubber stopper of the vacuum tube. Vacutainer holder The Vacutainer holder is a plastic sleeve into which the phlebotomist screws the double pointed Vacutainer needle. Vacutainer tubes Vacutainer tubes are glass tubes sealed with a partial vacuum inside by rubber stoppers. The air pressure inside the tube is negative, less than the normal environment. After inserting the longer needle into the vein, the phlebotomist pushes the tube into the holder so that the shorter needle pierces the stopper. The difference in pressure between the inside of the tube and the vein causes blood to fill the tube. Collection tubes for phlebotomy 6 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ Red Top ADDITIVE Plain MODE OF ACTION Blood clots, and the serum is separated by centrifugation Gold Top ADDITIVE None MODE OF ACTION Serum separator tube (SST) contains a gel at the bottom to separate blood from serum on centrifugation Purple Top ADDITIVE EDTA MODE OF ACTION Forms calcium salts to remove calcium Light Blue Top ADDITIVE Sodium citrate MODE OF ACTION Forms calcium salts to remove calcium Light Gray Top 7 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ ADDITIVE Sodium fluoride MODE OF ACTION Antiglycolytic agent preserves glucose up to 5 days Yellow Top ADDITIVE ACD (acid-citrate –dextrose ) MODE OF ACTION Complement inactivation Uses HLA tissue typing paternity testing , DNA studies Black Top ADDITIVE Sodium citrate MODE OF ACTION Forms calcium salts to remove calcium Light Green Top ADDITIVE Plasma separating tube (PST) with Lithium heparin MODE OF ACTION Antiglycolytic with lithium heparin : plasma is separated with PST get at the bottom of the tube SAFETY and infection control Protect yourself ■ Wear gloves and a lab coat when handling blood/body fluids. ■ Change gloves after each patient or when contaminated. ■ Wash hands frequently. 8 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ ■ Dispose of items in appropriate containers. ■ Dispose of needles immediately upon removal from the patient's vein. ■ Do not bend or break needles to avoid accidental needle puncture or splashing of contents. Clean up any blood spills with a disinfectant What you do If you stick yourself with a contaminated needle? ■ Remove your gloves and dispose of them properly. ■ Squeeze puncture site to promote bleeding. ■ Wash the area well with soap and water. ■ Record the patient's name and ID number. ■ Follow guidelines regarding treatment and follow-up. Protect the patient ■ Place blood collection equipment away from patients, especially children and psychiatric patients. ■ Practice hygiene for the patient's protection. When wearing gloves, change them between each patient and wash your hands frequently. Always wear a clean lab coat. Vein puncture Definition venipuncture (also known as phlebotomy, venesection, blood draw, drawing blood or taking blood) is the process of obtaining a sample of venous blood. Steps of venipuncture • Preparation for blood collection ‫هذه الخطة هامة جدا في سحب العينة النها سوف يتحدد على اساسها العديد من االمور التي ستؤثر في طريقة السحب او‬ : ‫كمية الدم المطلوبة نوعية االنابيب الت سيوضع عليها الدم وترتيب مراحل هذه الخطوة يكون كما يلي‬ • Read the request of the patient. • Verify the patient's condition. Fasting, dietary restrictions, timing of the test, and medical treatment • Estimate the blood volume to take • Prepare the suitable tubes for each test 9 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ ‫‪Chemical pathologist‬‬ ‫فرع الكيمياء الطبيه والحياتيه‬ ‫ملحوظة ‪ :‬تحضير االنابيب قبل سحب العينة هام جدا خاصة في تحاليل التجلط مثل البروثرومبين الن التاخير في‬ ‫تحضير االنبوبة بعد سحب العينة قد يؤدي الى تجلط الدم قبل وضعه في االنابيب ‪.‬‬ ‫شروط خاصة لعينات الدم ‪:‬‬ ‫اوال ‪ :‬عينات يشترط فيها الصيام‬ ‫‪ .1‬عينات الدهون تشترط صيام ‪ 12‬ساعة على االقل‬ ‫‪ .2‬عينة السكر الصائم تشترط صيام من ‪ 6‬الى ‪ 8‬ساعات ويجب اال تزيد على ذلك حتى ال يقل مستوى السكر في‬ ‫الدم ويكون التشخيص غير دقيق ‪.‬‬ ‫‪ .3‬بعض التحاليل االخرى الخاصة بمرض السكر يشترط فيها الصيام مثل ‪ Insulin‬و ‪c-peptide‬‬ ‫‪ .4‬تحليل صيام من ‪ 6‬الى ‪ 8‬ساعات في التحاليل االتية ‪ESR , URIC ACID , UREA :‬‬ ‫ثالثا ‪ :‬عينات تؤخذ في اوقات محددة ‪:‬‬ ‫‪ .1‬تحاليل الهرمونات االنثوية تؤخذ في مواعيد محددة اثناء الدورة الشهرية ‪.‬‬ ‫‪ .2‬عينة الدم لتحليل ‪ CORTISOL‬و ‪ ACTH‬البد ان تؤخذ في وقت محدد ‪ 9‬صباحا و ‪ 9‬مساءا‪.‬‬ ‫‪• Reassuring the patient‬‬ ‫‪You must get the patient confidence before sampling‬‬ ‫ملحوظة ‪ //‬البد من اكتساب ثقة المريض قبل البدء في سحب العينة خاص في عينات االطفال والثقة ليست في االخصائي‬ ‫الذي سيسحب العينة فقط بل في عملية السحب نفسها وانها لن تسبب ضرر او ام للمريض ‪.‬‬ ‫‪Note : Stress cause high glucose and enzyme activity due to adrenal stimulation‬‬ ‫‪Positioning the patient‬‬ ‫‪• The patient should sit Comfortable in a chair, lie down or sit up in bed.‬‬ ‫‪• Patients should not stand or sit on high stools because of possibility of fainting‬‬ ‫‪• The patient should sit 15 min in his place.‬‬ ‫‪Non controllable variables‬‬ ‫‪Biological influence‬‬ ‫‪1. Age‬‬ ‫‪Concentration of bilirubin increase after birth and peaks about the 3-5 days Glucose level in‬‬ ‫‪neonates is low due to low glycogen content and due to adrenal immaturity TSH is secreted‬‬ ‫‪10‬‬ ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ after birth leading to increased T3, T4 (this physiological hyperthyroidism gradually decline over the first year of life) 2. Sex Until puberty few differences in lab tests exist between boys and girls After puberty the activity of ALP, GOT, GPT, and CK are greater in man than women (due to increased body mass) 3. Race Serum TP level is known to be higher in blacks than in white The activity of CK and LDH are much higher in both black men and women than in white • Environmental factors 4. Altitude Individuals living at high altitudes have marked increase in blood Hb due to reduced atmospheric PO2 Ex: ► Moderate exercise will increase concentration of glucose ►Strenuous exercise will increase the activities of GOT, CK ► Increased lactic acid production will compete uric acid for renal excretion so serum uric acid concentration increases. Physical training Athletes generally have higher level of serum activity for enzymes of skeletal muscular origin at rest than do non athletes Serum creatinine, urea, uric acid and T4 are higher in athletes than in non athletes Circadian variation Circadian variation or diurnal rhythms are body fluid and analyte fluctuations during the a certain period (day, month,) This cyclical variation in body chemicals caused by activity, stress, light / dark and sleep / wakefulness 11 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ In some cases the variation is large so the time of sample must be controlled as in case of cortisol Some hormones are secreted in bursts Ex: • LH and FSH in women during menstrual cycle • Testosterone in men increase 20-40% during night • GH secretion is greatest shortly after onset of sleep PRE ANALYTICAL VARIABLES It is the factors that affect specimen before test is done • Controllable variables • Posture ►Postural changes (supine to sitting etc.) are known to vary lab results of some analytes. ►Because only the protein free fluid passes through the capillaries to the tissues the change in position results in reduction of plasma volume and increase in plasma protein concentration. ►Significant increase in K concentration occurs with 30 min of standing due to release of intracellular K from muscles. ►Also lipids, iron, and calcium are significantly increased with changes in position. Prolonged bed rest Note: With prolonged bed rest fluid retention occurs and serum proteins may be decreased, also the concentration of protein bound constituents will decrease. Notes : Although Ca will he decreased due to albumin decrease but mobilization of Ca from bone will increase free ionized (*a so compensate for the reduced protein bound Ca Exercise Its effect is related to the duration and intensity of the activity 12 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ Effects of hemolvsis ►Hb color interfere with colometric measurement ► Glucose decrease due to glycolysis by RBCs enzymes ► Increased k due to leakage from RBCs ► Increased p as organic esters in the cell is hydrolyzed ► Increased got LDH and GOT activity • Hyperbilirubinaemia Dark yellow to greenish serum cause color interference Lipemia Cause • Physiological after food (chylomicrons) • Pathological hyperlipidemia Effects ► Enhance hemolysis ► Turbidity cause color interference ► Interference with kinetic measurement ► False decrease of Na, K measured by flame due to false decrease of aqueous phase by lipid Management ► Fasting for 12 h ► Lipid precipitation using: • Freezing • Ultracentrifugation • Chemical precipitation by Ethylene glycol ► Heparin injection 15 min before collection will dissolve lipids (Heparin acts as a cofactor to the lipoprotein lipase enzyme which hydrolyze excess lipid this is called clearing effect of heparin) 13 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ ► Basal plasma insulin is higher in the morning than later in the day Blindness With blindness the normal stimulation of the hypothalamus pituitary axis is reduced Ex : Na usually low in blind patient due to reduced aldosterone secretion Diet Glucose and lipid are increase by food digestion Ingestion of protein rich meal in the evening may cause increase in urea, uric and phosphate (until 12 h later) Caffeine ingestion stimulates adrenal medulla and cause slight increase in plasma glucose Obesity Serum LDH and glucose concentration increase in both sexes as body weight increase In men AST, creatinine, and Hb increase as body weight increase In women serum Ca increase as body weight increase Serum P decrease as body weight increase in both sexes Fainting or coma ‫االغماء او فقدان الوعي له اسباب كثيرة وفي الغالب عملية سحب العينة نفسه ال دخل لها في باغماء المريض وفي‬ : ‫حالة وجود انسان فاقد الوعي اتبع االتي‬ ‫• تاكد من وعي المصاب وذلك بالنداء عليه او هز كتفه‬ ‫• تاكد من ان المصاب يتنفس‬ ‫• تاكد من وجود النبض‬ : ‫اذا كان النبض والتنفس موجودين اتبع االتي‬ ‫• افتح مجرى الهواء للمصاب وحافظ عليه مفتوحا‬ ‫• ارفع قدمي المصاب بوضع وساده او اي شي تحتهما‬ . ‫حافظ على تدفئة المصاب بتفطيته‬ 14 • ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ ‫اطلب المساعدة باالتصال باالسعاف ( ابلغ االسعاف بحالة المريض اذا كنت تعرفها مثال اذا كان مريض قلب او‬ • . ‫اي مرض اخر تعرفه عن المريض‬ : ‫ملحوظة هامة‬ . ‫ال تعطي المريض ماء او ماء بسك ليشربه فدلك يؤخر من استرداد المريض لوعيه‬ Factors influence blood sampling Site effect Blood samples obtained from different site differs in composition For example: Venous blood > capillary blood Bilirubin, Ca, TP, Na Capillary blood > venous blood glucose, K • Hemolvsis It means librations of Hb after RBCs have ruptured (in this case plasma appear pink or red) How to reduce the risk of nerve puncture? ► Don't perform venipuncture within 3 inches (7.5 cm) of the wrist. ► Minimizing the number of attempts you make. ► Using good venipuncture technique: First, retract the skin and stabilize the vein; then, insert the needle. What you do if you nick a nerve? The patient will feel an immediate, pain that he may describe as an electrical shock going down the arm. If your patient has this type of pain, immediately remove the needle , removing the needle immediately minimizes the chance of nerve damage. 2. Nerve compression injury This results from compression by a hematoma in subcutaneous tissue. When blood compresses a nerve, the patient will feel tingling and numbness in the affected area. These symptoms-which may be the only signals of a nerve injury-usually don't occur until 24 to 96 hours after the hematoma occurs and may not resolve after the blood is evacuated. Compression injuries may result in permanent disability, but injury can be minimized if it's recognized promptly. 15 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ 3. Neuroma In some cases, a nerve puncture may result in a neuroma-an enlarged area of the nerve at the point where the needle was inserted. Some patients who develop neuromas must have them surgically excised and need physical therapy. Depending on the degree of injury, the patient may have residual symptoms, such as decreased sensation in the affected area. Excessive bleeding This is may be caused due to: 16 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ 1. Anticoagulant therapy 2. High dosages of anti-inflammatory medications Note: never leave patient until bleeding stopped. Collapsed veins This is may be caused due to: • needle misplacement • excessive vacuum • excessive force on syringe plunger • withdrawing too quickly with syringe • inappropriate needle selection How to avoid hematroma? ► Use the major superficial veins ► Don not penetrate the vein from the side ► Penetrate into two steps skin then the vein Remove tourniquet before removing the needle ► Do not bend arm after sampling ► Apply pressure by cotton to the venipuncture site What is the management of hematoma? Don't worry--it will go away. If you're so concerned about it, apply ice off and on after any blood-draw and use the affected arm as little as possible. Hitting an artery Rarely a small artery, which contains blood at much higher pressure than in veins, will lie unusually close to or underneath a vein. In this situation the artery may be accidentally punctured at the time of venipuncture. If this happens you generally become aware of it within a few minutes because a painful deep swelling can be felt and sometimes seen near the area of the venipuncture. Tell the collector if this happens as it is necessary to apply firm pressure to the area for at least 5 minutes. Infection 17 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ There is the possibility of infection developing at the site of the venipuncture. This is extremely rare and would be noticed as developing redness and pain at the site of the venipuncture. • Problems to the patient Bruise The most common complication of venipuncture is bruising at the site of the needle puncture. This usually appears within 24 hours and may range in size from a small spot to a large purple bruise. It is caused by blood leaking from the punctured vein out into the tissues just under the skin. Bruising is not dangerous and will slowly disappear over a few days or weeks, depending on how extensive it is. Hematoma What is hematoma? A hematoma is just a bruise like any other bruise (known as obvious bruising), and can appear even with the most skillful of phlebotomists. What are the causes of hematoma? ► Penetration of the vein ► Partial penetration may allow blood to leak into the soft tissue surrounding the vein by way of the needle bevel ► Slow flow may cause hematoma ►Bending the arm after sampling ► Patients with bleeding disorders and those taking anticoagulants or long-term steroids are at particular risk for hematoma formation. 18 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ Complications of blood sampling • Problems in the sample ■ Hemolysis What are the causes of blood hemolysis? 1. Using wet syringe 2. Collecting blood from a narrow vein 3. when a vein collapses can also cause some hemolysis. 4. Alcohol don't dry (it cause lysis to RBCs membrane because it is lipid solvent) 5. Using excess EDTA as anticoagulant 6. Using moisten EDTA 7. Slow drawing blood from the vein 8. Forcing blood through the needle into tubes 9. When doing finger sticks, squeezing the finger is the main cause of hemolysis 10. Using wet tube or stopper 11. You do not force on the wall of the tube 12. Shaking the tube very hard 13. Repeated freezing and thawing cause hemolysis 14. Holding blood over two hours before centrifuging can and usually does cause some hemolysis. 15. Holding blood in too warm area 16. Patient with hemolytic anemia 17. Drugs as quinine may cause RBCs hemolysis What are the types of hemolysis? It may be: Slight Hemolysis The serum in this case appear pink and this hemolysis do not affect the results severely and most tests can be done using this serum Server Hemolysis 19 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ The serum in this case appears reddish and this hemolysis affects the results severely and the sample must be discarded, (see the effects of hemolysis later) Blood clotting: What are the causes of blood clotting? 1. Take too much time in obtaining blood 2. Using fewer amounts of anticoagulants 3. You do not shake the sample well ■ Blood dilution: What are the causes of blood dilution? 1. Using too much fluid anticoagulant 2. Making tests as Hb from ESR tube Hemoconcentration: What are the causes of hemoconcentration? 1. Prolonged tourniquet application (no more than 2 minutes) 2. Massaging, squeezing, or probing a site 3. Long-term IV therapy 4. Sclerosed or occluded veins Notes • When you hit the artery there will be a flash of blood into the hub of the needle. • If you are in the artery the syringe should fill under arterial pressure, confinning your placement (also arterial blood appear bright red). 20 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ • If you have had the flash of blood but the syringe has failed to fill you need to advance the needle a little further. What do I do if I hitting a vein? It is true that sometimes a superficial vein overlies the artery. If you are sure it is venous blood you are aspirating then choose a site more proximal and try again. Risks of arterial puncture • It is more painful than vein puncture • You may fell faint, dizzy while the blood is being collected • You may develop a small bruise at the puncture site • Femoral artery in leg (it is not • recommended due to heavy bleeding) • Umbilical artery in neonates. Arterial capillary blood By capillary puncture if arterial blood is impossible to obtain Procedure: 1. Prepare the pre-heparinised syringe as follow: 21 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ 2. Draw up 1 ml of 1:1000 heparin into a 2 ml syringe and then squirt out all the heparin so that the syringe has just been coated in heparin. This will be sufficient to prevent the sample clotting. 3. Attach the butterfly needle to the preheparinised syringe 4. Clean the skin with an alcohol swab 5. Palpate the radial artery to determine the point of puncture. 6. Then with your dominant hand holding the syringe and needle 7. Puncture the area at 45 degrees to the skin. 8. Advance the needle slowly a millimetre at a time. 9. Once you have taken 1-2 ml of blood withdraw the needle and immediately place a cotton wool swab over the site and press hard. 10. Pressure should be maintained for 5 minutes to prevent bleeding and bruising. 11. Place the sample in the bag containing ice and send it to the lab immediately. Arterial puncture • Purpose and indications It is used for special tests as blood gas, the purpose of arterial blood gas sampling is to assess patient's respiratory status as well as acid base balance. Also it may be used for laboratory testing when venous blood is unavailable, and is frequently requested for seriously ill patients. Note : taking arterial blood require more skills than venous blood 22 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ Site of collection Arterial blood Usually the artery is deeper more than the vein, and the most common arteries that may be used are: • Radial artery of wrist (it is preferable because it is easiest to fell and more accessible) • Brachial artery of elbow 23 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ Procedure 1. Make sure baby is lying in a safe position 2. Prewarming the infant's heel (42 C for 3 to 5 minutes) is important to increases the flow of blood for collection of specimens 3. Clean the site to be punctured with warm water sponge (no alcohol to avoid hemolysis). 4. Dry the cleaned area with a dry cotton sponge. 5. Hold the baby's foot firmly to avoid sudden movement and to apply slight tension to the skin. 6. Using a sterile blood lancet, puncture the side of the heel 24 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ 7. Make the cut across the heel print lines so that a drop of blood can well up and not run down along the lines. 8. Wipe away the first drop of blood with a piece of clean, dry cotton. 9. Since newborns do not often bleed immediately, use gentle pressure to produce a rounded drop of blood. Note: Do not use excessive pressure or heavy massaging because the blood may become diluted with tissue fluid. 10. Collect blood sample using: ► Filter paper ► Capillary tube ► Test tube ► Automatic pipette 11. When finished, elevate the heel, place a piece of clean, dry cotton on the puncture site, and hold it in place until the bleeding has stopped. Neonatal blood sampling Also known as heel prick • Purpose and indications The neonatal heel prick is a common procedure for taking a blood sample from the heel of newborn. The blood samples can be used for a variety of tests, including: • Bilirubin to detect jaundice 25 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ • TSH to detect hypothyroidism and hence prevent cretinism. • Trypsin to detect cystic fibrosis. • Detection of phenylketonuria, • A test for galactosemia • Site: The recommended location for blood collection on a newborn baby or infant is the heel, only lateral parts are used not central part to avoid medial calcanous nerve, injury to this nerve can cause necrotizing osteochondritis. Note : avoid puncturing a finger that is cold or cyanotic, swollen, scarred, inflamed or covered with a rash. • Advantage Easy specially for infants • Disadvantage: ► Small blood volume that is obtained ►RBCs hemolysis ►Hb, RBCs and PCV are less than those of venous blood because capillary blood is more arterial in composition ► It may give erroneous results so it shouldn't use unless venous blood cannot be obtained • Procedure 1. Prepare equipments of the test as sterile lancet, slides, cotton, stop watch, and so on. 2. Verify the patient's condition. 3. Position the patient. 4. Chose the site • The best locations for fingersticks are the 3rd (middle) and 4th (ring) fingers • Do not use the tip of the finger or the center of the finger (it is more sensitive and cause much pain). • Side of the finger is less sensitive than the tip 26 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ 5. Clean the finger but don't use ethanol to avoid hemolysis (you can use warm distilled water) 6. Let skin dry (wet skin can cause hemolysis) 7. Using a sterile lancet, make a quick skin puncture • Puncture should be 2mm deep (it should be deep enough so that blood flow freely with out squeezing the stabbed site) • It should not exceed 2.5 mm to avoid bone 8. Gently pressure will aid if blood do not flow freely 9. Do not squeeze the finger Note: Squeezing lead to sample dilution with interstitial fluid and also help blood clotting How to improve blood flow from the finger? The finger may be warmed by applying warm wet cloth 3 min before collection 10. Wipe away the first drop of blood, which tends to contain excess tissue fluid. 11. Collect drops of blood into the collection device by gently massaging the finger. Note: The blood can be sucked up by a capillary glass tube usually passively or sometimes by indirect suction. 12. The patient holds a small piece of cotton over the puncture site for a couple of minutes to stop the bleeding. 27 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ Capillary puncture Also known as Finger stick or Finger prick •Uses ► In children and neonates ► If sample is limited as Rh, blood group, BT, CT ► If patient have been burned so veins are unavailable ► If repeated vein puncture cause vein damage ► If vein is preserved for intravenous chemotherapy ► Special tests as filarial blood film Note : capillary blood is better than venous in differential count • Site Adult Finger tip and ear loop Newborn Heel and big toe ► Only 3 h allowed for PCV and ESR ► Only 20 h allowed for Hb, RBCs, WBCs ‫هل يوجد شروط خاصة اخرى للحفظ‬ 28 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ ‫‪Chemical pathologist‬‬ ‫فرع الكيمياء الطبيه والحياتيه‬ ‫بالطبع هذا ال ينطبق على جميع التحاليل ولكن في بعض التحاليل يكون هناك شرط اضافي لحفظ العينة بطريقة سليمه‬ ‫ويمكن معرفة الشرط من ورق الكيمياويات الخاص بالتحليل‬ ‫ومن امثلة ذلك ‪:‬‬ ‫‪► Bilirubin must be stored at dark (because light can degrade bilirubin ) ► Calcium is‬‬ ‫‪preserved at plastic tube‬‬ ‫ملحوظة هامة ‪/‬‬ ‫• ال ترمي عينات الدم مباشرة بعد انتهاء التحاليل النك من الممكن ان تحتاج العينة العادة مرة اخرى ( مثال لتقييم‬ ‫باقي النتائج )‬ ‫• ايضا ال ترمي عينات الدم في نفس اليوم بل احتفظ بها في الثالجة يوم اخر على االقل النك ربما تحتاجها العادة‬ ‫او اضافة تحليل اخر لنفس المريض ‪.‬‬ ‫• كلما كانت درجة الحرارة التي تحفظ عندها العينة اقل كلما زادت مدة بقاء العينة‬ ‫‪• Specimen transport‬‬ ‫عند نقل العينات خارج المعمل (الرسالها الى فرع اخر او معمل اخر لعمل تحليل معين ليس عندك في المعمل ) البد من‬ ‫مالحظة االتي ‪:‬‬ ‫• فصل العينة وحفظها بطرقة صحيحة ‪.‬‬ ‫• البد من توضع العينة في انبوبة محكمة الغلق‬ ‫• تكتب كل البيانات الخاصة بالعينة على االبوية وكذلك في ورقة مع العينة ‪.‬‬ ‫• اذا كان للمريض وضع خاص يفضل ابالغ المعمل بذلك ( مثال ان يكون صائما او ياخذ ادوية معينة مثل‬ ‫مضادات التجلط )‬ ‫•‬ ‫اذا كان البد من ارسال العينات مجمدة نستخدم لها الثلج الجاف (‪)solid co2 dry ice‬‬ ‫• يفضل االحتفاظ بعينة احتياطي في المعمل من العينات الخارجية ( قد تحتاج الخارجية الرسالها معمل اخر او‬ ‫اعادتها في نفس المعمل او عمل تحليل اخر لنفس المريض للتاكد من النتيجة التي وهذه العينة االحتياطية توفر‬ ‫الوقت بدال من انتظار المريض وسحب عينة اخرى منه ‪.‬‬ ‫‪Preservation of sample‬‬ ‫ال يتم حفظ العينات في العمل بعد اخر العينة بصورة عشوائية ولكن البد ان يتم ذلك على اساس علمي بناء على نوع‬ ‫التحليل والبد من معرفة االمور عن كل تحاليل حتى تمون عملية الحفظ سليمة ‪:‬‬ ‫هل البد من ان تفصل العينة سريعا ام ال‬ ‫‪29‬‬ ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ ‫بعض المواد تتاثر بمدة بقاء البالزما او السيرم مع كرات الدم الحمراء دون ان تفصل‬ Glucose and ammonia must be separated quickly : ‫ما هي درجة الحرارة المالئمة لحفظ العينة‬ : ‫ويختلف ذلك من نوع تحليل الخر فمثال‬ ►Troponine and D — dimmer must be preserved at room temperature and don't freeze it ►Enzymes as GOT, GPT, ALP ... , must be preserved at refrigerator at 4°C the test may be done in this case within 7 days. ►If it is anticipated that analysis can not occur within 7 days, samples should be frozen immediately at -20°C. ► Coagulation factors must be preserved at freezer at -70°C : ‫هناك بعض التاثيرات التي تطرا على العينة عند حفظها في درجة حرارة الغرفة مثال‬ ► High temperature may affect enzyme activities ► MCV increase due to swelling of RBCs ► Prothrombin time slowly increase ► ESR, PLT, WBCs decrease ‫ما هي اقصى مدة زمنية لحفظ العينة بحيث تظل العينة محتفظة بنشاطها‬ ‫وهذا يعني ان لكل تحليل مدة زمنية اذا تاخر عمل التحاليل عن هذ المدة فان المادة المراد قياسها يمكن ان تتكسر في حالة‬ : ‫تحاليل الكيمياء او تموت الخاليا في حالة تحاليل الهيماتولوجي وبذلك تكون نتيجة خاطئة و من امثلة ذلك‬ ► Most enzymes are stable at refrigerator for about 1 week ► Reticulocytic count must be made during 6h after blood collection ► Prothrombin time must be analyzed within 12h ► Only 1 h allowed for platelets and blood smear before changes starts to occurs ►Coagulation tube ► ESR tube ► Heparin tube ► EDTA tube ► Fluoride tube ► Plain tubes 30 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ vacuum system ‫ هذا الترتيب يختلف عن ترتيب االنابيب في نظام‬: ‫الحظ‬ ■ Separation of serum or plasma For plasma: ► For plasma samples, blood should be cooled and centrifuged as soon as possible and separated immediately after centrifuging. Stoppers should not be opened during the centrifuging. ► For plasma, blood should spin 15 minutes at 2000g to 3000g. ► The plasma should be separated from cells and transferred to a clean tube. For serum: ► The blood samples are allowed to clot at 15-24 °C. ► The clotting time should be minimally 30 minutes and maximally one hour. ► For serum samples, blood should be centrifuged within one hour after blood collection. ► For serum preparation blood should spin for 10 minutes at 1500 g. ► The serum should be separated from clot and transferred to a clean tube. ‫ ينبغي اال يغادر المريض المعمل حتى تتاكد من سالمة العينات وذلك بعد فصلها في السنترفيوج والتاكد من عدم‬/ ‫ملحوظة‬ . ‫) او التاكد من عدم وجود جلطة في العينات الماخوذه على مانع تجلط‬hemolysis( ‫وجود تحلل‬ 2. Loosen the tourniquet. It may have been applied too tightly, thereby stopping the blood flow. 3. Re-anchor the vein. Veins sometimes roll away from the point of the needle and puncture site. 4. Try another tube. There may be no vacuum in the one being used. If a blood stops flowing into the tube what I do? The vein may have collapsed. Collapsed veins occur when there is excessive vacuum in the syringe. Smaller veins and veins of geriatric patients are prone to collapse. Note: Reduce the aspiration rate during your blood draw to avoid vein collapse. In this case resecure the tourniquet to increase venous filling. If this is not successful, remove the needle, take care of the puncture site, and redraw. 31 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ • Handling of specimen ■ butting samples in test tube ► Be sure that the tube is dry ► Remove the needle from the syringe ► Pour the blood gently from the syringe at the wall of the tube ► Mix well blood with anticoagulant if found ► Label the tube and write the name, tests and date Order of the tubes ‫المبدا العام هنا ه البدء بالعينات التي تحتاج الى كمية محددة من الدم مثل سرعة الترسيب ثم العينات الماخوذة على مانع‬ : ‫للتجلط ثم اخيرا العينات السيرم‬ : ‫وبناء على ذلك يجب ان يكون ترتيب العينات كاالتي‬ ■ Non-additive tube (red top) ■ ESR tube ■ Additive tubes in this order: Sodium heparin (green top) EDTA (lavender top) Fluoride (light gray top) Can I use local anesthetic? If a patient is really needle phobic cream containing local anesthetics may help. This should be applied for at least 30 minutes to allow the local anesthetics to take effect. If a blood sample is unobtainable what I do? 1. Change the position of the needle. If the needle has penetrated too far into the vein, pull it back a bit. 32 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ If it has not penetrated far enough, advance it farther into the vein. Adjust the angle (the bevel may be against the vein wall). 4. After entry into the vein push the tube all the way into the holder; vacuum is broken, and blood flows freely into the tube. The vacuum in the tube causes blood to move through the needle and into the tube. 5. Remove the tourniquet once the blood flow has begun. 6. Fill the tube until the vacuum is exhausted. 33 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ 7. Remove tube from adaptor and insert subsequent tubes. ensure the needle is not moved while tubes are being changed. 8. Place a sterile cotton piece over the point where the needle entered the skin. 9. Remove the needle quickly 10. Patient press cotton for 5 min to stop bleeding and do not bend arm Order of the tubes When we use evacuated system tubes must be drawn in a specific order to avoid crosscontamination of additives between tubes. The recommended order of draw is: • Coagulation tube (light blue top) If there is a concern regarding contamination by tissue fluids or thromboplastins, then one may draw a non-additive tube first, and then coagulation tube 10. Dispose of contaminated materials/supplies in designated containers. 1. Insert the stopper of the first tube to be drawn into the adaptor. 2. Do not pushes too far as that might cause premature loss of vacuum if it is punctured by the needle. Before using the tube it should be tapped gently to dissolve any additives from stopper and this prevents aspiration of additives into vein 3. Insert the needle into the vein. 34 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ 5. Withdraw blood gradually by gently pulling upon the syringe plunger (don not use negative pressure) Notes : Vigorous pulling on the plunger of the syringe can collapse the vein, produce hernolysis of the blood specimen 7. Place a sterile cotton piece over the point where the needle entered the skin. 8. Remove the syringe quickly 9. Patient press cotton for 5 min to stop bleeding and do not bend arm 35 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ Veninuncture with needle and syrinae 1. Check the syringe • Check to be sure that the syringe works smoothly by pressing the piston. • The syringe must be dry to avoid hemolysis of the red cells. • The plunger must be pushed firmly to the bottom of the cylinder to prevent injection of air into the vein, this can be fatal. 2. Feel the vein by left hand 3. Fix the vein by drawing skin tight over the vein to prevent vein from moving 4. Enter by the needle at 45 degree angle (under the skin and then into the vein) When the needle enter the vein there is sudden lose of resistance and blood come in the head of needle Notes : Do not probe or move the needle horizontally, as discomfort and possible nerve damage may result. 5. Remove the tourniquet once the needle has been inserted Note: if the needle were removing prior to the Tourniquet being removed, blood would be forced out of the venipuncture site, resulting in hematoma. yaw ■ Alcohol Decontamination Use 70% alcohol as disinfectant. Let it to dry to avoid hemolysis and burning sensation. 36 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ Notes : if sample is collected for ethanol concentration determination use another disinfectant other than alcohol ■ Apply tourniquet Apply the tourniquet 3-4 inches above the selected puncture site. Tourniquet obstructs the venous return so it helps to distend the vein. As a rule, the tourniquet should not be placed too tightly (as to stop arterial flow) or left on the patient for more than 2 min. What are the effects if it left for more than 2 min? Prolonged application of the tourniquet results in partial stasis of blood and changes many quantitative values of blood components as: Concentration of total protein, lipids, iron, GOT and bilirubin will increase Concentration of potassium will decrease. Notes Check the pulse at the wrist to make sure that arterial circulation is not cut off ■ You can force blood into the vein by massaging the arm from wrist to elbow ■ Palpate and trace the path of veins with the index finger. ■ Feel vein using the tip of finger and think in four things during feeling the vein Bounce, direction, depth and size of needle ■ Lower the extremity over the bedside to allow the veins to fill. ■ Apply a warm, damp washcloth to the site for 5 minutes this increases your blood circulation, which makes it easier for the phlebotomist to find a vein. ■ Drinking water helps blood flow better and makes the veins more likely to stick up and be found easily (even if the patient is fasting) What I do if I can't find the vein? 37 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ After all these methods if the phlebotomist does not succeed in reaching the vein, another phlebotomist may step in to complete the procedure. This keeps both the patient and the phlebotomist from becoming flustered and should be seen as a reassuring step. Blood collection from Cannula In general, blood should not be drawn from an arm with a cannula because intravenous therapy (IV) fluid may dilute the specimen, so collect from the opposite arm if possible. Otherwise. follow these procedures: ■ Turn off the IV for at least 2 minutes before venipuncture. ■ Apply the tourniquet below the IV site. Select a vein other than the one with the IV. ■ Draw 5 ml of blood and discard before drawing the specimen tubes for testing. If the vein is difficult to be seen: 38 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ For most people, venipuncture is quick, easy, and relatively painless. But some people, however, have veins that are quite small or difficult to access. The following methods can be done to help.  Ask the patient to make fist this helps make the vein more prominent.  Apply tourniquet to distend the vein  Arm that cannot be extended well  Edematous extremities (tissue fluid accumulation alters test results).  Arm with sclerosed or occluded Veins (this results from frequent puncture leading to scarring, or inflammed veins, these veins are hard when palpated and generally have minimal flow.)  Arm on the side of a previous mastectomy why? A patient with a mastectomy may have lymphostasis on the effected side, making the patient highly susceptiple to infections , also lymphostasis affect blood composition and test results. Notes :Patients with bilateral mastectomies should have venipuncture performed on the hand.  Collection of blood sample Vein selection The vein selected should be large, readily accessible, and sufficiently close to the surface to be seen and palpated. For adults: Although the larger and fuller median cubital and cephalic veins of the arm are used most frequently, the basilic vein on the dorsum of the arm or dorsal hand veins are also acceptable for venipuncture. Foot veins are a last resort because of the higher probability of complications. Note: the elbow area does not have many nerves so this is a good site Note: Avoid using dorsal hand veins in diabetics and patients with poor circulation 39 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ Due to position change may cause increase as much as 13% in the concentration of plasma proteins and protein bound constituents due to redistribution of fluids in the EC space.  The patient hand extend from shoulder to wrist  The patient arm should not bent at elbow  If the patient cannot hold the arm in position, a pillow may be used for additional support.  If the patient lies down arm should extend and supported. Choosing the arm Note : Right arm is better due to working with right hand make its veins larger and fuller Avoid the following:  Arm with burn area  Arm with hematoma (may cause erroneous test results. If another site is not available, collect the specimen distal to the hematoma)  Arm with scaring (it is difficult to puncture the scar tissue and obtain a specimen)  Arm with IV line  Arm with recently injected or withdrawn syringe 40 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ Increased RBCs count lead to increased turn over of nucleoproteins and excretion of uric acid 5. Ambient temperature Acute exposure to heat cause plasma volume to expand by influx of interstitial fluid to vessels so plasma proteins may decrease by up to 10% If prolonged exposure to heat leads to excessive sweating , hemo-concentration occurs rather than hemo-dilution 6. Menstrual cycle Normally the concentration of the female sex hormones are affected by the cycle Also other hormones are affected by the cycle for example corticosteron concentration is much as 50% higher in leuteal phase than in follicular phase Medical condition 7. Fever Hyperglycemia occurs soon after onset of fever and stimulates the secretion of insulin Fever also appear to reduce the secretion of thyroxin 8. Injury After injury a loss of fluid to extravascular tissue occurs with a resulting decreased plasma volume If this decrease is enough to impair GFR diminished renal functions lead to urea and creatinine increase 9. Burns Serum TP decrease due to loss to extravascular spaces and loss to catabolism of protein 10. Surgery The stress of surgery reduce the serum T3 by 50% in individual without thyroid disease Certain enzymes as CK, LDH, and GOT show increased activity after surgery 11. Transfusion 41 ‫د رياض حنيوه‬ ‫جامعه وارث األنبياء‬ ‫كليه الطب‬ Chemical pathologist ‫فرع الكيمياء الطبيه والحياتيه‬ Transfusion of whole blood or plasma raises the plasma protein concentration Serum LDH will rise with the breakdown of transfused RBCs Blood transfusion rise serum iron 42