بسم الله الرحمن الرحيم 285 PHL Lab# 1 Introduction Blood

blood is a tissue that circulates in the closed system of blood vessels. Functions: Respiration Nutrition Excretion Defense mechanisms Regulation of acid base balance Regulation of body temperature Regulation of body water.

Composition: Cellular elements- RBCs, WBCs, Platelets Plasma- Water + Solids Solids: 1) Diffusible constituents: a) Anabolic e.g. glucose, amino acids, and vitamins b) Catabolic e.g. urea, uric acid, and creatinine. 2) Non diffusible constituents: e.g. albumin, globulin, and fibrinogen.

Purpose of blood analysis: Serum: serum = Plasma- Blood clotting factors. Purpose of blood analysis: Diagnostic: any change in the normal constituent, or presence of abnormal constituent indicate disease state. 2) Prognostic.

Preparation of serum sample: Obtain venous blood using empty centrifuge tube. Allow the blood to clot. Rime the clot. Centrifuge. Transfer the clean specimen tube using Pasteur pipette, don’t disturb the clot.

Preparation of plasma sample: Put some anticoagulant in a clean dry centrifuge tube. Add the venous blood and mix. Centrifuge. Transfer the top clear plasma to a clean specimen tube.

are chemicals which prevent clotting of blood. Anticoagulant: are chemicals which prevent clotting of blood. 1.Heparin: MOA. antithrombin “ prevent conversion of prothrombin to thrombin” Disad. High cost. 2. Oxalates: Na, K, Li, or NH4 salts are used. MOA. Forms insoluble salts with Ca ions. Disad. - Inhibit lactate dehydrogenase - Na, K salt should not be used in determination of electrolytes as this lead to significant error.

3. E.D.T.A: (ethylene di amine tetra acetic acid) MOA. Bind to Ca++. Adv. Prevents clumping of platelets. 4. Na Fluoride: (enzyme poison) MOA. Inhibit glycolysis, therefore its used in sample for determination of blood sugar. ”can’t be used for determination of enzymes” 5. Tri Na Citrate: used to determine ESR. 6. Acid Citrate Dextrose solution: used mainly to study platelets and to preserve blood.

Deproteinization: Precipitate the proteins, Why? → because the proteins have certain U-V absorption and could give false reading. Also, proteins are colloids which make the solution turbid and difficult to read on the spectrophotometer.

Deproteinization agents: Trichloroacetic acid Tungestic acid Zinc hydroxide Organic substances.

Diabetes mellitus: Type: IDDM NIDDM Diagnosis of Diabetes mellitus: 1) if the patient has symptoms: measurement of fasting plasma glucose level measurement of two hours postprandial plasma glucose level 2) if the patient has no symptoms: Oral glucose tolerance test (OGTT)

Glucose conc. (mg/dl) Two hours after 75g glucose Fasting 65-140 mg/dl Normal 140-200 mg/dl 110-126 mg/dl Impaired glucose tolerance >200 mg/dl > 126mg/dl Diabetes mellitus

Investigations for diagnosis of diabetes mellitus: Glucose in the urine (glucose urea) > 180 mg/dl. 2) Glycated proteins: a) glycated hemoglobin b) glycated albumin ( fructosamine) 3) Microalbuminuria:

Determination of blood glucose Serum glucose is determined by tow methods: 1) Oxidation method “Enzymatic method”. 2) Alkaline copper reduction method “ Asatoor and king method”

Oxidation method “Enzymatic method”. Principle: Glucose is oxidized according to the following reaction: Glucose glucose oxidase Gluconic acid + H2O2 H2O2 +phenol + amino-4-antipyrine peroxidase Quinoneimine + H2O

Procedure: Mix and incubate in the water bath at 37°C for 10 min, then Read at wave length 505nm. Standard Sample - 0.1 ml serum standard 1 ml Working reagent

Calculation: Serum glucose (mg/ dl) = A sample X standard conc. A standard Normal serum level: 75- 110 mg/ dl.

Clinical Significance of determination of blood glucose: Hyperglycemia: blood glucose level > 110 mg/dl. Causes: 1) Diabetes mellitus 2) Hyperactivity of thyroid, adrenal, pituitary gland 3) Acromegaly 4) Asphyxia 5) Exercise or stress 6) Pancreatic carcinoma 7) Acute pancreatitis.

Clinical Significance of determination of blood glucose: Hypoglycemia: blood glucose level <70 mg/dl. Causes: 1) Overdose of insulin. 2) Hypoactivity of thyroid, adrenal, pituitary gland 3) Glycogen storage disease.

Renal glucose threshold: The ability of renal tubules to reabsorb glucose is 180 mg/dl “renal glucose threshold” If blood sugar level rises above this value the glucose appears in the urine “Glucosurea”