Clinical Pathology (Hematology & Instruments) PDF

What is Clinical Pathology ? Clinical Pathology Introduction to Clinical Hematology  Blood The biological fluid most routinely analyzed in the laboratory.  Blood Blood is a fluid tissue that circulates through the vascular channels carrying the necess...

What is Clinical Pathology ? Clinical Pathology Introduction to Clinical Hematology  Blood The biological fluid most routinely analyzed in the laboratory.  Blood Blood is a fluid tissue that circulates through the vascular channels carrying the necessities for life to all cells of the body, and it receives the waste products of metabolism for transport to the organs of excretion.  Blood Composition The Cellular portion The Liquid portion ”Clinical Hematology” “Clinical Biochemistry”  Aim of Blood Examination 1. Screening procedure for general health. 2. Diagnosis of different diseases. 3. To asses the body's ability to fight infection (cellular immunity). 4. Evaluation of certain disease condition (prognosis) 5. Follow up the response to certain therapy.  General Considerations 1- Instrument for collection should be clean and dry. 2- The blood should be drawn gently. 3- Use a suitable anticoagulant and in proper amount. 4- Removal of needle of the syringe at time of evacuation. 5- Quick transfer of blood into the anticoagulant. 6- Blood should be collected with minimal tissue injury.  Physiological Action Altered test Response Emotional Epinephrine Hyperglycemia Serum Glucose test stress: Lecucytosis T.L.C. (Fear, Lymphocytosis in cats D.L.C excitement) Exercise: Hypoxia induce Release of blood cells into PCV splenic contraction circulation TLC Prolonged increases activity of muscular enzymes CK, AST, LDH exercise: Diet: after lipemia Hyperglycemia Serum Glucose test meals lipemia Lipids test Starvation: Decreased BUN & serum albumin Drugs: TLC, DLC,BUN Glucocorticoids Liver enzyme activity ALP, ALT alter tests to detect immune-mediated diseases.  Physiological function Altered test response Drugs: decreases serum Blood glucose test exogenous insulin glucose and serum potassium Antibiotics : increased creatinine Cephalosporins concentration Age, sex and breed Significantly influences some of the blood values. Collection and Handling the Blood Specimen The selection of the proper container and the proper anticoagulant must be made prior to specimen collection. 2.Anticoagulants: 1.Containers ❖ Heparin (equipment): ❖ EDTA ❖ Na Citrate ❖ Syringes ❖ Na Flouride ❖ Vaccutainers ❖ Double oxalate  Syringes : precautions ✓Are of variable sizes. ✓Should be chemically clean and dry. ✓ Using calibrated syringe to ensure the collection of an exact volume of blood suitable to the amount of the anticoagulant contained in the sample vial. Old metallic syringe Plastic disposable syringe  Vacutainers (Vacuum Tubes) Glass or plastic vials fitted with rubber or polyethylene stoppers are recommended. Plain, anticoagulated, or containing clotting accelerator. The stoppers' color indicates the anticoagulant and use.  Technique and Precautions of Blood Collection I. Precautions before blood collection: 1. Select the site of sampling; free from cyanosis, inflammation, or edema. 2. In long-haired animals, shave the site of sampling. 3. Sterilize the site of sampling by 70 % alcohol. (Rubbing with alcohol makes the vein more clearly outlined). 4. Anesthesia and skin incision may be required; in the rat, guinea pig, and rabbit.  II. Amount of Blood Required: Depends on the requested test(s), and the method used for conducting the test. As a general role; it is usually safe to take about 0.5 ml blood/kg body weight in all species. 5 ml in large animals and 2 ml of blood in small animals are sufficient for routine blood study.  1. Small amount of blood: Site: Capillary bed of the skin, Clipping the toe nail, or Pricking the marginal ear vein. Aim: Small amount of blood is collected if only RBCs count, hemoglobin concentration, PCV, leucocytes count, or blood smear examinations are needed. Technique: Sudden, sharp stab using an automatic lancet, an ordinary lancet, or sterile needle. Exclude the first drop (contaminated with the tissues) and collect the second. Lancets Automatic lancet  2. Large amount of blood 1) Large blood samples are drawn from major veins (Phlebotomy) 2) Occlude the vein using digital pressure or tourniquet. 3) Stretch the skin across the vein. 4) Insert the needle, while the bevel is directed upward. 5) Apply gentile suction of blood to avoid collapse of the vein. 6) Release the digital pressure or remove the tourniquet. 7) Gently withdraw the blood by the plungers.  Sites of Blood Collection Horse, Sheep, Goat and Camel: Jugular vein (middle third; more superficially exposed). Cow: 1- Jugular vein (middle third; more superficially exposed) 2- Tail venipuncture (coccygeal vein) 3- Mammary vein. Jugular Vein Jugular Vein Coccygeal artery in cow  Dog: 1- Cephalic vein (most commonly) 2- Jugular vein(occasionally) 3- Saphenous vein 4- Tibial veins. Cat: 1- Cephalic vein (most commonly) 2- Jugular vein (occasionally) 3- femoral veins 4- Clipping of the toe nail. Cephalic vein in cats Jugular vein in dog  Rabbit, Guinea pig: 1- Heart 2- Ear vein 3- Jugular vein (occasionally). Pig: 1- Anterior vena cava 2- Ear vein 3- jugular vein (rarely). Rat, Mouse: 1- Retro orbital venous plexus 2- Tail amputation 3- Heart. Birds: Wing vein, cutaneous ulnar vein, or brachial veins, or the heart.  Ear vein in pig Ear vein in rabbit Anterior vena cava in pig Sampling in mice  Wing vein in bird Caudal vein in fish Handling of the Collected Blood Sample Blood samples should be processed as soon as possible after collection. On standing; the cells settle down and separate from the plasma, so it is necessary to mix the sample thoroughly each time a portion is removed for a test. Handling of the Collected Blood Sample The blood film is best made immediately from fresh blood. As soon as possible from anticoagulated blood, preferably within an hour of sampling, as WBCs undergo degenerative changes due to aging of the cells. Handling of the Collected Blood Sample Blood can be kept at room temperature for 1-2 hours. If blood examination is to be postponed for several hours or overnight; make blood films immediately, perform ESR, and then refrigerate the sample. The cell remains stable at 4o C for 24 hours. An ice box or cold packs should be used to transport blood samples to a distant laboratory.  Identification of the Collected Blood Sample Specimens to be sent to the laboratory should be completely identified. A complete history should be included with each sample.  Anticoagulants Hematological examination requires blood in liquid form. Immediately after withdrawal; blood must be thoroughly mixed with the anticoagulant to prevent clotting. The common used Anticoagulants are: Heparin, EDTA, Sodium fluoride, Sodium citrate and double oxalates.  Causes of Specimen Spoilage 1. Hemolysis: Breakdown of the cellular elements of the blood. Effect of hemolysis on laboratory results: Hemolysis interferes with the laboratory investigations which are measured colorimetricaly; either by: a) Changing the optical density. b)Releasing intracellular components; causing false increase or decrease in the concentration of the analytes.  Causes of hemolysis A) Before withdrawal: Using wet syringe or needle. Using needle of small bore. B) During withdrawal: Rapid withdrawal of the blood. Moving the needle inside the vein. C) After withdrawal: o Emptying of blood rapidly into the container. o Dispensing the blood without removing the needle. o Vigorous mixing of the blood with the anticoagulant (Rough handling of specimen). o Centrifuging blood for longer time and at higher speed. o Exposing blood to extreme heat or cold. o Storing whole blood samples in freezing temperature. o Bacterial or chemical contamination.  Causes of Specimen Spoilage 2. Lipemia: The presence of high concentration of lipids in the blood. Effects of lipemia on laboratory tests: Lipemia enhances hemolysis. Lipemia produce false-high values of Hb, and so incorrect MCH and MCHC.  Lipemia Lipemia falsely decrease serum Na and K by flame photometry (lipid displacement of the aqueous phase) Serum turbidity impairs OD. Lipemia falsely increase plasma proteins (measurement by refractometry).  Causes of Specimen Spoilage 3. Clotting: The blood loses its liquid form and becomes clotted. Causes of clotting: Taking too much time in drawing the blood; so it has already started to clot before being mixed with the anticoagulant. Using no anticoagulant. Using inadequate dose of the anticoagulant. Failure to mix the blood with the anticoagulant.  Causes of Specimen Spoilage 4. Decomposition: due to overgrowth of bacteria or mold. The blood become unfit for examination. Causes of decomposition: High temperature. Bacterial contamination. Contamination with the soil or fecal materials.  Causes of Specimen Spoilage 5. Desiccation (Drying): Drying of blood sample. Causes of sample drying: Failure to pack the specimen properly. The principal packing faults are: a) too small sample in a too large container. b) leaving the container open to air. Clinical Pathology  Laboratory Instruments  Laboratory Instruments Basic Advanced – Microscope – Coulter counter – Centrifuge – Autoanalyzer – Colorimeter – Flame photometer – Spectrophotometer – Ion selective electrode – Balance – Blood gas analyzer – pH meter (pH paper) – T.S. refractometer – Distiller – Electrophoresis equipment – Temperature controllers & Denistometer – Vortex  Basic Instruments  Microscope Light microscope  Basic equipment Microscope Micro = small Scope = to see Lens system Object ex:: slide Illumination system Basic equipment Microscope  Microscope Practical note ! ◼ Wet preparation ◼ Stained preparation (Reduce the light) (Increase the light) ◼ ex: fecal smear ◼ ex: blood film ◼ Diaphragm ◼ Diaphragm ◼ Semi-closed ◼ Widely open ◼ Condenser ◼ Condenser ◼ Down position ◼ Upward position Monocular microscope Binocular microscope Trinocular microscope Centrifuge Basic equipment Centrifuge Basic equipment Centrifuge  Basic equipment Centrifuge Used for: Separation or Sedimentation (Blood) (Urine)  Basic equipment Centrifuge ◼ CONSIDER !!! ◼ Speed: RPM Revolution Per Minute (rpm) ◼ Time ◼ Temperature  Basic equipment Centrifuge Speed: RPM Revolution Per Minute (rpm) Centrifuge can be: Ordinary (maximum 6000 rpm), High speed (12,000 to 15,000 rpm) or Ultra speed (50,000 to 100,000 rpm) Time Temperature The force of centrifugation results in extreme heat that may results in denaturation of some biological substances like hormones, so the cooling centrifuge was invented. Colorimeter  Basic equipment Colorimeter Uses: Estimation of different chemical constituents in different body fluids with the concept of Colorimetry Colorimetry  Colorimetry I0: Original light I: Light absorbed = OD = Absorbance I1: Light transmitted ↑ Colour of the solution → ↑ the Optical Denisty (Absorbance)  Colorimeter and Spectrophotometer InfraRed Visible Light UltraViolet Colorimeter Spectrophotometer Balance  Basic equipment Balance ◼ Single pan balance ◼ Double pan balance are used for weighing materials in the lab Single pan balance Double pan balance pH paper and pH meter  Basic equipment pH paper ◼ Used for measurement of pH of different solutions in the lab.  Basic equipment pH meter ◼ Used for measurement of pH of different solutions in the lab. Water Distiller  Basic Equipment Water Distiller ◼ Used for distillation of water to obtain distilled water Temperature Controllers  Basic equipment Temperature controllers ◼ Liquid nitrogen (-196°c) ◼ Water bath or incubator ◼ Deep freezer (-70°c : -20°c) (37°c : 65°c) ◼ Refrigerator (0 : 5°) ◼ Boiling water bath (100°c) ◼ Hot air oven & Autoclave (> 100°c) Room Temperature : 15°c : 25°c  B. Advanced(Accessory) Equipment 1. Coulter counter 5. Blood gas analyzer 2. Autoanalyzer 6. T.S. refractometer 3. Flame photometer 7. Electrophoresis unit 4. Ion selective electrode 8. Vortex Coulter Counter = Electronic Cell Counter Coulter Counter Coulter Counter Sensing zone  Coulter counter When the blood cell passes through the sensing zone aperture, it impedes the current flow from the cathode to the anode The number of deflection in current voltage is the number of cells counted when passed through the sensing zone. The magnitude of deflection is proportional to the cell volume. Flame photometer  Flame photometer Used to measure certain metals concentrations based on the fact that metallic elements burn on hot flame emits light of characteristic wave length specific for each one. E.g. Na, K, and sometimes lithium. Electrophoresis Unit Cellulose paper with the separated serum protein stained with Ponsesu S Stain _ + Normal Serum Protein Electrophoresis Diagram Polyacrylamide Gel electrophoresis T.S. Refractometer T.S. Refractometer Beakers Beaker Mug, easily handled Glass funnels Volumetric Flasks Conical Flasks Cylinders Pipettes Glass pipettes: 1. Volumetric pipettes 2. Graduated pipettes 3. Pipettes with air pulp Pump for glass pipettes  Pasteur pipettes Glass Pasteur pipettes Plastic Pasteur pipettes  Automatic and micropipettes Eppendorf Cuvette ‫عينات ُمصورة‬ Vacutainers (Vacuum Tubes) Basic equipment pH paper Venoject Lancet Lancet T.S. Refractometer  (Illumination system) Condenser and diaphragm