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    34 views53 pages

    Physiology Manual

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    physiology manual

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    © All Rights Reserved
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    TABLE OF CONTENTS Practical Title of Practical Page Number | Number Table of Contents WW Instructions for Students 1 Safety Measures in Physiology Laboratory UVAS, Lahore 2 Introduction to Physiology 3 A. Introduction to Microscopy 4 2. Pipetting 2 8 3. Introduction to Hematology 10 4. Blood Collection and Preservation 15 3. ‘Complete Blood Count (CBO) 22 6 Principle of Hemocytometry 26 3 Counting of Erythrocytes = 29 38. Counting of Leukocytes 32 9. Platclet Count 35 10, | Differential Leukocyte Count 37 i. Red Blood Cell Osmotic Fragility Test 42 12. | Determination of ABO Blood Groups and Rh Type 46 13, | Hematocrit/ Packed Cell Volume 50, 14. | Hemoglobinometry 54 152 | Red Blood Cell Indices 38 16= | Determination of Bleeding Time 60 1% | Determination of Coagulation Time, fi 63 18, | Erythrocytes Sedimentation Rate 66 19. | Determination of Arterial Blood Pressure 69 Heart Sounds Instructions for Students istructions This manual contains guidelines for students t¢ consists of following headings: Title of experiment Date performed Principles ‘Apparatus Methodology Results/ Observations Interpretation Important Symbol: 10 conduct their experiments. Each experiment Serial Number Symbol Explanation Wear Gloves Wear Safety Glasses Poisonous Health Hazard Radioactive ®/+ De D ft Bleeding Hazard Safety Measures In Physiology Laboratory UVAS, Lahore Always wash your hands before and after the performance of an experiment in the lab. . Never eat, drink, smoke, or apply cosmetics in the laboratory. . Switch off mobile phone before entering thie lab. Ifyou have long hair or loose clothes, tie or confine them. Never do unauthorized experiments. Never store any type of eating or drinking materials in the refrigerator of lab. . Do not pipette any material (chemicals, blood and urine) by mouth. Always perform the procedures causing splash, spray and airborne droplets behind barriers (bio-safety cabinet). 9. Always put on your apron / lab coat before entering the laboratory. 10. Wear gloves during blood collecting procedures and samples handling. 11, Do not forget to change gloves between patients 12, Use face masks where necessary. 13. Read labels carefully 14, Never "smell" a solvent!! The contents of a bottle can be checked by reading its label. 15. Always check your glassware for cracks and chips every time before using it. 16. Do not handle any equipment unless you are trained and authorized by trainer/instructor. 17. Disinfect all work place before and after conducting experiment, and instantly after any spill or splash of possibly hazardous material, 18. After blood collection, the needles should be discarded and disposed of properly and never be re-used. 19. Pay attention to waming and caution signs displayed in the lab. 20. Follow standard safety instruments in case of any emergency. 21. Inform your instructor instantly after any breakage, injury, fire or any spill. PI AMERY Introduction to Physiology’ > Physiology: . ‘The word “Physiology” is derived from two Greek words; physio mean naturelorigin and Jogia mean study of. “It is a branch of biology which deals with scientific study of normal functions of an organism.” OR “The biological study of the normal functions of living organisms and their pars is known as Physiology.” ; ; The branch of Biology that deals with the functions and activities of life or of living matter and of the physical and chemical phenomena involved. Physiology has various disciplines like: > Human Physiology: is the study of normal functions of the human body. > Animal Physiology: is the study of normal functions of animals. OR > Animal Physiology: is the scientific study of the life-supporting properties, functions and processes of animals or their parts. . Plant Physiology: is the study of normal functions of plants. Microbial Physiology: is the study of various normal functions of microbes. Parasite Physiology: is the functional study of different parts of parasites. Veterinary Physiology: is the study of normal body functions of domestic animals is called veterinary physiology. vvvv Physiological Examination: It helps us in; 1. Understanding of the normal functions which are going on at cell, tissue, organ, system and organism level. 2. On basis of normal function, abnormal function (pathology) can be identified and diagnosis of disease can be made easy. 3. Provides a base for understanding the veterinary pharmacology and pathology. Exercise: 1._ Differentiate between Physiology and Patholo 2. Differentiate between Physiology afd Anatomy” ? Differentiate between Physiology and Histology. 5. . Differentiate between Physiology and Pharmacology, Differentiate between Physiology and Theriogenology, Experiment: 1 Introduction to Microscopy “Microscopy is the technical field of using microscope to see objects that are not visible with the naked eye.” Parts of Microscope: Eyepiece: Contains the ocular lens (lens at the top that you look through). They are usually 10X. Tube/Eyepiece Tube: Joins the objective and eyepiece lenses. Nosepiece: The point which hold the different lenses. Is can be rotated to view the specimen at appropriate resotution. Objective lenses: Different magnifications, normally ranges from 4X to 100X. Stage: The platform where you place the sample. Stage clips help fix the sample at the stage. Light source: Normally near the base and projects light upwards through the diaphragm to the sample to the lenses. Coarse adjustment knob: Moves the stage at comparatively more pace for focusing. ine adjustment knob: Moves the stage slightly to improve the image. Diaphragm and iris: Adjust the quantity of light to be placed on the specimen. Condenser lens: ft focuses the light beam onto the stage where the specimen is placed. ‘Arm/frame: Used to support the microscope when carried. Base supports the microscope. Alci - condenser Principles of Microscopy: Magnification is simply the measure of how big an object appears such as when light from specimen is collected via a lens. The ability of microscope to magnify and cnable to study fing details is the resolving power. It is defined as the minimum distance between two objects that can be visualized. It is a function of the wavelength of the light used and the quality of the optics, ‘A beam of light with a shorter wavelength will enable higher resolution of the microscope, Working distance is the distance between the objective lens and the sample. Working distance is relatively long at lower magnification and decreases when microscope is adjusted at higher resolution. Understanding the Limits of Resolution: In microscope we make use of the wavelength of the light to visualize an object. A light with a shorter wavelength enables us to study finer detail of the specimens. Light microscope uses visible light which has a minimum wavelength of 400 nm. So, any object smaller in size by about half of this wavelength (i.e. 200 nm) will not be visible under light microscope. Electron Microscope: Electron microscope use beam of electrons. They have wavelength much shorter than light microscope and can be used to visualize two objects one-twentieth apart. Therefore, electron microscopes can be used to visualize viruses, molecules and even atoms. Resolving power of microscopes Am tem tom teen 100m 10mm tem «em itm +m 109m 10m ‘© Goommam. 2012 Umaretyo wena 1 ments he How to Focus the Microscope: Tum the revolving, uureet to move the lowest resolution lens (4x) into position © Place the sample (or slide) on the stage and secure its position with stage clips. © Look at the stage from the side and move to the appropriate position till light falls directly on the samples contained in the slide © Look through the objective lens and use coarse adjustment to move the stage upward till the sample start to appear Use fine focus and adjust so that the sample ean be clearly viewed through objective lens. Use light adjustment to view sample more clearly. © After getting clear view of the sample with the lowest magnification, you can switch to the next objective lens. If required, you can readjust view by using fine adjustment or lighy intensity © Repeat the previous step if you want to view the sample at higher resolution. Use oil immersion to view sample at 1000X resolution. Ceadoa Wes Label The Diagra el Precautions: T. Do not touch the slide with objective lens 2. Keep the microscope covered when not in use 3. Always use both hands to grab the microscope. Use one hand to hold the microscope arm while place the other hand at the base. Exercise: > What is the magnifying power of the compound microscope? Experiment; 2 Pipetting A micropipette is used to transfer small amounts of liquid, usually in micro liters. The volumeter is used to adjust the amount to volume to be transferred. It consists of number dials whose which show the volume of liquid to be used. They are read from top to bottom. Some pipettes also have additional locking mechanism to prevent accidental change in volume. The simple procedure to operate a micropipette is given below. Set the desired amount of volume by tuning the volumeter adjustment. Attach the suitable tip to the end of pipette. Press the volumeter adjustment knob to the first stop. Immerse the tip just inside the surface of the sample Slowly release the volumeter knob (o the up position. (Do not snap it up) Wait for one or two seconds to confirm that the required volume of sample is taken in. Dispense the sample into required container by inserting the tip against the wall of receiving tube, Release the plunger to first stop. Wait for the sample to drain properly. Then depress the knob to the second stop. Remove the tip from the receiving tube with the knob still fully depressed Eject the used tip into the waste receptacle using the tip ejector. Precautions: Do not use a pipette outside its range. Do not let knob to snap while removing the tip out of sample. ‘Use appropriate tip according to the requirement, Do not depress the knob while afler dispensing the sample while itis still in the receiving container. Experiment: 3 4 Introduction to Hematology Hematology: The study of blood components and coagulation is called hematology. It includes. 1. Study of the concentration, structure and function of blood cells and their precursors in the bone marrow. 2. Study of chemical constituents of plasma or serum. 3. Study of functions of the platelets, procoagulant and anticoagulants in blood. Blood: Specialized fluid connective tissue, which circulates in a closed system of blood vessels and heart, is known as blood (circulatory fluid). Blood Composition: Blood has two components 1. Plasma: pale yellow/straw colored fluid (54-55%) 2, Formed Elements: like erythrocytes, leukocytes and platelets (45-46%) Functions of Blood: Blood has an important role in various functions of the body. 1, Role of Blood in Respiration: Blood transports oxygen from the lungs to the tissues and carbon dioxide from tissues to the lungs. 2. Excretory Function of Blood: Blood collects the metabolic wastes (urea, uric acid, CO3) from all over the body and transports them to the kidneys and lungs for excretion. 3. Regulation of val Acid- nce: Blood keeps the acid base balance within normal limits. 4, Transport of Nutrients: Blood is responsible for absorption and transportation of Various nutrients (fatty acids, glucose and amino acids). 5. Regulation of Water Balance: Water content of the body is also regulated by the blood. 6. Body Temperature Regulation: Blood dissipate extra heat from the body to environment. 7. 8. % ‘Transport of Hormones: Blood transports different hi Transport of vitamins and electrolytes: Various types of vitamins and electrolytes (sodium, potassium, calcium, chlorides, phosphates, sulfates and carbonates) are transported by the blood. jormones to their target areas, Immunity: Blood provides immunity against infection in the form of antibodies ang activated T-lymphocytes. ‘Hemostasis: Prevents the blood loss with the help of clotting factors. Hematology Laboratory: It deals with: yeaa aeny Counting of total number of red blood cells, white blood cells and platelets in circulation under microscope. The hemoglobin estimation. ‘The Packed cell volume (PCV) or hematocrit determination. Erythrocyte sedimentation rate (ESR) determination. Differential leukocyte count (DLC). Coagulation time determination. Bleeding time determination. Blood grouping/typing Blood biochemistry Basic Requirements for the Hematology oratory: Glassware: The following glassware’s are required: 1. Hemoglobin pipettes (Sahil'spipette) 2. Red blood cell (RBC) pipettes (Thoma diluting pipette) SerAnwew 10. 1. 12, 13. . White blood cell (WBC) pipettes (Thoma diluting pipette) }. Hemocytometer (Neubauer counting chamber) Wintrobe’s tubes Westergren’s tubes Pasteur pipettes }. Glass slides Cover slips Disposable syringes Needles etc. Capillary tubes Vacutainers Reagents: ‘The reagents used in hematology laboratory includes 1, Hemoglobin Diluting Fluids a) N/10 hydrochloric acid b) N/10 NaOH 2, RBC-Diluting Fluid 2) Normal saline . b) Heyem’s solution (ini 2/100 ml): (sodium chloride 0.5, sodium sulphate 2.5 and mercuric chloride 0.25) ©) Toisson solution: (NaCI- 1.0g, Na Sulfate- 8g, Glycerin- 308, methyl violet- 0.025 ml, distilled water- 180 ml) 4) Gower solution: (Sodium Sulphate 12.5 g. Glacial acetic acid 33.3 ml. Distilled water 100 ml) °) 3. WBC-Diluting Flui ‘Natt-Herrick’s solution (Avian blood) (995 yl Natt-Herricks Solution, contains NaCI 3.88 g/l, Na2SO4 2.5 g/l, Phosphate Buffer, Formalin 37% 7.5 ml/, C.1. 45535 0.1 g/l) 4, Turk’s Solution (Mammalian Blood) Staining Solutions a) Methyl alcohol b)Methylene blue ©) Eosin d) Leishman’s stain €) Modified Wright’s stain ) Gram stain g) Wright’s stain 5. Platelet Diluting Fluid Rees Ecker fluid: (sodium citrate, formaldehyde and brilliant cresyl blue) 6. Anticoagulants: a) Sodium citrate (2g/ml of blood) b) EDTA (2-3g/ml of blood) ©) Heparin d) Oxalate 7. Reticulocyte Count Fluid (New methylene blue or brilliant cresyl blue) 8. Distilled and De-lonized Water Equi inocular with camera “1. Compound/student Microscopes «| 2, Centrifuge Machine a) Macro-hematocrit/clinical centrifuge machine b) Micro-hematocrit centrifuge ‘machine (10000-12000rpm) ‘ 3, Water bath 4, Hemoglobinometer / hemometer. 5. Tube stands for Westergren and Wintrobe methods 6. Slide-staining racks 7. Stop watch 8, Automatic hematological analyzers 9. Student Spirometer 10. Spectrophotometer 11, Pipettes 12. Micropipettes 13, Pipette fillers TA, Glass test tubes 15. Test tube holder 19. BP apparatus Exercise: 1. Mention the ifTerent glass w i i Meare nm of different glass ware, equipment and instruments you observed at 2. Write down the composition and then prepare in i i N/10 HCI solution, 10% NaCI solution and Tus elation? selun, Hayes soiion Experiment: 4 Blood Collection and Preservation Blood Collectio rmed in such a manner that the animal’s health is The collection of blood must be perfor i not compromised. Collection of excessive volumes of blood can lead to severe decreases in blood pressure, shock, and death. Hematocrit are recommended for chronic blood collection protocols slood Collection by Speci Site of Collection - = Cardiac (terminal only), retro-orbital sinus* (anesthetized only), tail vein, Mouse saphenous vein, facial vein, distal tail transection (1-3mm, anesthesia required) Rat Same as mouse Guinea Pig Caras (terminal only), anterior vena cava/subelavian vein, saphenous Gerbil, Hamster __| Jugular vein (anesthesia required), caudal vena cava (anesthesia required, Rabbit Dog/Cat saphenous, and jugular veins ‘Non-human Primate ‘saphenous, femoral, brachial, and jugular veins Ruminants Jugular vein Swine Jugular vein , anterior vena cava (anesthesia recommended), car veins Chicken Brachial wing vein, right jugular vein, cardiac (anesthetized only) ‘Table 4.2 Total Blood Volume and Sample volume for Species of specific body weight (Volume estimates are based on the example body weight) —— Species ial Example Animal —_| Total Blood 10% (mirigy* Weight Volume 15% (Max. Single Mouse _| 72 25 Sample) Rat | 64 a 1.8 mi Old ml [0.18 mi Rabbit_| 56 oe 16 mi 1.2 ml 1.6 ml Cat__[37 aie 168 mi 12.6 ml | 16.8 ml Dog [85 3h 171 mi 12.8ml__| 17.1 ml [Pie es ra 187. 140 mi__ [187 ml For mature, aly animals wit av adequate plans of atifon 2am _[285. Blood Preservatioi 7 Blood clotting happens when blood is taken out of the body. However, blood should be required in liquid form for various hematological tests. For this different anticoagulant are used. The choice of an anticoagulant depends upon the type of hematological test desired and time following collection. Principle Citrates, oxalates, and ethylene diamine tetra acetic acid (EDTA) prevent coagulation by complexing calcium ions which are needed for coagulation. Heparin prevents clotting by complexing with anti-thrombin III, thus potentiating the ability of anti-thrombin IJ! to inhibit the action of thrombin and clotting factors XIla, Xla, IXa and Xa. A. Oxalates: The different salts of oxalates used as anticoagulants are potassium oxalate, sodium oxalate, lithium oxalate and ammonium oxalate. The necessary concentration of ammonium oxalate alone causes swelling of red cells, while potassium oxalate causes shrinkage. Therefore, the mixture of these two in the proportion of 60:40 is used to keep the red cells in their normal state. This mixture, called Heher and Paul's mixture, consists of 1.2 g of ammonium oxalate and 0.8 g of potassium oxalate in 100 ml of distilled water. It is used at the optimum concentration of 2mg/ml of blood. Advantage: 1. Itis cheaper than EDTA. Disadvantages: 2, Platelets tend to be clumped. 3. Blood with oxalates is not useful for the estimation of NPN, blood urea nitrogen (BUN) and electrolytes. B. Ethylene Diamine Tetra Acetic acid (EDTA) It is a chelating agent and is available as disodium and dipotassium salts, however, dipotassium salts are more soluble. It is an excellent anticoagulant, commonly used at the concentration of 0.5-2 mg/ml of blood or as one drop of a 10% solution for 5 ml of blood. Advantages: | 1. It is used for thrombocyte count as clumping of platelets does not occur in collection. 2. Normal staining is not affected. — 3. Blood containing EDTA is suitable for PCV &Hb. estimation and cell count to 24 hours post collection. 4, Suitable for total protein and blood urea nitrogen (BUN) estimations, isadv: 2 1. It is not suitable when blood is to be transfused. 2, Not suitable for estimation of electrolytes. 16 C. Heparin ant found in body. It is available as sodium, calcium and the lithium salt is preferred. The anticoagulant activity ‘concentration is 0.1 to 0.2 mg/ ml of blood: Itis a naterally occurring anticoagul: ammonium salts, but for clinical tests, ranges from 100-150 IU/mg. The optimum Advantages: 1. Itis the best one for blood pH studies. . “ne 2. Lithium salt does not interfere in estimation of calcium, sodium or urea levels in samples. 3. Hemolysis is very unlikely to occur as it does not alter the osmotic pressure. 4, It can be used in blood transfusion. Disadvantages: L. Itis relatively more expensive. 2, Inorganic phosphorus is elevated when heparin is used. 3, It causes clumping of leukocytes. 4. It gives poor stainability of leukocytes in smears. D. Sodium Citrate It can’t be considered as a routine anticoagulant although used for special purposes. It prevents clotting by inactivating calcium ions. It is used at the concentration of 3 mg/ ml of blood. The di-sodium hydrogen citrate is used to produce acid citrate dextrose (ACD), used to preserve the blood for transfusion. Two grams of this citrate and 3gm of dextrose in 120 ml of water are autoclaved and are used in the ratio of I part of ACD and 2 parts of blood. E. Fluoride-Oxalate Mixture Sodium fluoride-potassium oxalate mixture is‘effective in inhibiting glycolytic enzymes it acts both as preservative as well as apticoagulant. A 0.5ml of 2.25% solution, after evaporation, is engugh for 5 ml of blood. Disadvantages: I. Itis poisonous. 2. Itis not used for BUN estifhation with a method using enzyme. 3. Fluoride inhibits SAP and acid phosphatase. Questions: Q. Why EDTA is not used for the estimation of seru Q. What is the use of calcium EDTA? ~ Q. What happens when higher concentration of * Q. Why ammonium oxalates, sodium cit estimation of blood pH? im alkaline phosphatase (SAP)? EDTA is used in PCV? trate or EDTA are not used as anticoagulants for ‘Table 4.3 Types of Vacutainers Order of ‘olor of Stopper ‘Addit Comments! Draw Common Tests 1 Clear No additive Used ONLY as a 2 Blood Culture Bottle Bacterial growth | When a culture is i) medium and activated charcoal ordered along with any ther blood work, the Blood Cultures MUST is drawn first. 3 Yellow with Clear Label ‘Sodium Polyanethol | For Mycobacteria Sulphonate (AEB) blood culture. 4 Royal Blue (With Red Band on Label) No additive For Copper and 5 Light Blue < | Sodium citrate For coagulation studies. 6 Black Glass Sodium citrate For ESR only. Mt 7 Red Clot activator and no | For serum tests, anticoagulant | which cannot be separation tube (SST). such as tests performed by Tissue Typing? 8 Gold Gel separator and | Usually referred to clot activator |as SST. After centrifugation, the gel forms a barrier between the clot and the serum. Dark Green GLASS (With Rubber Sodidin heparin | Forantimany: Stopper) i in | For Amina Adi Dark Green PLASTIC | Sodium heparin ids "0 - and Cytogenetic Light G int Lithium heparin | Usually referred to " agi Green (mint) anticoagulant and | as “PST” (plasma gel separator | separator tube). After entrifugation, the gel forms a barrier between the blood cells and the plasma. 12 Royal Blue (With Blue Band on Label) K, EDTA For Trace Elements. 13 Royal Blue (With Lavender Band on Na; EDTA For Lead. Label) 14 Lavender EDTA For CBC, pre- xo) transfusion testing, Hemoglobin AIC, and anti-rejection potassium oxalate drugs. =7 +. Yellow (With Yellow Banded White Acid citrate dextrose | For Tissue Typing Label) solution ‘A’ and some Flow (ACDA) Cytometry testing. Gray Sodium fluoride and | For Lactate. Types of Vacutainers without Anti-Coagulants Plan tubeno | Piaintube. EDTA Linum [7 Fitonde” ~~ Hepannzed anteoagulant | contamsSST anicoagulart —epann | oxalate syngas Cot forms, gel anticcagulant | | | | 8. a | | | 1 i} | } | S “| 4 dey | ft | j 2 | | | 4 | | I | | + Whole blood | | | + General * General analysis General | + Glucose cAnonal | * Red cell | «Lactate ' | sampling | pids and | Ipoproteins OBSERVATI Exercise: Preserve blood by anticoagulants. 20 Experiment: 5 \ Complete Blood Count (CBC) CBC is a frequently used terminology in the hematology lab. This term was invented by Antony van Lecuwenhoek in 1674. During (1852-1952) the manually performed CBC, were as follows: 1. Hemoglobin determination 2. Erythrocytes count 3. WBC count 4. Blood film examination Later, replaced with automatic counting with the invention of automatic blood cell counter in 1952, Now days many types of analyzers are present that counts the mammalian as well as animal blood cells in short time, making the counting easy and save time. Hematology Analvzer Princip Automatic hematology analyzers utilize one of the basic techniques of flow cytometry: using fluorescent dyes, electrical impedance or light scatter method to differentiate cell types. Using one of these principles, hematology analyzers classify single cell types based on size or shape or > biochemicaV/antigenic configuration. The final blood count result in a laboratory is a result of cell-by-cell examination of the blood routed through the examination chamber. Complete Blood Count Tests Complete Blood Count Includes: 1. Hemoglobin Concentration 2, Hematology Analyzer 3. Total Leukocyte (WBC) Count 4, Total Erythrocyte (RBC) Count 5. Examination of blood film (smear) for: a) Study of Red Blood Cell Morphology b) Platelet Count c) Differential White Blood Cell Count 22 d) Blood Parasites Detection 6. Erythrocyte sedimentation rate (ESR) Complete Hemograt ‘Another imporant terminology used is complete Hemogram, includes the following 1, Complete Blood Count: - . ‘a, Hemoglobin (Hb) Concentration b. Total Leukocyte (White Bleod Cell) Count (WBC) c. Total Erythrocyte Count (RBC) d. Examination of Blood Fim (Smear) 2. Packed Cell Volume (PCV): Relative proportion of RBCs to Blood plasma. 3, Mean Corpuscular Volume (MCV): Average Volume of corpuscle (RBC). 4. Mean Corpuscular Hemoglobin (MCH): Average amount of Hemoglobin in » corpuscle. 5. Mean Corpuscular Hemoglobin Concentration (MCHC): Saturation of RBCs with hemoglobin (ratio of hemoglobin to RBC cytoplasm). 6. Platelet Count: Numerical count of Thrombocyte (platelets). Other Routine Hematological Tests are: Red cell osmotic fragility test Exercise: 1. Define hematology? 2. Enlist other hematological tests? 3. Whatis the composition of blood? 4, What is the role of blood in respiration? 23 Blood Reference Ranges: [Blood Parameters |Dog [Cat [Cow [Horse [Sheep | Goat | Rabbit PCV (%) 35-57 [30-45 [24-46 [27-43 ___[ 27-45 22-38 [33-50 High (g/dL) We 9.8-15.4 | 8-15 10.1=16.1 [9-15 $12 10-17 RBCs (* 10"/AL) is 5.0-10.0 | 5.0-10.0 | 60-104 [ 9-15. ce Reticulocytes (%) 0-1.0 0-06 im ‘Absolute reticulocyte | <0 <60 count (x 10°/jiL) ‘MCV (fL) 66-77 39-55 [40-60 _ [3749 28-40 16-25 | 58-67 ‘MCH (pg) 21.0- W417 [P=17 [13.7182 | 8-12 52-8 _|262 ‘MCHC (g/dL) 2 30-36 | 30-36 | 35.3-39.3 | 31-34 30-36 | 29-37 Platelets (x 107uL) 211-621_| 300-800 | 100-800 | 117-256 | 800-1,100_| 300-600 [ 250-650 MPV (iL) 61-101 [1218 [55-65 [40-60 WBCs (x 10%uL) S041 [55-195 [40-120] 56121 [48 ‘Neutrophils (%) 58-85 [45-64 [15-33 [52-70 | 10-50 (segmented) 29120 [25-125 [0640 [2585 [07-60 («10% uL) Newrophis (7) 3 02 [02 oO 0 (band) (« 10°7uL) 0-045 [003 [oor [oor Lymphocytes (%) $21 27-36 | 62-63 [21-42 | 40-55 (C107 uL) 04-29 [1570 [25-75 [12-51 [2-9 Monocytes (%) 2-10 | 0-5 os [06 06 10°7L) ol-14 [0-09 [009 [007 0-075 Eosinophils (%) 0-9 4 [o20 [07 10 10 [uL) O13 [008 (024 [008 [Or -[Basophils (%) oI oa o2 [02 3 Plasma proteins (g/dL) [60-75 | 60-75 [60-80 [60-85 [67.5 Data on various species compiled and adapted in part from multiple sources, including Latimer KS, Duncan &Prasse's Veterinary Laboratory Medicine: Clinical Pathology, Sth ed., Wiley-Blackwell, 2011; and Weiss DJ, Wardrop KJ, Schalm's Veterinary Hematology, 6th Ed., Wiley-Blackwell, 2010. Reference ranges vary between laboratories. Values provided by the reference laboratory should be used, MCV = Mean Corpuscular Volume MCH = Mean Corpuscular Hemoglobin MCHC = Mean Corpuscular Hemoglobin Concentration MPV = Mean Platelet Volume Experiment: Principles of Hemoc ome Introduction: Hematological diagnostic tests are normally divided into two groups: (a) Chemical tests that are concemed with Hb and other biochemical measurements on blood..cells. (b) Morphological tests include qualitative as well as quantitative tests. The hemocytometry deals with qualitative tests like counting ofblood cells. Principle: 45. The blood is diluted with an appropriate diluent and a specific volume of difitted! blood is placed in a special counting chamber to calculate the number of cells. From thisCount, the number of cells/micro liter of blood is computed. Description of Hemocytometer Ki: i * The kit used to count the blood cells is called hemocytometer kit which consists of following parts: a) Thoma Diluting Pipette for RBCs Tt is a long glass pipette having a bulb at one end. The bulb contains a red cblor bead for mixing of blood with diluent. There are 10 divisions on capillary end with 0.5 and 1,0 points are numbered prominently. Beyond the mixing bulb, a mark ‘101” is numbered. All thé:graduation markings are arbitrary“as they give only the ratio of resultant dilution and docs not‘indicate the actual volume used. If blood is drawn to the 0.5 mark and pipette is filled with diluting fuid up to mark 101, the resultant dilution is 0.5: 101-1 or 1:200. b) Thoma Diluting Pipette for WBCs. ‘y Like Thoma pipette for RBCS, it has also 10 divisions on the capillary end with 0.5 and I marks’ are prominently scored. Beyond the mixing bulb, that contains a white bead, there is another mark, 11, which indicates the level to which diluting fluid is drawn. If blood:is drawn to the 0.5 mark the resultant dilution is 0.5:11-1 or 1:20. : c) Improved Neubauer Counting Chamber Tt consists of a thick glass plate upon which bright lines are present, permitting the accurate measurement of volume. The central platform contains 2-counting chambers separated by a groove. The depth of counting chamber is |/ 10 mm below the two lateral bars upon which cover-glass rests. This gives it an ‘H’ shape. Each ruling area consists of nine primary squares. ‘The four comer primary squares are used for white cell counting (marked W). Each primary square is sub-divided into 16 secondary squares, each measuring | / 16 mm?. The total volume held in all white cell corner (Primary squares) is 0.4 mm®, The central primary square (designated as R) is used for counting RBCs. The central square is divided into 25 secondary squares, each having an area of 1/25 mm?, Each secondary square is further sub divided into 16 tertiary squares (1/400mm?). The 5 secondary squares marked “R” are used for counting RBCs. The total volume held in these 5 secondary squares is 0.02mm. 26 Calea Tee ye is the volume of blood actually measured, “D” is the dilution of the bloog sample, D is depth of the counting chamber and SA is the surface area counted. ‘No. of cells per cubic mm of blood = N/V “N? is the number of cells counted. . Questions: . Q. How much blood is diluted in anemia? 7 Q. Why we use Thoma pipette for WBC” for counting erythrocytes in anemia? Q. Why squeezing of finger gives false counting? Q. Under which power of lens the RBC are counted? . Q. Why we use diluting fluid for counting the blood cells? Q. Why we discard first 3 to 4 drops of mixture before charging the hemocytometer? Q. How will you differentiate between secondary squares of RBCs and WBCs under microscope? Q. How many tertiary squares are present in comer primary square for WBC. Q. What will be dilution if blood is sucked up to mark’ I’ in Thoma pipette for RBCs? Q. How many primary squares are present in one hemocytometer? Q. What is the difference between Neubauer and improved Neubauer counting chambers? Counting of Erythrocytes “” int ieton function of erythrocyte is to serve as & carrier of Hb. The mammalian erythrocyte is a-nuclear. Species differences can be found in the erythrocyte shape. The number of erythrocytes is computed to diagnose the anemia or polycythemia. equi ts: | . , ; ® e emoeytometer kit, microscope, diluting fluid (e.g. Normal saline, Heyem’s solution, Gower's solution, Toisson’ssolution), Veeder hand counter, cover-slip. Technique: A) Filling of Thoma Pipette: 1. Draw the blood in Thoma pipette up to mark’0.5' 2. Wipe the tip of pipette without touching the opening of capillary. 3. Draw the diluting fluid steadily into pipette up to mark 101. 4. Remove the aspirator tubing and mix the blood with diluents either by flicking movement of wrist up-and-down or with a mechanical shaker (Automatic Yankee Shaker) for 45 seconds. B) Charging of Counting Chamber: 1. Place the cover slips in its proper position and examine the assembled instrument for squares under low power of microscope. (10 x) 2, Discard the first 3 or 4 drops of diluted blood and place the tip of the pipette at an open end of the chamber. 3. The fluid enters in the chamber by capillary action. Allow the mixture to settle in the chamber and locate the ruling area under microscope. C) Making the Count: In a routine, enumerate the cells in all the five secondary i ‘ squares. Under a microscope, the erythrocytes appear as small circular cells. Counting is normally started in the upper left of cach square and proceeds as follows. Count first row of tertiary squares to the right, drop down count across to the right and then 29 Calculations: RBCs (per micro liter) = Average count x 200 x 25 * 10 Where 200 = Dilution Factor 25 = Area Multiplication Factor 10 = Depth of Chamber The central grid comprises 25 secondary squares having Imm x Imm area, 0.1 mm deep and 0.1 ul of blood. . Since only 1 (average of 5) out of 25 is counted so multiply your count by 25 Since you have diluted your sample 200 times, so add this value in multiplication factor to get answer in the original concentration. The count you obtained was in 0.1 il, so multiply the count by 10 to express the answer in cubic millimeter. ot 30 Experiment: 8 Counting of Leukocytes Introduction: \ The leukocyte count usually rises well above its normal range in different inflammatory processes and infections. Therefore, counting of the total number of leukocytes is an important clinical measurement, which helps to establish capacity of blood for performing their defense functions. There are also many physiological factors which alter the count, e.y. age, sex, breed, time of collection, exercise etc, Requirements: Veeder root hand counter, « Hemocytometer kit Microscope Turk’s fluid (3 m! of acetic acid + 97 ml of distilled water+ crystal violet Gr aqueous methylene blue stain) + Cover slip Procedure: A) Filling of Thoma Pipette: Draw the blood up to mark 0.5 in the Thoma pipette, Fill the pipette with diluting fluid up to mark 11 on it, thus making a dilution of1:20. B) Making the Count: Focus under low-power dry magnification preferably with condenser removed or lowered. After adjusting light, the leukocytes appear as slightly iridescent round bodies with a very definite outline. (Fig, A & Fig. B) For routine counting, enumerate the cells in 4 comer "W" squares described earlier. Caleulati WBC = Average x 20 x10 Where 20 = dilution factor 10 = depth of chamber No. of WBCs per cubic mm (micro liter) of blood Differential Leukocyte Count Introduction: The DLC are made to determine whether the health of the patient is normal or abnormal, ‘The DLC should be interpreted in relation to the total number of leukocytes present. This is because the finding of an increase in the proportion of one type of WBC may be due to either an increase in absolute number of that type, or a decrease in the absolute number of other types, From both the TLC & DLC, the absolute number of each type of leukocyte can also be calculated e.g. cattle in severe bone-marrow depression may have 96-98% lymphocytes in DLC, One might conclude that animal has marked lymphocytosis and a marked neutropenia. The latter is correct, but lymphocyte number may be normal. On contrary, in some diseases, there is a proportionately equal change in the absolute number of each type of leukocytes. e.g. in canine panleukopenia, the relative proportions of each type remain as normal, although the TLC is markedly reduced. Pri I Differential leukocyte count is rhade for determining the percentage of normal and abnormal leukocytes in the blood. From the percentage obtained in the DLC, the actual number of cells of each type per cubic millimeter of blood can be determined.” : Requirements: Blood sample, anticoagulant, microscope, cover slip, 70% alcohol, Wright's stain, marble cell counter. . ‘The counting of different leukocytes can be carried out as: Preparation of blood smears. Staining of blood smear. Identification of cells under microscope. Counting of different types of leukocytes. vvVV A) Preparation of Blood Smear Slide Method 1. Place { or 2 small drops of blood about 0.5 inches at one end of a slide. 2. Place the second slide (spreader) in the middle of the first slide. Draw the spreader at 25° angle towards the drop of blood. 3. Through capillary action, blood spreads along the lower edge of the spreader 4. Push the spreader to the other end of the other slide uniformly, swift and smooth movement. The blood will follow and form a thin, uniform smear. Let it dry in air and label it. . Add 2-3 drops of methanol for smear fixation and allow to air dry again. sow 37 Cover Slip Method: 1. Hold two cover-slips by their edges with thumb and forefinger of each hand. 2. After placing a small drop on cover-slip, superimpose the second cover-slip diagonally at about 45°. 3. The blood spreads by capillary action between two cover-slips. Then gently and smoothly move the cover-slips apart in a horizontal plane. 4. Dry the both smears in air. B) Staining of The Blood Smears The different staining techniques used are: (i) Romanowsky-staining im > The slide should be clean. Place a small drop of blood, or one side about 1-2 em from one end. > Without delay place a spreader at an angle of 45° from the slide and move it back to make contact with the drop. The drop should spread out quickly along the line of contact of spreader with the slide. The ideal zone to examine the blood film is the areas between tail and body. Place the dried slide on a staining rack. Flood the blood smear with Giemsa stain for 5 minutes, After 5 minutes add distilled water in a gentle stream to float off the stain. Drain water from slide and allow it to dry in air. ©) Identification of Different Types of Leukocytes Inspect the blood smear under low power to observe the distribution of cells and select a Portion of the smear near the thin end where the erythrocytes do not over-lap, Then switch to the oil immersion objective (100X). : The leukocytes are spherical in shape except monocytes which are oval to irregular shaped. The cytoplasm stains blue except neutrophils cytoplasm which is stained light pink. Neutrophils 3-5 lobed nucleus 12-15 um in diameter ‘Small, neutral cytoplasmic granules Eosinophils 2-3 lobed nucleus About 10-12u1m in diameter Reddish, eosinophilic granules in cytoplasm Basophils 2-3 lobed nucleus About 12-15um in diameter Bluish, basophilic granules in cytoplasm Lymphocytes ‘Very large, almost spherical nucleus 10-15pm in diameter : Non-granular cytoplasm Monocytes Horse-shoe shaped nucleus 15-20 ym in diameter Non-granular cytoplasm vvvvvv 38 C) Counting of Different Types of Cells istribution of different types of leukocytes is not even, more cells at the margin San canta aon err gr Method’s also known as Battlement of slide than central area, therefore, Four-Field Meander Me a Count, is usually employed which is as: Start the examination along the outer margin of the smear for about 3 fields, move inward a short distance (1 mm or 3 fields), parallel the margin after 3 fields, then move back to the edge of the smear. Report the procedure for as many times as necessary to identify the required number of cells. After calculating the required number, the percentage of each can be computed by the formula: Percentage of a type = No. of that type x 100 Total no. of WBCs The shape of blood film Precautions: 1. The drop of blood should not be too large. 2. Movement of spreader should be even smooth and rapid, 3, Smear should be of good quality. 4, Gently wash the slide by tap water running over the stain. DO NOT put smear directly under tap water, 5. Do not touch the slide with objective lens while counting cells, Questior Q. Why cover-slip method is preferred over slide method for DLC? Q. Why the counting is carried out at the margin of the slide? Q. What is the effect of age and exercise on DLC? Q. Why a separate fixative is not used in Wright's staining? Q. What is the role of cedar-wood oil in DLC? Q. What is the difference of polymorph nuclear leukocytes of birds and mammalian? 40 , Experiment: 11 Red Blood Cell Osmotic Fragility Test introduction: Cell membrane is a partially permeable barrier where osmotic gradient is established between extracellular and intracellular fluids and maintain flow of water and materials into and out of the cell. The concentration gradient decides the amount of osmotic pressure and the flow of diffusible ions on either side of the membrane. The intracellular environment of red blood cells contains salts, protein, glucose and hemoglobin. A solution of 0.9% NaCl is considered isotonic; when erythrocytes are kept in such a medium, the intracellular and extracellular fluids are said to be in osmotic equilibrium across the plasma membrane and there is no net efflux or influx of water. When cells are placed in a hypertonic environment, for example 1.5% NaCl, there is rapid efflux of water and cells lose normal biconcave shape and, undergo collapse, leading to crenation. Contrariwise when cells are placed in a hypotonic environment, for example 0.5% NaCl, an influx of water occurs, the cells swell up leading to disruption of plasma membrane, escape of hemoglobin into vicinity and hemolysis. . In this experiment, we make use of the property of RBCs that their osmotic fragility is not uniform and the susceptibility of cells to undergo hemolysis is not uniform. The concentration of hemoglobin released in the hypotonic environment gives an index of the degree of osmotic hemolysis. In this experiment your task in to determine the association between the extent of hemolysis and the osmolarity of the environment in which red blood cells are suspended. When subjected to Aypertonic media (e.g. 1.8% NaCl), the cells lose their normal biconcave shape, Purpose: . © Toaid diagnosis of hereditary Spherocytosis & Thalassemia. Procedure: 1. Prepare six labelled test tubes containing 10 ml of 0.9%, 0.6%, 0.5%, 0.4%, 0.3% and 0.0%NaC! solutions 1 2 3 4 5 6 2. Add 0.1 ml of blood in each tube and mix gently to obtain a uniform suspension of RBCs. . 42 » 9 ‘The tubes are capped and inverted 1-2 times and allowed to stand for 15 minutes, : 5 00 rpm. : Centrifuge the test tubes for 10 minutes at 50 5. Transfer | mil of supernatant in spectrophotometer cuvettes and measure absorbance values at 540 nm. (Take first reading for blank tube, containing’ distilled water only) BY At this point, generate a calibration curve.100% hemolysis occurs in 0% NaCl tube and 0 hemolysis in tube 1 (isotonic to cells). Draw a linear relationship plot between % hemolysis and OD values. Calculate the percent hemolysis for each sample from the calibration curve. Enter the OD values for each sample and get the corresponding% hemolysis from the curve. Osmolarity is dependent upon the number of impermeant molecules in a solution, not on How to Calculate Osmolarity? IM solution of NaCl: 58.5 g/L the Weniy ofthe molecules, For example, a solution of a non-ionizing substance suct 09 BH NaCleomesponds10 9 siLof NaCl | 3°" Facose is m losmolir soluion: «IM Tube [NaCl [NaC ——Osmotarny | solution of NaCi=2 Osmoles (The NaCl salt gl00mL | g/L | osmoleet, || Particle dissociates fully in water to become 109 7 7 two ,separate particles: a Na"ion and a ee Z Clin. Therefore, each mole of NaCl 3—58 7 a becomes two osmoles in solution, one mole of i—9 2 Tort Na’ and one mole of CI) sts $ at Osmolarity in current situation is calculated as 6 0 7 0 €.g, for 0.3 % NaCl ) Molarity of NaCl = No. of Moles/Liter Where Moles = Amount of substance in Grams/ Molecular Weight 2) Osmolarity of NaCl = 2* Molarity of NaCl ity test is designed to esti ; survive increasing internal pressures me ntait the capacity of the red cells membrane to 's brought about by the diffusion of water into the cell. 43 _ Normal Ran: The following is a sample set of reference values: 0150 g/100mL NaCl (Unincubated): 09%-47.8% +60 g/100mL NaCl (Incubated): 18.7%-67.4% +65 g/l00mL NaCl (Incubated): 4.4%-36.6% | Hemolysis Hemolysis lemolysis {075 g/100mL NaCl (Incubated): 0.896-9.1% Hemolysis Tube | NaCl e/100mL | Nat Osmolarity | Hemolysis ea, Osmotes/1, T 09 9 03 0 0 r z 06 6 02 10 0.05 3 05 5 0.17 90. 0.45 4 04 4 0.14 96 0.48 5 03 3 O1 98 0.49 E 6 0 0 0 100 05 4 Experiment; 12 Determination of ABO Blood Groups & Rh Type Introduction: 7 : A 'SO present in the serum e.g. The blood group A human contains antibody against antigen B (antibody-t) The blood a B human contains antibody against antigen A (antibody-a). The blood group AB will neither contain antibody-a nor antibody- b while blood group “O” contains both types of antibodies, The work of Levine and Stetson (1939) and Landsteiner and Weiner (1940) led to the discovery of another important antigen called antigen D, on the RBCs. This antigen, also called Rh, occurs in 85% of Caucasian population while 15% are negative to this. Its name, Rh, was derived from the fact that it was first discovered in Rhesus monkey. In addition to this, there are also other blood group systems like MNS, Kell, Lewis, Duffy, Kidd, Diego etc. In animals, the blood group antigens are more complex than man. e.g. the major recognized blood group systems in cows are 12, sheep 7, horses 9, pig 16, dog 7, cat 2, minks 5, Rhesus monkey 6, rat 4, mouse (C 57)4, chicken I and rabbit 4. The blood groups are typed to aid in cross-matching donor's blood with the recipient's blood in transfusion, also aids tor identify the breeding pairs potentially at risk of hemolytic diseases. This * system can also be used to settle the disputes of parentage. ‘The identification of blood group antigen can be carried by either slide method or test tube method. Slide Method: Principle: / The RBCs agglutinate if they are mixed with antibodies (antisera) corresponding to the ‘antigen(s) present on erythrocytes, . Requirements: . . i Slides, sterilized lancet, 70% alcohol, mixing match sticks, antisera A and B, antiserum Rh. (antiscrum D). 1. Using a wax pencil, draw slide (the circles prevent bl 2 der one circle and Bunderthe other. ; 3. Place one eo enti

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