Final record


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Final record

  1. 1. Name:__________________________ Reg no:__________________________ Semester:_______________________ Year:____________________________
  2. 2. SAPTHAGIRI INSTITUTE OF MEDICAL SCIENCES & RESEARCH CENTER NO.15,CHIKKASANDRA, HESARAGATTA MAIN ROAD, Bangalore-90 DEPARTMENT OF PATHOLOGY CERTIFICATE This is to certify that this is the bonafide Pathology Practical Record of Mr/Ms/Miss _________________________________________ He/ She has satisfactorily Completed the Pathology Practical as prescribed by Rajiv Gandhi University Of Health Sciences, Bangalore during the year 201 - 201 His / Her University Registration No. is_____________________________________ Lecturer/Demonstrator Professor and Head Department of Pathology Date________________
  3. 3. INSTRUCTION TO STUDENTS 1. Students should come to the practical class with buttoned apron and long hair should either be tied up or covered by apron. 2. Students will sit in the serial order of their roll numbers and shall work in small groups allotted under one member of the teaching staff. 3. Students should bring with them their practical record books and also either the class notes, or the text books. Protocols for practical classes are announced in advance in each term. 4. Students should come prepared with theoretical knowledge of practical exercise of the day. 5. Students must possess lead-pencil and variety of coloured pencils. 6. Before starting the day’s work every student must check up the microscope given to him; clean the eye piece and objectives and adjust the tube length, condenser, diaphragm and mirror correctly. After the work the lenses should be cleaned, particularly the oil immersion, and see that the lowest objectives is left in alignment. Any defect found should immediately be reported to the teaching staff. 7. Sketches and diagrams should be neatly drawn in the same colours as seen in the microscope filed, paying attention to relative size of cells etc., 8. As there is little time for repetition of practical work, the two hours should be fully utilized by repeating exercises several times and drawing as many diagrams as possible. 9. Hands contaminated with infected material should immediately be washed with antiseptic solution and soap provided. Contamination of work tables should immediately be reported. 10. Every student should spend the major time in the given class work and the reminder can be spent by turns on the demonstration arranged at the side table. 11. Every student should show his day’s work including, diagrams in the practical record book and must get them signed by the teacher with date, before he can get his attendance for the day’s practical class. It is student’s responsibility to earn the required percentage of attendance. 12. Loud talking and unnecessary movement of students from place to place should be avoided. 13. At the end of this session the record book completed in their own handwriting should be submitted for scrutiny and certification by the Professor for having completed the course in pathology.
  9. 9. PART – I GENERAL
  10. 10. ANTONY VAN LEEUWENHOEK (1632 – 1723) Antony Van Leeuwenhoek (Pronounced Layu-wen-hook) was born on October 24, 1632 in Delft, Holland. Leeuwenhoek received only an elementary education. After reading the book, Micrographia, he became interested in microscopes and made over 500 microscopes. In 1674, he made the first observation of microbes, becoming one of the greatest seminal discoveries in history. He described many types of bacteria and protozoa and calculated their sizes. In 1677, he became the first person to describe spermatozoa and was one of the earliest to describe red blood corpuscles. In 1680, he was elected a fellow of the Royal Society of England and was also a corresponding member of the Academy of Sciences in Paris. Antony a Leeuwenhoek died on August 30, 1723.
  11. 11. ORGANISATION AND FUNCTIONING OF THE DIAGNOSTIC LABORATORY Of better functioning of laboratory it is organized in to sub divisions 1) Histopathology; it deals with the study of tissue removed from the living body which includes study of tissue by paraffin embedding technique and by frozen section for rapid diagnosis. This technique includes study of structural changes observed by naked eye i.e gross or macroscopic changes. The changes detected by light microscopy and electron microscopy are supported by numerous special staining methods which includes histochemical and immunological techniques, to arrive at most accurate diagnosis. 2) Cytopathology-it includes study of cells shed off from the lesion-exfoliative cytology and fine needle aspiration cytology (FNAC) of superficial and deep seated lesions for diagnosis. 3) Haematology-deals with disease of the blood, work with whole blood to do full blood counts and blood films as well as many other specialsed tests. 4) Clinical pathology-analysis of biochemical constituents of blood, urine, semen, C.S.F, and other body fluids. 5) Immunology-Detection of abnormalities of immune system of body comprises immunopathology. 6) Molecular pathology-detection and diagnosis of abnormalities at the level of DNA of a cell, is included in molecular pathology. EXERCISE-VISIT TO CENTRAL LABORATORY
  13. 13. THE MICROSCOPE Microscope is essentially an optical instrument used for magnifying minute objects in order to study the details of the structure. The simplest form of a microscope consists of a single convex lens mounted onto a ring. The lens is held at an appropriate distance from the object and thus magnification is brought out. Usually a magnification of 10x is obtained. The compound microscope, which is widely used in the laboratory, consists of the eye pieces, objectives and a graduated tube which holds them in proper separation. The instrument is focused by a rack and pinion with both the coarse and fine adjustments. The tube is mounted on a stand with a mechanical stage to support the specimen. There is a mechanism under the stage consisting of a condenser, an iris diaphragm and a mirror for receiving and regulating the amount of light from a particular source. Objectives are usually three in number, and they are designated according to their focal lengths as under: 1. 16 mm.or2/3” - Low power 2. 4 mm. or 1/6” - High power – dry 3. 2 mm. or 1/12” - High power – oil immersion The eye-pieces usually give 5x to 15x magnification. Magnification of the microscope depends on. (1) The focal length of the objective. (2)Magnifying power of the eye piece and (3) distance between the objective and the image produced. Optical tube length is usually kept at 160 mm. Thus the total magnification can be calculated by a formula: Optical tube length (160 mm) X Eye piece magnification Focal length of the objective Oil Immersion objectives: In case of oil immersion objective, the space between the outer lens of the objective and the object is filled with and oil like cedar wood oil which has the same refractive index as that of glass (1.51). The rays of light do not undergo refraction but pass into the objective giving a bright, clear image. For critical study of bacteria, blood cells etc., where in high magnification and resolution is required, the oil immersion lens is of much help. Definition is the capacity of the objective to render the image distinct, eliminating chromatic and spherical aberration. Spherical Aberration: The rays passing through the periphery of the lens focus on the axis at a shorter distance than those passing through the central portion of the lens with the result that the image is distorted. Chromatic aberration: It is caused by splitting of a white ray into its component colour when it refracts through lens the violet-blue rays focusing nearest and red rays farthest from the lens rendering the image fringed with colours.
  14. 14. Both these aberrations can be corrected by the combination of lenses of different dispersive powers. Resolution is the ability of an objective to distinguish minute structures. Limit of resolution is a minimum distance between two points an objective can form separate images of and this interval with the visible spectrum is about 0.2 µ. Resolving Power of an objective depends upon the wave length of the light used for illumination. The shorter the wave length the greater is the resolution. Ultra violet rays increase the resolution and although the image is invisible, it can be photographed by fluorescence. Numerical Aperture indicates the amount of light which enters an objective from a point in the microscope field. It is defined as the ratio of the diameter of the lens to the focal lengths and is expressed as: NA = n sine µ Where ‘n’ is refractive index of the medium, 2 µ= angle of aperture or angle formed by the two extreme rays of light which starting from the centre of the objective reach the eye. Thus ‘n’ being constant, NA depends upon half the diameter of the lens. The theoretical limit of 2µ is 180 in case of a dry objective where the refractive index of air is ‘1’ and NA 1 sine 900=1. When oil is used, n = 1.5 X sine 900 = 1.5 In general practice, 16 mm, objective should have N.A. of 0.25: 4 mm. Objective N.A. of 0.65; and 2 mm. Objective N.A of 130. Condenser is used for focusing light on the object. The commonly used type condenser consists of a system of convex lenses. Lowering of the condenser diminishes light where as raising increases. The Iris Diaphragm: Controls the angle of light which passes into the condenser. Mirror: The plane side of the mirror is used along with condenser and concave without condenser. METHODS OF ILLUMINATION 1. Transmitted illumination or `direct illumination: The object and its structural details will be visible if it is coloured naturally or artificially stained. 2. Reflected illumination: When examining opaque specimens the light is thrown onto the specimen from above. The light reflected from the specimen enters the objective. 3. Dark Ground illumination: In case of living bacteria, spirochete etc., which are almost invisible when examined by transmitted light, dark field illumination, is used. By means of a special type of condenser, the specimen is illuminated from below by oblique
  15. 15. light. Hence, only the rays that are reflected from the object enter the objective. As a result, the organisms appear bright in a dark back ground. CARE AND USE OF MICROSCOPE Clean the microscope with a clean soft cloth. The objectives and the eye piece must be cleaned with a lens paper and xylol. Alcohol should not be used as it dissolves the cement that binds the lenses. Set up the microscope in a convenient position, facing the source of light. Place the object on the stage and adjust the mirror to the illuminant. Always try to focus the blue sky if artificial light is not available. Direct sunlight should not fall one the mirror. For prolonged work it is advisable to use artificial light of a particular wave length. Adjust the various parts as follows: 1. For Unstained Preparations : a) Lower the condenser. b) Close the iris diaphragm c) Use concave mirror. d) Focus under low power and then turn on to high dry power. 2. For stained preparations : a) Lower the condenser. b) Use concave Mirror. c) Adjust the iris diaphragm to give an even illumination of the microscopic field. 3. For oil immersion examinations : a) Raise the condenser completely. b) Open the iris diaphragm. c) Use plane mirror. d) Study the object under low power first and then place a drop of the cedar wood oil on the object and turn on the oil immersion lens. Rack the objectives down until the tip of the objectives dips in the oil. Then using the fine adjustment focus the object. e) After the use remove the oil from the objectives and the object with a cloth dipped in xylol. f) Turn the nose piece until the low power objectives is in position. Eye Pieces: 1. Simple eye pieces with an eye lens at one end and field lens at the other. The latter collects the image and throws into the focal length of the eye lens in order to get a virtual image of the object. 2. Demonstration eye pieces in which a small hair or bristle is incorporated which acts a pointer, and it is used to point out particular cells in the field. 3. Double Demonstration eye pieces: In this, ordinary eye piece is attached with a side tube at the end of which there us another lens. Parts of the rays
  16. 16. of light that fall on the eye piece are diverted through a prism into the side tube, so that another observer can see the field at the same time. The side tube is provided with an adjustable pointer. 4. Micrometer eye piece: In this, a small, circular glass disc on which graduations in microns is made is kept between the eye lens and the field lens. The size of the object can be measured directly using this scale. OTHER TYPES OF MICROSCOPES I. DARK GROUND MICROSCOPE: PRINCIPLE: The light is allowed to fall on the object in an oblique direction so that it gets reflected from the object and thus enters and objective. This microscope is used to study delicate organisms like spirochetes which are not visualized by the ordinary compound microscope. It has special types of condenser which does not allow the light to pass through the central portion but the light is reflected with the help of the mirrored surface and emitted out at an objective angle. Where there is no object in their path these rays pass out of the objective. If a reflective object is introduced, viz., spirochetes, in their path, those rays that are reflected by the object only will pass into the objective and thus they are seen as bright objects against dark back ground. USE: To demonstrate spirochetes. II. PHASE CONTRAST MICROSCOPE: PRINCIPLE: Light is assumed to be due to wave motion, to the upward and downward vibration of other particles. They move at right angle to the direction of the light. Since there is supposed to be a homogenous medium, the particles can move in any direction at right angles to the path of the ray. But when the light rays pass through a Nicol’s prism it becomes polarized and the direction in which the particles move is known as the ‘optical path’. Now if another prism is introduced in the path of the ray, it will pass unchanged provided the optical path of the second prism is in alignment with the former and if the second prism is rotated through 900, so that the optical paths are crossed, the ray will be totally reflected out of the second prism. Thus no light will pass through crossed prisms. In the polarizing microscope two prisms are used, one below the condenser and the other above the objective. When both are crossed no light will pass through the upper. Now, if a birefringent object, such as cholesterol crystals, be placed at the object plane, those light rays that rays that pass through these crystals will pass through the upper prism because the prisms are no longer crossed from these rays. The rays that do not pass through these crystals are absorbed by the upper prism. Thus the crystals will be seen as bright object against a black background. USE: To visualize the birefringent objects such as cholesterol crystals in atherosclerosis.
  17. 17. III. POLARISING MICROSCOPE : PRINCIPLE: Light waves differ from one another in their (1) amplitude (2) wave length & (3) phase. The phase difference cannot be made out by the eye. If by some means, the phase difference could be converted into difference of amplitude, different tissue components in an unstained preparation could be made to appear of different degrees of brightness and so be distinguished easily from one another. This aim may be achieved in a phase contrast microscope by introducing a phase retarding a phase retarding plate in the objective. USE: To study the living tissue in an unstained preparation. IV. FLOURESCENT MICROSCOPE : PRINCIPLE: Since the visible light is too great a probe to see minute particles, ultraviolet rays which have shorter wave-length are utilized in this microscope. The resolution power of this microscope will be accordingly increased. But since the ultraviolet rays are not visible to the human eyes, fluorescent screen is placed at the eye piece, which when struck by ultraviolet rays will glow and thus the magnified image of the object is visualized. The resolution power of this microscope will be between 0.1u, and 0.2u. USE: To study inherent or induced fluorescence in tissues for various clinical purposes. V. ELECTRON MICROSCOPE: Since the light microscope have a limited resolution power, greater magnifications to yield more cell details are not possible with these microscopes. The electron microscope uses the electron as the probe, which has a very small wavelength, and thus the resolution power of the microscope will be greatly increased. Construction: Here the lenses are made up of electromagnetic fields whose strengths can be varied. The object to be examined should be ultra-thin, since electrons have a poor penetration power. The electrons that are generated at the tip of the cathode tube will be converged by means of an electromagnetic field (condenser) on the object. The objective E.M. field will produce an enlarged image of the object, which is either photographed or visualized by using a fluorescent screen. Those parts of the tissue that are electron dense, will allow less number of electrons to pass through and correspondingly a less change in the photo graphic plate and vice-versa. When the positive print of the photographic plate is made, the electron denser objects appear as dark bodies and electron-rare objects as lighter objects.
  18. 18. USE: Since the resolution power of the microscope is extremely great, ultra microscopic objects like viruses can be seen with great ease. The cellular structures are also studied in greater detail in infections and neoplastic diseases.
  19. 19. RAPID DIAGNOSTIC METHOD The clinical significance of many micro organisms has urged scientist to find out the accurate and rapid diagnostic methods to identity them. The rapid diagnostic method must be accurate, simple, quick and affordable for the population for which it is needed. It must also provide a result in time to institute effective control measures and particularly treatment. The rapid diagnostic method tests are 1) Urine reagent strip test – for Glucose, Ketone, Blood, Protein, PH & Urobilinogen 2) Pregnancy test – HCG, Combo, Urine/serum rapid strip test. 3) Blood Glucose Level – Glucometer 4) Infection disease – a) HIV rapid serum cord test b) Dengue NSI rapid strip test c) Malaria rapid strip test d) HBV & HCV rapid test serum rapicord Etc… AUTOANALYSER An autoanalyser is an instruments/machine with which a large number of samples can be analysed daily by partial or complete automation. Automated syrings deliver fixed quantities of raegents to a series of samples or aliquotes. Dilution, dialysis, separation of interfering substances and also treatment with appropriate reagents to bring about the colour reaction are all done by the machine.The colorimetric measurements are also carrried out automatically and the results are recorded in a computer. Advantages; (a) Large number of samples can be examined on a very short period. (b) Personal errors are minimised. (c) A wide number of estimation can be done with one instrument. (d) Small quantity of sample is required. (e) Most suitable for laboratories handling large number of samples daily. Disadvantages: (a) The cost of the equipment is high, so not suitable for small or medium sized laboratories handling a limited number of samples . (b) Skilled persons are required to operate the machine.
  21. 21. MAXWELL M.WINTROBE (1901-1986) Maxwell Wintrobe is a name almost synonymous with hematology. He has taught, diagnosed, researched and wrote about hematological disorders for over five decades. His name goes with the tube used for finding out erythrocyte sedimentation rate. He was born in Halifax, Canada & trained in Canada and the United States. He has served as a faculty member in prestigious institutes such as John Hopkins University, University of Utah and Salt Lake County General Hospital. He was also one of the first to pioneer treatment of cancer through chemotherapy.
  22. 22. METHODS OF COLLECTION OF BLOOD The blood should preferably be collected early in the morning before the patient has eaten, and stored in a clean and sterile container in order to avoid bacterial contamination. A. Capillary Blood: It is collected by pricking the finger tip or ear lobe in adults and heel or great toe in children. Sterile needles, disposable lancets, spring lancet, B.P. knife blades or capillary glass pricker can be used for pricking. The part should be sterilized with spirit and the surrounding area gently pressed to produce venostasis. A free flowing, large drop of blood should be obtained. The capillary blood is used for the following tests: 1. Estimation of Hb% and R.B.C., W.B.C and Platelet counts; 2. Preparation of peripheral smears. 3. Blood grouping. 4. Estimation of blood glucose level by Somogyi’s micro-method. B. Venous Blood: This is collected by using a dry, sterile syringe and needle. Anti cubital vein in adults and external jugular vein in children are the usual sites of veni-puncture. Blood should be drawn. In order to minimize haemolysis, a clean puncture is essential. Blood should be stored in a chemically clean container with or without the anticoagulant as the case may be. Venous blood in necessary for the following tests: 1. Estimation of E.S.R., P,C,V. etc., 2. Estimation of blood constituents like sugar, urea etc; 3. Bacteriological and serological examinations; 4. Blood grouping and cross matching. C. Arterial Blood: Sometimes it may be impossible to collect blood from veins. In such case arterial punctures can be attempted. Brachial artery and radial artery are the usual sites. Sometimes arterial blood gives positive culture results when venous blood is negative in case of sub acute bacterial endocarditic. Arterial blood is collected most importantly for blood gas level studies. ANTICOAGULANTS: The blood has to be kept in the fluid state for many of the hematological and chemical examinations. In order to achieve this, anticoagulants have to be added in appropriate proportions. Following are the various anticoagulants commonly used:
  23. 23. A. Oxalates: These salts combine with the calcium in the blood to form insoluble compounds and thereby deplete the blood of its calcium which is necessary for the coagulation of blood. 1. Dried Potassium Oxalate: For 10 ml of blood, 1 ml of 2% solution of potassium oxalate dried in a container for blood at 800 C. Disadvantages-causes shrinkage and destruction of cells. 2. Lithium Oxalate: Used in case of estimation of blood constituents since is does not introduce an element which is tested in the blood. 2 mg of dried salt is sufficient for 1 ml. of blood. 3. Double Oxalate or Wintrobe’s Salt: Combination of Ammonium and Potassium oxalates (1.2 g and 8 g of these salts respectively and distilled water 100 ml.) is used. 1ml of this solution dried in a container at 800c is sufficient for 10ml of blood. Advantages: Ammonium oxalate causes swelling of the cells where as potassium oxalate shrinks. The action of both these salts is counterbalanced and thereby the cells retain their original shape and size Uses: Used in the estimation of E.S.R by Wintrobe’s method and P.C.V. 4. Saturated Potassium Oxalate: It is a 20% soln. of Potassium oxalate. One drop of the supernatant solution is sufficient for 10ml of blood. It mixes more readily with blood than dry salt. B. Citrates: 1. Sodium Citrate: 1 part of 3.8% solution of the salt and 9 parts of blood mixed for coagulation studies. Used in blood transfusion as the salt is relatively non toxic and is excreted by the kidneys or utilized by the body. 3.8% solution in 1:4 ration with blood is used in ESR (Westergren’s method). 2. Acid Citrate Dextrose Soln. (ACD): Erythrocytes are better preserved in the ACD than in Tri-Sodium Citrate alone. Tri-Sodium Citrate 1.32 g Citric Acid 0.42 g Dextrose 1.40 g Dist. Water add to 100 ml 1 ml of ACD is sufficient for 4 ml of blood. This is used in blood transfusion. C. Heparin: It has an affinity for blood proteins and acts as an antithrombin and antithromboplastin. 1 mg of dry heparin or 1000 I.U. of liquid Heparin suffices for
  24. 24. 10ml of blood. Used in haematocrit studies and blood transfusion especially exchange and rapid transfusion as in case of thoracic surgery; E.S.R and fragility tests. It keeps for only three days. D. Ethylene Diamine Tetra Acetate (EDTA): Its di-sodium and di-potassium salts are used. They act as chelating agent and thus separate the calcium ions from the blood. Used for most of the hematological and chemical tests except for the estimation of N.P.N and electrolytes. DETERMINATION OF HAEMOGLOBIN PERCENTAGE’ (Sahli’s Hellige Method) Sahli’s Haemoglobinometer Consists of (1) Comparator-Which consists of a rack with standard fixed in front of ground glass. (2) A graduated tube with markings in gms (2-22gms) and in percentage (10-160%) (3) Haemoglobinometer Pipette – glass pipette with 20cu mm capacity. Used to pipette blood for hemoglobin estimation. Used: For Hemoglobin estimation. Normal Range - Males 14-16g% Female’s 12-14g% Methods used for Hb estimation Sahli’s Acid Haematin Method Procedure: 20µ 1(0.02ml) of blood is placed in a specially calibrated tube containing N/10 HCI, to the 20 mark. Let the mixture stand at room temperature for 10 minutes. During which time haemoglobin is converted to acid haematin. The solution is diluted with distilled water until the colour matches exactly with the standard of the comparator block. The percentage of Hemoglobin is read directly from the calibration on the tube in which the solution is diluted.
  25. 25. Advantages: (1) Simple Method (2) Small quantity of blood is needed. (3) No sophisticated equipment is needed. (4) Can be repeated often Disadvantages: (1) Visual Error (2) Other forms of Hb cannot be estimated (sulfhaemoglobin, Methaemoglobin) (3) Fading of the standard-false reading (4) Time for 100% conversion takes 30 minutes. (5) Affected by hyperbillirubinemia. Other methods 1) Alkali haematin method 2) Hb electrophoresis 3) Photo – electric colorimeter method – using cyanmethaemoglobin. 4) Spectroscopic method. Conditions in which Hemoglobin is increased 1) Polycythemia Vera 2) Polycythemia secondary to hypoxia dehydration Conditions in which Hemoglobin is decreased 1) Anaemias 2) Haemorrhagic disorders
  26. 26. HAEMATOLOGICAL INDICES The Colour Index The colour index compares the mean haemoglobin content of one erythrocyte with that of a normal erythrocyte which is taken as unit. In a normal individual both haemoglobin level and R.B.C count are 100% of normal. Therefore the colour index is 1.0. A positive colour index greater than 1.0 indicates that each erythrocyte contains more than the normal amount of haemoglobin, and a negative colour index less than 1.0 indicates lowered heamoglobin content per cell. Colour Index = Hb% of normal Erythrocytes% of normal Normal Hb% - 14.5g Normal R. B.C Count – 5 millions / cu mm Calculation of ‘absolute values’ for mean cell size, Heamoglobin concentration: 1. Mean corpuscular haemoglobin gives the average weight of haemoglobin in each red cell in picograms M.C.H. = Hb grams per 1000ml of blood R.B.C. count millions per cu. mm of blood Normal Value: 27 to 32 pg (Picogram) (MCH: decreased in iron deficiency anemia) 2. Mean Corpuscular volume is the mean value for the corpuscular volume as determined by the haematocrit value and is recorded in Femto litres. M.C.V. = Volume of packed erythrocytes per 1000 ml R.B.C.count millions per of blood Normal Value: 80 to 94 fl (Femto liters) MCV: Increased in all macrocytic anaemias and decreased in hypochromic anaemias. 3. Mean corpuscular haemoglobin concentrations given the average concentration of haemoglobin in the erythrocyte expressed in percentage. M.C.H.C. = Hb grams per 1000ml of blood x 100 Volume of packed R.B.C per 100 ml of blood Normal Value: 28 – 32% MCHC: is either normal of decreased but not increased.
  27. 27. ESTIMATION OF ERYTHROCYTE SEDIMENTATION RATE (ESR) WINTROBE OR HAEMATOCRIT TUBE It is a thick walled tube with narrow lumen, one end of the tube is closed. Total length is 11 cm, diameter 2.5 mm. The tube is graduated from 0-100 mm. Markings on one side are above downward and on other side below upwards. The below upward markings are used for haematocrit and above downward for ESR. Anticoagulant used is double oxalate, dry ammonium oxalate and potassium oxalate mixture (2mg/ml). Special pipette, Wintrobe’s pipette or Pasteur pipette. Procedure for Haematocrit / PCV: Blood is filled in the pipette and tip of the pipette is introduced into haematocrit tube touching the bottom and blood is slowly released in the tube, no air bubble should be allowed to remain in the tube. Tube should be filled up to the upper mark. This is centrifuged for 30 mins at 3000 revolutions per min and column of packed RBC is recorded. Uses 1) For PCV 2) ESR A. Wintrobe’s Method of ESR The E.S.R. denotes the velocity of sedimentation of R.B.C. per unit of time and is expressed in mm at the end of one hour. 1. Wintrobe’s Method: Deliver 5 ml of venous blood into a tube containing 10 mg of dry potassium and ammonium oxalate. Mix the samples well and fill a Wintrobe’s haematocrit tube to the 100 mm mark by means of a capillary pipette. Place the tube in exactly vertical position and observe the point on the scale to which the red cells fall during one hour = ESR. Centrifuge the tube for 30 minutes at 3000 r.p.m. and read the volume of packed cells. Correct the ESR for anemia using Wintrobe’s chart. Normal Range: Men : 0 to 6.5 mm in one hour Women : 0 to 15 mm in one hour B. Different layers observed in Haematocrit Tube 1. Uppermost clear fluid i.e. plasma layer-colour is yellow in jaundice – brown in intravascular haemolysis – milky in the case of increased lipids. 2. Just below it is thin layer of platelets. 3. Next to platelet layer which looks buffy is WBC.
  28. 28. The thickness of buffy coat gives an idea of WBC (1 MM = 1000/ Buffy coat smear is diagnostic in: 1) Aleukemic Leukemia 2) Kala – Azar and Trypanosomiasis 3) LE Cells 4) Myeloma 5) Nucleated RBC Colour of the Plasma 1. Orange, green or yellow colour suggest-increased bilirubin 2. Pink or red suggest – Haemolysis 3. Cloudy / Opalescent suggest – nephrosis or abnormal hyperglobulinemia C. Westergren’s Pipette 1. Length of the pipette – 300 mm (30 cm open at both ends. The lower 20 cms are marked from 0 – 200mm. Diameter – 2.5mm. 2. Uses: 1. Estimation of ESR 3. Anticoagulants Used: 1) Sodium citrate 3.8% 0.4ml with 1.6 ml of venous blood. 2) If EDTA is used, the blood must be diluted with trisodium citrate prior to testing (4 volumes to 1 vol.citrate) 4. Procedure: the anticoagulated blood is sucked up to the mark 0 and placed vertically in a Westergren’ stand. The reading is taken of the end of the column of sedimenting red cells. Normal Ranges: Sex Male 3-5 mm/1 hour Women 4-7 mm/1 hour Advantages: Most sensitive ESR method for serial study of chronic diseases e.g tuberculosis.
  29. 29. Disadvantages: 1. Large amount of blood is needed. 2. Involves dilution when collected in EDTA. Precautions: 1. The tube should be grease free. 2. Vertically placed in the rack. 3. Should be placed on a non-vibrant surface. 4. Should not be exposed to sunlight and heat. Diseases in which ESR is raised: 1. Tuberculosis 2. Multiple myeloma 3. Rheumatoid arthritis 4. Collagen vascular diseases 5. Anemia’s 6. Renal insufficiency Diseases in which ESR is decreased: Polycythemia Normal PCV : 45% + 2 Children : 35 – 47% Other Method : Microhaematocrit method – Heparinised capillary tube centrifuged and compared with the graph. PCV is used to estimate severity of anemia and to calculate absolute value to classify anaemia as microcytic, macrocytic and normocytic. PCV is decreased in : Anaemias PCV is increased in : Polycythemia Dehydration Burns
  30. 30. THE RETICULOCYTE COUNT Supravital staining Brilliant cresyl blue. – 0.15 g. Citrate saline solution -100 ml. (1 part of 3% sodium citrate + 4 parts of 0.85 sodium chloride). BrillIant cresyl blue in citrate saline solution is delivered in 1 ml amounts into 80 x 1 mm tubes (Kahn Tubes). Two to three drops of blood are added to each and mixed with the diluent. The tubes are corked and allowed to stand for 10 to 15 minutes. Then the tubes are centrifuged for 2 minutes, at 1000 revolutions per minute. The supernatant fluid is removed by pipette leaving a volume of fluid about twice the volume of cells below. The cells and supernatant fluid are well mixed and a drop taken on a clean slide and a thin film is drawn. Number of reticulocytes per 1000 R.B.C’s may be counted in the unfixed film or after fixation in methyl alcohol for 3 minutes and subsequent staining with 1% aqueous methylene blue for 30 seconds. Alternatively the blood films may be stained by Leishman’s method. This is done if permanent preparation is necessary. To facilitate counting, a circular piece of thin card board or paper with a square slit cut in the centre may be attached to the eye piece. Count about 500 cells in the unfixed film and calculate the percentage of reticulocytes in the given sample of blood. Normal Values: Infants at birth 2.0-6% Children upto 5 years 0.2-5% Adults 0.2-2% Interpretation: Reticulocyte counts are low in ineffective erythropoiesis e.g.,myseloscelerosis, aplastic anemia, megaloblastic anaemia, thalassemia, erythroleukemia and sideroblastic anaemia. Reticulocytosis occurs after blood loss or effective therapy for certain kinds of anaemia, e.g., therapy of iron deficiency or megaloblastic marcocytic anaemias. Reticylocytosis occours in hemolytic anaemias. An increase of 15% to 20% of reticulocyte count is considered as index of therapeutic efficiency.
  31. 31. TOTAL RED CELL COUNT Instruments used: a) R.B.C. Pipette b) Haemocytometer R.B.C. Pipette 1. Consists of a pipette with bulb & a red bead in it. Red bead helps in mixing. 2. Markings on the pipette are 0.5 and 1.0. Marking on the stem above bulb is 101. 3. Rubber tubing attached to the pipette should be thick walled to resist collapse during suction & should be long enough (at least 10 inches) to permit easy reading. 4. Blood is drawn upto 0.5 mark and R.B.C diluting fluid up to 101 marks. (1 in 200 dilution). 5. Constituents of R.B.C. diluting fluid. a) Dacie’s Formal Citrate 40% formalin – 10ml made upto 1 liter with 32g/l trisodium citrate. Formalin acts as a preservative. In Case of auto – immune hemolytic anaemia, only trisodium citrate 32 g/l is used. (Formalin is not used as it prevents the clumps of agglutinated red cells from breaking up) b) Hayem’s Fluid Mercuric chloride 0.5gm Sodium chloride 1.0gm Sodium sulphate 5.0gm DH2O to 200ml (Distilled water) The other diluting fluids used are as follows: c) Sodium Citrate 3.0gm d) Toisson’s Fluid e) Gover’s Solution 10
  32. 32. PROCEDURE: Filling the Pipette: The pipette must be clean and dry. Suck the blood to mark 0.5 holding the same almost horizontally. If the blood goes slightly beyond the mark, draw it back by touching the tip of the pipettes to a moistened towe. Quickly wipe off the blood adhering to the tip, plunge it into the diluting fluid and suck the fluid up to the mark 101, slightly rotating the pipette meanwhile. Close the ends of the pipette with the fingers, and shake vigorously for about a minute or two. Charging the Counting Chamber: Clean the counting chamber and cover glass. Adjust the cover glass properly. Shake the pipette well. Now quickly blow 2-3 drops of fluid and wipe them off. Holding the pipette in an inclined position, touch the tip of the pipette to the angle between the edge of the cover glass and one of the floor piece. The fluid must nearly or entirely fill the space beneath the cover glass, none into the depression and there must be no air bubbles. Counting of Erythrocytes: Allow atleast 5 minutes for the red cells to settle. The RBC’s must be evenly distributed over the whole disk. Count the erythrocytes in 10 small squares under the dry high power objective. Count the cells which touch the lower and left sides and omit those touching the upper and right sides. Calculation: In the Neubauer counting chamber each small square is 1/20 mm, on each side, the depth of the chamber is 1/10mm, the dilution is 1:200 and when 80 squares are counted the number of erythrocytes per, is calculated from the formula: (1/20 x 1/20 x 1/10 x 1/200 x 80) = 10,000 i.e., multiply the number of cells in 80 squares by 10,000. Sources of error: (1) Inaccurate dilutions; (2) Slow manipulation, allowing some blood to coagulate in the capillary portion of the pipette; (3) Imperfect application of the cover glass; (4) Uneven distribution of erythrocytes and presence of air bubbles; (5) Counting insufficient number of cells (less than 500) ; (6) Personal bias in counting. HAEMOCYTOMETER: 1. Counting chamber is “improved Neubauer’s. 2. Consists of a thick rectangular slide which has a central H shaped depression on either side of which are two stages. Each stage consists of a ruled area of 9 sq mm divided into 9 squares. The central square is further divided into 25 small squares by triple lines. Each of these 25 squares is further divided into 16 squares. Depth between cover slip and ruled area – 0.1 mm.
  33. 33. Improved Neubauer counting chamber (above) and as it is seen under low power objective (below)
  34. 34. 3. PROCEDURE: a) Blood and corresponding diluting fluids drawn in the pipettes. b) First 3-4 drops from the corresponding pipettes discarded. c) Cover slip placed over Haemocytometer. d) Pipette held at an angle of 350 and the tip should touch the groove at the edge of cover slip. e) Fluid runs under the cover slip by capillary action- till the stages are covered with fluid. f) Cells in the chamber to settle for several minutes. g) Counting done with condenser diaphragm of microscope partially closed to make the cells stand out clearly. h) Calculation – Number of cells counted are multiplied by a factor i.e., W.B.C.Count - W x 50 R.B.C.Count - R x 10000 Platelet Count - P x 1000 Errors 1. Due to nature of sample – coagulated blood 2. Operator’s error – faulty technique 3. Errors due to equipment – inaccuracies in graduations. 4. Inherent or field error. Uses: 1. RBC Count- Central square- 5 big squares in centers with small squares. 2. WBC Count – corner square – (4) 3. Absolute eosinophil count – all central and corner squares.(9) 4. Cell counts of cerebrospinal fluid and other body fluids- all central and corner squares(9) 5. Semen analysis – count-corner squares.(4) 6. Platelet Count – 25 big squares in center with 16 small squares each. Normal Values: R.B.C Count Adult Male 4.5 – 6.2 millions/µ1 Female 4.0 – 5.5 millions/µ1 Increased RBC Level Decreased Level a) Age- at birth more a) Old age b) Cases of Haemoconcentration b) Anaemias c) Central cyanotic states c) A plastic Anemia d) Polcythemia Vera
  35. 35. THE TOTAL LEUKOCYTE COUNT Instruments used: W.B.C.PIPETTE 1. It contains of a pipette with small bulb with a white bead in it. White bead helps in mixing. 2. Markings on the pipette are 0.5 and 1. Marking on the stem above bulb 11. 3. Rubber tubing attached to the pipette – heavy walled to resist collapse during suction and should be long (at least 10 inches) to permit easy reading. 4. Blood is drawn into 0.5 mark and WBC diluting fluid upto 11 mark (1 in to 20 dilutions). 5. WBC Diluting Fluid Glacial acetic acid – 1.5 ml. 1% aqueous Gentian Violet – 1 ml. Distilled water – 98 ml. Acetic acid lyses erythrocytes, Gentian violet stains the nuclei Draw blood into the WBC pipette to the Mark 0.5.Suck the diluting fluid to the mark 11 and mix at least for 2 minutes by tilting and rotation. Charge the counting chamber with the fluid and allow to settle. Using 1/6 objective, count in four 1 mm. square Areas. Take usual precaution as in R.B.C. Count. Calculation – Let the No. of cells counted in 4 WBC Squares = X cells Volume of 1 large square = 1 x 1 x 1 10 4 large squares = 4 x 1 = 2 10 5 2/5 contain – X cells. =X x 5/2 cells 2 Dilution = 1:20 =X x 5 x 20/2=50 x X cells 2 Total WB.C.COUNT = X x 50 cells/ white 11
  36. 36. W.B.C 1. Leukocytosis Leukopenia 1. Infections: - Pyogenic – staphylococcoi- 1. Infections :- Bacterial-Typhoid nonpyogenic – Acute rheumatic fever viral – Typhus 2. Haemorrhage – especially haemorrhage 2. Drug induced – chemotherapy of into cavities malignancy 3. Trauma 3. Aplastic anaemia 4. Malignant disease 4. Irradiation 5. Cardiac disorders – following myocardial infarction 6. Drugs and chemical poisoning–phenacetin 7. Metabolic disturbances 8. Collagen vascular disorders 9. Leukemias. 2. Eosinophilia Eosinopenia 1. Allergic disorders – asthma, hay fever 1. Administration of hormones or drugs – adrenalin, insulin, steroids 2. Parasitic infestations – hookworm 2. Response to stress 3. Drug administration’s – pencillin 3. Endocrine disorders–Cushing’s disease streptomycin and Acromegaly. 4. Skin disease – exfoliative dermatitis 4. Aplastic anaemia 5. Pulmonary eosinophilia 6. Bloods dyscrasias and malignant lymphoma 7. Familial eosinophilia 3. Lymphocytosis Lymphopaenia 1. Tuberculosis 1. Acute Anaemia 2. Viral Infections
  37. 37. ABSOLUTE EOSINOPHIL COUNT Clinical Significance Increased eosinophil count is often associated with allergic reactions, parasitic infections, brucellosis and in certain leukemias. Increase in the adrenal function (hyperadrenalism or Cushing’s syndrome) is associated with a fall in eosinophil count. Normal Range 40-440/cu mm (µ I) Specimen EDTA or heparinized blood Principle Blood is diluted with a special diluting fluid, which removes red cells and stains the eosinophils red. These cells are then counted under low power (10 X) in a known volume of fluid by using Neubauer counting chamber. Instruments 1) Microscope 2) Improved Neubauer chamber of Fuch-Rosenthal counting chamber 3) Diluting fluid: (Hingleman’s solution) It is prepared as follows: (a) Yellow eosin : 0.5 g (b) 95% phenol : 0.5 ml (c) Formalin : 0.5 ml (d) Distilled water : 99 ml Procedure 1) Pipette 0.36 ml of diluting fluid in a test tube. 2) Add 0.04 ml of blood (use Hb pipette, twice). 3) Mix and keep for 10 minutes 4) Mix the diluent and charge the counting chamber, 5) Let is stand under a moist petri dish for about 2 to 3 minutes. 6) Count the cells under low power objective in all nine squares of Neubauer counting chamber. Calculations Total number of eosinophils , cu mm (µ I) Number of cells counted X 10 =-------------------------------------- 0.9 Note a) Dilution = 10 b) Volume of fluid = area counted X depth = 9 sq. mm X 0.1 = 0.9
  38. 38. MAKING OF BLOOD FILMS Thin Film: Clean the finger tip. After allowing the skin to dry, puncture it with a sterile blood lancet or needle with a firm quick stab. Wipe away the first drop which appears. Take a small drop of blood a clean slide about ½” from the end, taking care that the slide does not touch the skin. Place the edge of a second slide against the first slide at an angle of about 350 and draw it up against the blood drop which will immediately run across the end filling the angle between the slides. Push the upper slide back along the other slowly to get a thin film. Dry the blood film in air. Thick Film: Take 2 to 3 drops of blood close together on the slide, spread them out enough to show hands on watch through the film. Dry in air for ½ to 1 hour. Fixation: Blood films are dried rapidly in air and fixed by immersing in methyl alcohol for 3 minutes or in ethyl alcohol for 5 minutes. Staining Blood Films: The Romanovsky stains depend for their action on the compounds formed by the intersection of methylene blue and eosin. The stain gives a reddish purple colour to the chromatin of malaria and other parasites. This colour is due to substance which forms when methylene blue is ripened either by age as in polychrome methylene blue or by heating with sodium carbonate. There are several modifications of the original Romanovsky stains, of which well-known are LEISHMAN’S AND GIEMSA’S STAINS. I. Leishman’s Stain : (a) Pour the undiluted stain on a dry, unfixed blood film. Allow 3 minutes. Methyl alcohol in the Lieshman’s stain fixes the film. (b) By means of pipette with rubber teat add buffer solution equal in quantity to the stain added. Mix by gentle blowing. Allow seven minutes.
  39. 39. c) Wash the film in distilled water allowing the preparation to differentiate until the film appears bright pink in colour about half a minute. (d) Dry the film in air. II.Giemsa’s Stain: (1) Rapid method: Fix the blood film in methyl alcohol for 3 minutes. Pour the diluted stain (1:2) and allow to act for 3 minutes. Wash with water for a minute. Dry in air. (2) Slow Method: Fix the film in methyl alcohol for 3 minutes. Take a dish with diluted stain (1:10).Place a piece of glass rod in it. Lay the slide film downwards in the fluid with one end of the slide resting on the rod. After 24 hours wash the slide and dry. This particular method is also useful in spirochaetes. Differential count: Count the cells as arrows indicate the directors of movement of the slide. Note: As the larger leukocytes, particularly granulocytes have a tendency to be more abundant at the margin and at the end of the smear, while counting use Turrel’s method.
  40. 40. Normal Leukocytes Percentage Abnormal Leukocytes (Precursors) Granular: Polymorphs : 65% to 75% Myeloblasts Promyelocyte Eosinophils : 2% to 5% Myelocytes Basophils : 0.5% to 1% Metamyelocytes Non-granular: Lymphocytes: 20% to 25% Lymphoblasts Prolymphocytes Monocytes : 3% to 8% Monoblasts Exercise: Draw a thin film of your blood. Stain the film with Leishman’s stain. Make the following counts under oil immersion objective
  41. 41. STUDY OF LEUKOCYTES I. Granulocytic Series: 1. Myeloblast: 15-20µ in diameter. The nucleus is round or oval, eccentrically placed with a delicate network of chromatin containing 2 to 3 nucleoli. The cytoplasm is abundant, non-granular and basophilic. 1. Myelocyte: 12 to 20µ in diameter. The nucleus is large, round or oval but is frequently flattened on the medial side. Nucleoli are absent and the chromatin is coarser. The myelocytes are classified according to the medial side. Nucleoli are absent and the chromatin is coarse. The myelocytes are classified according to the colour of the granules in the cytoplasm they take with Leishman’s stain, into neutrophilic, eosinophilic and basophilic myelocytes. The cytoplasm is less basophilic and more extensive. 2. Neutrophilic metamyelocyte-Juvenile cell: 14 to 16 µ. The nucleus in smaller, the strands of chromatin are dense and more deeply staining. The cytoplasm is eosinophilic with neutrophilic granules. 3. Stab-cell Band neutrophils: 12 to 14 µ. The nucleus elongated and rounded at the ends and curved. The chromatin forms a coarse network. The cytoplasm is eosinophilic and contains fine dust like neutrophilic granules. 4. Segmented Neutrophils: 10 to 12 µ. The nucles is lobulated, having 2 to 5 lobes of irregular size. The cytoplasm is eosinophilic with fine neutrophilic granules. 5. Eosinophil: 10 to 12 µ. The nucleus is bilobed, connected by a thin chromatin thread. The coarse highly refrigent granules in the cytoplasm are eosinophilic. 6. Basophil: 9 to 10 µ. The nucleus is bilobed, elongated and usually bent in the form of ‘S’. The granules in the cytoplasm are larger and take a strong basic stain. As the granules are very coarse and numerous they may obscure the nucleus. II. Non-Granulocytic Series: 1. Lymphoblast: (12 to 15 µ). The nucleus is large, round or oval with coarse chromatin network. The cytoplasm is slightly basophilic. No granules. 1 or 2 nucleoli are present. Lymphocytes: (6 to 10 µ). The nucleus is oval or round, dark blue with dense chromatin network. The cytoplasm is slightly basophilic, abundant in the large lymphocytes and scanty in the small lymphocytes.
  42. 42. 2. Monoblasts:(6 to 10µ). The nucleus is large, has a fine chromatin network which stains light purple and contains nucleoli. The cytoplasm is basophilic and darker than that of the lymphoblasts. Monocytes : ( 9 to 12µ). The nucleus is large, oval or kidney shaped. The chromatin strands are fine and dense. The cytoplasm is slightly basophilic, appears foamy. 3. Plasma Cells :( 0 to 15µ). The nucleus is eccentrically situated. The cell is round or oval with chromatin in the form of triangular clumps giving the nucleus a ‘cartwheel’ appearance. The cytoplasm is basophilic with a few acidophilic granules. A clear crescentic zone,the para nuclear demilune can also be seen. Study: Leukocytosis: Leukopenia: Agranulocytosis: Leukaemoid Reaction: Leukemia: 1. Acute : 2. Sub acute : 3. Chronic : Aleukemic Leukemia :
  43. 43. BONE MARROW BIOPSY Indication: A. For Diagnosis: 1. High colour index anaemias – (a) Megaloblastic anaemias marrow shows typical megaloblastic red cell formation with the presence of giant metamyelocytes. Such a picture occurs in pernicious anaemia, nutritional megaloblastic anaemia usually secondary to steatorrhea ,megaloblastic anaemia of pregnancy and megaloblastic anaemia due to anti-convulsant drugs, (b) hemolytic anaemia – suggested by cellular normoblastic marrow. (c) Aplastic anaemia, few cells mostly lymphocytes, monocytes and plasma cells. (d) Myxoedema and renal failure- hypoplastic marrow with moderate diminution of all cell types. 2. Tropical diseases – (a) Kala-azar – L.D. bodies may be readily distinguished within the monocytes. (b)Malaria-Marrow tissue for identifying the malaria parasite. 3. Aleukemic leukaemia – 50% or more cells are primitive leukocytes though abnormal cells are absent or too few for diagnosis in peripheral blood. 4. Myelomatosis – infiltration with plasma cells. 5. Lymphomas 6. Malignancy – at times, cells from malignant tumors occur in marrow smears. 7. Gaucher’s disease – reticulum cells stuffed with abnormal lipid. B. For prognosis : 1. Primary form of thrombocytopenic purpura – if the marrow shows increased activity of normoblasts and megakaryocytes. In this type, the response to therapy is good but not in the secondary type. 2. Chronic myeloid leukemia- if myeloblasts are 10% or less, response to treatment is likely to be good, if 50% or more the reverse. CONTRAINDICATION - Hemophilia and allied disorders of coagulation. Bone marrow study can be facilitated either by bone marrow aspiration are by biopsy
  44. 44. BONE MARROW NEEDLE Parts- Wide Bore Needle Stilette Adjustable guard Types :( 1)Klima needle Aspiration (2)Salah’s needle (3)Jamshidis needle-both aspiration and biopsy. Salah’s needle Jamshidis needle Sterilization- Autoclaving Dry sterilization in hot air ovens. Site of BM Biopsy 1) In Infants – Tibial tuberosity 2) Grown up children and adults-Anterior superior iliac spine Anterior Surface of sternal body membrane.
  45. 45. TECHNIQUE: (1) Marrow aspiration is done with marrow aspiration needle. The skin, subcutaneous tissue and periosteum over the posterior iliac crest (or manubrium sterni) are infiltrated with 2% procaine. The marrow needle is pushed through the bone with a boring motion, the guard being kept at a distance of about 1 cm above the surface of the skin. When the needle has entered the marrow the sylette is withdrawn and a long 10 ml syringe attached, 0.2 – 0.3 ml of marrow fluid is aspirated. The needle with trocar is removed and the site of aspiration is sealed with benzoin tincture. Marrow juice drawn off the smeared onto glass slides. In selected patients, residual aspirate is placed in appropriate specimen containers for chromosomal analysis, microbiological culture, cell culture and electron microscopy. 2) Marrow trephine – immediately on completion of the aspiration a trephine biopsy (with a Jamshidi-Swaim needle) of an adjacent area of bone is performed through the same puncture site. After expulsion of 2 cm core of bone and its enclosed marrow from the needle, the biopsy specimen is smeared gently across three glass slides and then placed in fixative for subsequent histopathological processing and staining. Preparations of bone marrow smears - marrow material drawn off are smeared onto glass slides and extra material is collected in the EDTA tube. The marrow practical are picked by a forecep and put on glass slides and crushed smears are prepared. Bone marrow aspiration staining Routine – Leishman & wrights stain Special – Perl’s stain – Iron content Bone Marrow biopsy Routine – H & E – Stain Special - Reticulin stain - Myeloscelrosis
  46. 46. Cause of ‘dry’ or bloody tap - 1. Acute myeloblastic or lymphoblastic leukaemia (Preleukaemia Stage) 2. Acute promyelocytic leukaemia. 3. Hairy’ Cell leukaemia. 4. Myelosclerosis. 5. Refractory anaemia with cellular marrow 6. Malignant infilitration of marrow
  47. 47. NORMAL BONE MARROW Bone Marrow Differential Count Cell Present Cell Precent Myeloblasts 0.8 Segmented (Total) 10.2 Progranulocytes 2.8 Neutrophils 9.6 Myelocytes(total) 7.4 Eosinophils 0.4 Neutrophilic 6.7 Basophils 0.2 Eosinophilic 0.6 Prolymphocytes 0.4 Basophilic 0.1 Lymphocytes 6.2 Metamyelocytes(total) 16.0 Monocytes 1.2 Neutrophilic 15.6 Plasmacytes 1.8 Eosinophilic 0.3 Megakaryocytes 0.6 Basophilic 0.1 Normoblasts (total) 24.2 Band cells 28.0 Pronormoblasts 0.6 Neutrophilic 26.6 Basophilic normoblasts 4.2 Eosinophilic 1.4 Polychromatophilic Normoblasts 16.6 Orthochromic normoblasts 2.8 Reticulum cells 0.4
  48. 48. Normal Marrow- Nucleated cells 20,000 to 100,000 per predominant cells – granulocytes of normal types, and a few normoblast, both are showing some mitoses, megakaryocytes present, other cells few. No abnormal cells.
  49. 49. ORGANISATION AND FUNCTIONING OF BLOOD BANK The organisation of blood bank should receive utmost attention and a careful design and management for smooth functioning of various components. The goal of blood bank is to provide effective blood and blood components which are as safe as possible and adequate to meet the patients need. Registration and medical examination room for registration and selection of donors. Blood collection room (Bleeding area) where blood is collected from the voluntary and replacement blood donors. Room for blood group serology (serology room) – The blood which is collected from the donors and the recipient is tested for the blood groups and cross matching. Room for testing communicable disease like hepatitis, syphilis, malaria, HIV antibodies. The collected blood is tested for above mentioned antibodies. It should have ELISA test kits with reader. Component separation room - The blood bags which are tested negative for communicable diseases are taken and components of blood like RBC & WBC, platelets & plasma are separated in different blood bags and stored. Storage room – The blood which is tested and separated is stored at temperature 4- 6 degree C0 with recording thermometer and alarm device. Records room and store- Correct maintainance of record is very important in Blood bank. Records like blood donor register, blood stock register, issue register, etc. are maintained. Basic functions of blood bank. Recruitment & retention of voluntary and replacement blood donors. Collections, processing, storage and transportation of blood and blood components. Laboratory procedures (investigations.) Participating in clinical use of blood and blood components. Teaching and training of personnel. Research and developments. EXERCISE-VISIT TO CENTRAL BLOOD BANK
  50. 50. DETERMINATION OF BLOOD GROUPS Puncture a finger and collect 2 to 3 drops of blood in a concavity of a porcelain slab carrying about 1 ml of 1% sodium citrate solution. Mix gently by means of pipette. In a double paraffin ring slide, place one drop of ‘A’ serum in the centre of the ring and a drop of ‘B’ serum in the centre of the other. Use separate pipettes for each serum. Mark with glass pencil. Add one drop of corpuscle suspension to each and mix by gently rocking the slide. Allow to stand for about 5 minutes, occasionally rolling or tilting the slide to ensure thorough mixing. If definite agglutination has occurred, a reading and report may be made. If there is no definite agglutination, cover each ring with a cover glass and examine at the end of 30 minutes for final reading. The readings are made as follows: No agglutination by either A or B sera - Group ‘O’ Agglutination by both A and B sera - Group ‘AB’ Method of Cross Matching: Direct matching tests are always advisable before transfusion to guard against the possibility of transfusing with incompatible sub- groups [Sub –Groups A1, A2, A3, A2B and Rh). In one ring of a double paraffin ring slide, mix a drop of donor’s serum with a drop of suspension of recipient’s corpuscles. In the other ring mix a drop recipient’s serum with a drop of suspension of donor’s corpuscles. After 15 minutes take the reading.
  51. 51. HAEMATOLOGICAL DATA Normal Values Conditions causing variations 1. Blood: Increased in : Decreased in: Haemoglobin Men 13.5-18.0 Dehydration Anaemias (gms.per 100 ml) Women 11.5-16.4 First two weeks of infancy Primary and secondary Infants (full term cord Polycythaemia Blood) 13.6-19.6 Children, 1 year (mean) 12.9 Children, 10 year (mean) 12.9 Total Red Blood Cells Men 4.5-6.5 Polycythaemia Anaemias: (Million per cubic Women 3.9-5.69 Dehydration, Anoxia Primary and secondary Millimeter) Infants (full term cord Congestive cardiac failure Blood) 4.0-6.0 Congential heart disease Children, 1 year Congential heart disease (mean) 4.5 High altitude Children 10 years (mean) 4.7 Reticulocytes Adults 0.2-2.0% Perinicious anaemia Aplastic Anaemia Infants (full term) 2-6% During treatment of anaemia Haemolytic anaemias
  52. 52. Blood: Increased in Decreased in: Platelets 150,000-400,000 Hemorrhagic Thrombocytosis Pancytopenia (Hypers- After Splenectomy plenism) Purpura After exercise haemorrhagica Perinicious Anaemia. Erythrocyte Men 3-5 mm in 1 hour Many general diseases, Polycythemia Sedimentation Rate 7-15 mm in 2 hours Febrile conditions (useful for (Westergren Method) Women 4-7 mm in 1 hour prognosis in tuberculosis Sedimentaion Rate 10-12 mm in 1 hour rheumatism & Men 0-9 mm in 1 hour Coronary thrombosis Women 0-20 mm in 1 hour Bleeding Time 0-7 min. Essential and Symptomatic (lvy’s Method) Thrombo- cytopenia, Haemorrhagic state in infants. Prothrombin deficiency. Allergic purpuras. Alcholuric jaundice. Clotting time 5-11 min. Hemophilia, Christmas diseases, (Lee & White Method) Anti Coagulant therapy. Red Cell Osmotic No definite haemolysis at Spherocytosis Cooley’s and Sickle Cell Fragility. Concentrations higher than anaemias: polycythemia 0.45% Nacl.Sol. Vera. Complete haemolysis at Concentration below 0.35% Nacl. Sol.
  53. 53. Packed Cell Volume Men 40-54 Dehydration, Burns Anaemia (P.C.V) % Women 36-47 Polycythaemia Infants (full term cord Blood) 44-62 Children, 1 year (mean) 35 Children, 10 year (mean) 37.5 Mean Corpuscular Adults 76-96 Pernicous & Aplastic Iron deficiency anaemia Voiume (M.C.V) fl Anaemia, Liver Disease, Acholuric Jaundice (femto liters) Sprue, Tropical Macrocytic Polycythaemia Vera Anaemia Mean Corpuscular Adults 27-32 Haemoglobin (M.C.H.) pg (Picogram) Mean Corpuscular Adults 32-36% Normal in aplastic Iron deficiency anaemia Haemoglobin Con- anaemias and pernicious Acholuric Jaundice and centration (M.C.H.C.) anaemias. Anaemias. Total White Cells Adults 5,000-10,000 Pyogenic infections Typhoid, some virus (per cmm) Children 5,000-14,000 Leukamia, Infectious infections, kala-azar, Mononucleosis, Whooping Chronic Malaria, Cough, most allergic Splenic Anaemia, Conditions Agranulocytosis, Anaphylactoid shock
  54. 54. STUDY OF ANAEMIA MICROCYTIC HYPOCHROMIC ANAEMIA Lymphocyte Platelets Microcytic hypochromic cells HISTORY: A 60 yr old male complains of weakness,fatigue and bleeding per rectum(Haemorroids) since 8 months. Hb-4.8 gms/dl,TC-88OO cells/cumm,platelet-2.5 lakh.,MCV-56fl,MCH-14.4pg,MCHC-25.8% PERIPHERAL SMEAR RBC : Predominantly microcytic hypochromic cells WBC : Normal in count, morphology PLATELET : Adequate ABNORMAL CELLS: Nil HAEMOPARASITE : Nil IMPRESSION : MICROCYTIC HYPOCHROMIC ANAEMIA
  56. 56. MACROCYTIC ANAEMIA Macrocyte Lymphocyte Hyper segmented neutrophil Platelet HISTORY: A 20yr old female complains of anorexia, loss of weight and burning sensation in the leg. O/E-stomatitis and glossitis present. Hb-7.1gm/dl,TC- 4000cell/cumm,platelet-1.5 lakh, MCV-112fl, PERIPHERAL SMEAR RBC : Predominantly macrocytic cells WBC : Normal in count, hyper segment neutrophils present. PLATELET : Adequate ABNORMAL CELLS: Nil HAEMOPARASITE: Nil IMPRESSION : MACROCYTIC ANAEMIA
  58. 58. DIMORPHIC ANAEMIA Platelet Macrocytes Microcytic hypochromic cells HISTORY: A 25 yr old pregnant lady complains of vomiting since 15 days. O/E- pallor+.Hb-8.3gm/dl,TC-4300 cells/cumm,platelet-1.9lakh.,MCV-67fl,MCH- 19.1pg,MCHC-28.4% PERIPHERAL SMEAR RBC : Both macrocytes and microcytic hypochromic cells are seen. WBC : Normal PLATELET : Adequate ABNORMAL CELLS: NIL HAEMOPARASITE : Nil IMPRESSION : DIMORPHIC ANAEMIA
  60. 60. NORMOCYTIC NORMOCHROMIC ANAEMIA Normocytic normochromic cells Platelet HISTORY: A 20 yr old male was admitted with a history of minor accident. O/E- haematoma seen in the scalp.Hb-11gm/dl,RBC-2.9 millions, TC-5800cells,Platelet3 lakh. PERIPHERAL SMEAR RBC : Predominantly normocytic normochromic cells WBC : Normal PLATELET : Adequate ABNORMAL CELLS: Nil HAEMOPARASITE : Nil IMPRESSION : NORMOCYTIC NORMOCHROMIC ANAEMIA
  62. 62. STUDY OF HAEMOLYTIC ANAEMIA SICKLE CELL ANAEMIA Sickle cell Normoblast Target cell Platelet HISTORY: A 2 yr old male child presents with growth retardation and repeated respiratory tract infections. O/E pallor with splenomegaly PERIPHERAL SMEAR RBC : Normocytic norm chromic cells with sickled cells WBC : Normal PLATELET : Adequate ABNORMAL CELLS: Nil HAEMOPARASITE : Nil IMPRESSION : SICKLE CELL ANAEMIA
  64. 64. THALASSEMIA Normoblast Microcytic hypochromic cell Target cell Fragmented RBC Lymphocyte Platelet Basophilic stippling HISTORY: A 4 yr old child is suffering from growth retardation, irritability and anorexia till infancy. O/E, pallor-marked, huge enlargement of liver and spleen with sternal tenderness, depressed nasal bridge with malar prominence.Hb-6gm%. PERIPHERAL SMEAR RBC : Moderate anisopoikilocytosis, predominantly microcytic hypochromic cells along with target cells and fragmented RBC and normoblast WBC : Normal PLATELET : Normal ABNORMAL CELLS: Nil HAEMOPARASITE : Nil IMPRESSION : THALASSEMIA
  66. 66. SPHEROCYTIC ANAEMIA Spherocytes Lymphocyte Platelet Polychromatophilic RBC HISTORY: 34 year old male c/o slight pallor, jaundice, and sclera shows icterus. History of cholecystectomy five years back. O/E Spleen palpable 3 cm below the left costal margin. HGB-11.3g/Dl,MCV-84.1fL,MCH-28.8pg,MCHC-34.4g/dL. PERIPHERAL SMEAR: RBC : Normocytic Normocromic, spherocytes and Polychromatophilic RBC WBC : Normal PLATELET : Normal ABNORMAL CELLS: Nil HAEMOPARASITE : Nil IMPRESSION : SPHEROCYTIC ANAEMIA
  68. 68. HAEMOLYTIC ANAEMIA WITH G6PD DEFICIENCY Platelet Bite cells microspherocyte Polychromaticphilic RBC Fragmented RBC HISTORY: A 57 yr old male presents with passage of dark,smoky urine for 3 days. He has taken primaquine 2 days back. O/E, pale with jaundiced skin and scleral icterus. Moderate splenomegaly and hepatomegaly. PERIPHERAL SMEAR RBC : Normochromic normocytic cells with prominent polchromas ia Bite cells, microspherocytes and fragmented cells seen. WBC : Normal PLATELET : Adequate ABNORMAL CELLS: Nil HAEMOPARASITE : Nil IMPRESSION : HAEMOLYTIIC ANAEMIA WITH G6PD deficiency
  70. 70. EOSINOPHILIA Eosinophils HISTORY: A 60 yr old male admitted to the hospital with difficulty in breathing with past history of bronchial asthma. PERIPHERAL SMEAR RBC : Are normocytic normochromic WBC : Normal in count with increase in eosinophil count PLATELET : Adequate. ABNORMAL CELLS: Nil HAEMOPARASITE : Nil IMPRESSION : EOSINOPHILIA.
  72. 72. STUDY OF LEUKAEMIAS ACUTE MYELOBLASTIC LEUKEMIA Myeloblast with auer rod Myeloblast Nucleoli HISTORY: A 23 yr old male presents with bleeding gums for 5 days .O/E: Pallor, splenomegaly and gingival hyperplasia,petechial bleeding spots over trunk is seen. PERIPHERAL SMEAR RBC : Normocytic normochromic WBC : Myeloblast seen with auer rod PLATELET : Decreased ABNORMAL CELLS: Myeloblasts HAEMOPARASITE : Nil IMPRESSION : ACUTE MYELOBLASTIC LEUKEMIA
  74. 74. CHRONIC MYELOID LEUKEMIA Basophil Myeloblast Metamyelocyte Promyelocyte Myelocyte Neutrophil Platelets HISTORY: A 57 yr old male complaining of progressive weakness,weight loss and anorexia for last 3 months.O/E-moderate pallor with massive splenomegaly.Hb- 10gm/dl,TC-2-3 lakh/cumm.,platelet 3 lakh PERIPHERAL SMEAR RBC : Normochromic normocytic cells with presence of normoblasts WBC : Very high count, mature and few immature granulocytes seen. Basophil count is increased PLATELET : Adequate ABNORMAL CELLS: Granulocytes HAEMOPARASITE : Nil IMPRESSION : CHRONIC MYELOID LEUKEMIA
  76. 76. ACUTE LYMPHOBLASTIC LEUKEMIA Lymphoblast Normocytic normochromic cell HISTORY: A 3yr old girl is admitted into hospital with high fever,,generalized lymphadenopathy ,mouth ulcerations,petechial bleeding spots over skin,,pallor and sternal tenderness.Hb-4.2gm/dl,TLC-3,600/cumm,platelet-40000. cells/cumm PERIPHERAL SMEAR RBC : Normocytic normochromic WBC : lymphoblasts with 1-2 indistinct nucleoli PLATELET : Decreased ABNORMAL CELLS: Lymphoblasts HAEMOPARASITE : Nil IMPRESSION : ACUTE LYMPHOBLASTIC LEUKEMIA
  78. 78. CHRONIC LYMPHOCYTIC LEUKEMIA Platelets Lymphocytes Smudge cell HISTORY: A 66 yr old male presents with cervical and axillary lymphadenopathy for 6 monhts. O/E-hepatosplenomegaly present.TLC-1,67000/cumm PERIPHERAL SMEAR RBC : Normocytic hypochromic WBC : Increased mature lymphocytes along with smudge cells PLATELET : Adequate ABNORMAL CELLS: Leukemic cell HAEMOPARASITE : Nil IMPRESSION : CHRONIC LYMPHOCYTIC LEUKEMIA
  80. 80. MULTIPLE MYELOMA Abnormal Plasma cell Plasma cell HISTORY: 50 year old lady came to the hospital with c/o of fractured rib, O/E there is pain and mild swelling, X-ray showed pathological facture of 4th rib with osteolytic lesion PERIPHERAL SMEAR RBC : Normocytic normochromic WBC : Normal PLATELET : Normal ABNORMAL CELLS : Plasma cells HAEMOPARASITE : Nil IMPRESSION : MULTIPLE MYELOMA
  82. 82. STUDY OF MICROFILARIA Microfilaria HISTORY: 40 year male came to hospital with the c/o of swelling and pain in left leg. On examination there is massive swelling (elephantiasis). Peripheral smear showed above picture PERIPHERAL SMEAR RBC : Normocytic normchromic cells WBC : Normal PLATELET : Normal ABNORMAL CELLS: Nil HAEMOPARASITE : Microfilariae IMPRESSION : MICROFILARIAE
  84. 84. STUDY OF MALARIAL PARASITE Gametocyte HISTORY : A16yr old boy is admitted into medicine ward with irregular pyrexia for more than 3 months/E-Pallor-moderate, Spleen-moderatly enlarged,firm ,Liver-mild enlargement,firm PERIPHERAL SMEAR RBC : Normocytic normochromic cells WBC : Normal in count and morphology PLATELET : Decreased ABNORMAL CELLS: Nil HAEMOPARASITE : Malarial gametocyte seen IMPRESSION : MALARIAL PARASITE
  87. 87. WILLIAM BOYD (1885 – 1979) William Boyd trained in medicine in Edinburgh and was Professor of pathology successively in the universities of Manitoba, Toronto and British Columbia. He wrote textbooks which related clinical; disease to autopsy findings in lucid readable English. These books were popular worldwide as undergraduate and Post- graduate medical texts, and helped to give the Manitoba Medical School an international reputation in the 1930’s. “It has become the fashion to regard morbid anatomy both gross and microscopic as somewhat of an outworn creed, a science as dead as the material with which deals. But morbid anatomy is not dead, and never has been except in the minds of those whose dull minds would take the breath from the most vital subjects.” W.Boyd, a Textbook of Pathology: an introduction to Medicine, Lea dn Febiger, Philadelphia, 1932.
  88. 88. EXAMINATION OF URINE Urine should be examined soon after being passed. For routine examination random specimen is satisfactory, but for certain investigations urine passed at a particular time is valuable. Physical Examination: Volume of Urine: A healthy adult excretes about 1000 to 1600 ml of urine 24 hours. POLYURIA: Implies an increased volume of urine and occurs in (1) Increased ingestion of fluid; (2) Diabetes mellitus; (3) Chronic renal diseases; (4) Diabetes insipidus and (5) Certain hysterical conditions. OLIGURIA: Means a decreased urinary out-put (less than 500 ml/24 hrs) occurs (1) Restricted intake of fluid; (2) Excessive loss of fluid through extra-renal channels e.g. sweating, diarrhea, vomiting etc. (3) Reduced blood supplied to kidneys e.g., in hemorrhage, dehydration and shock. (4) Renal diseases like acute glomerulo-nephrits, nephritic syndrome, acute tubular necrosis such as in crush syndrome, incompatible transfusion, heavy metal poisoning, sulphonamide,anuria etc, (5) Addison’s disease. ANURIA: Means excessive suppression of urine formation due to severe impairement of venous blood flow, obstruction to the outflow of urine or severe pathological change within the nephron itself. In this condition the urine outflow will be less than 100 c.c. /24 hours. Colour: Normal urine is pale-yellow of light-straw in colour due to the presence of urochrome, the chief urinary pigment. In polyuria, urine almost becomes colourless. In oliguria, due to concentration, the colour is dark brown. Presence of blood, melanin and abnormal pigments and various drugs etc., may change the colour of urine. Transparency and Turbidity: Normal urine is usually clean when passed-fresh, but sometimes phosphates may produce turbidity in fresh urine. Urine may become turbid on standing due to the presence of mucus or the formation of amorphous or crystalline deposits or bacterial growth. Infections of bladder, prostate or urethra are sometimes associated with increased secretion of mucus. Pathological causes of turbidity of urine include pus, blood or bacteria which are identified by microscopy. Odour: Normal fresh has either no smell or some characteristic aromatic odour. Ammonia odour is detected in decomposed urine and smell of acetone in ketonuria. Reaction of Urine: In health, the pH of urine varies from 4.85 to 8.0 but usually it is slightly acidic (about pH 6). Technique: The reaction of urine is determined with blue and red litmus paper (pH range 5.8 to 7.4). Alkaline urine turns red litmus paper blue and acid urine turns blue litmus paper red. Both blue and red litmus papers turn reddish purple when the urine is faintly on the acid side of neutral (pH 7.0). The urine when examined must be fresh as it turns alkaline on standing due to bacterial decomposition
  89. 89. The acidity of urine is decreased after a meal and with a vegetarian diet the reaction may become alkaline. A strongly alkaline urine may also be due to infection with urea fermenting organisms. Ingestion of citrates and bicarbonate tends to make urine alkaline. The degree of acidity is increased in high protein diet, febrile illness, ketonuria and in leukaemia. Urine may be made acid with ingestion of ammonium chloride, mandelic acid or ammonium mandelate.
  90. 90. SPECIFIC GRAVITY URINOMETER It is an instrument by which the specific gravity of urine is determined. (a) Identification: It is a hydrometer adapted to measure the specific gravity of urine room temperature. It is a weighed glass cylinder, with a bulb containing mercury and a stem. The stem has a scale with readings from 1,000 to 1060 with divisions of 0.001 to 0.002 used at temperature of 150C. It floats in urine taken in the container. (b) Method: The urinometer vessel is filled three fourths full with urine (minimum volume required is 15 ml). The urinometer is inserted with a spinning motion to make sure that it does not touch the sides of the container and floats freely. It should also not touch the bottom of the cylinder. Results: The reading is taken at the bottom of the meniscus at eye level. (c) Precautions: One must see that there is no surface bubble in the urine. The reading is corrected as required for dilution, temperature and total protein. (d) Corrections: In case of the test, temperature being more than 150C, for every 30C rise in temperature, 0.001 is added to the reading and for each 30C below this, 0.001 is subtracted from the reading. (e) The urinometer should be checked every day by measuring specific gravity of distilled water which has a specific gravity of 1.000. If the urinometer does not give a reading of 1.000 an appropriate correction must be applied to all readings taken with that urinometer. The accuracy of a urinometer may be further checked in solutions of known specific gravity. Eg: Solution of potassium sulphate – specific gravity of 1.015.
  91. 91. (f) Normal and Abnormal values: Normally the value ranges between 1.003 to 1.030. Substances which influence specific gravity are urea sodium chloride, phosphates, albumin and sugar. = Urine of low specific gravity is called”Hyposthenuric” less than 1.003. eg. Diabetes insipidus. = Urine of fixed specific gravity are called “lsosthenuric” fixed at 1.010. eg. In end stage renal disease. = Urine of high specific gravity> 1.030 is seen in Diabetes mellitus, Nephrotic syndrome. The measurement of specific gravity gives an indication of urinary total solute concentration. Several other methods used are: 1) Refractometer 2) Reagent strip method.
  92. 92. CHEMICAL EXAMINATION (FOR ABNORMAL CONSTITUENTS) Proteinuria: It is not found in normal life although it is seen in some physiological conditions like severe muscular exercise, orthostatic postural proteinuria, exposure to cold etc. The pathological conditions due to pre – renal causes are toxicity etc. Those due to renal causes are acute or subacute glomerulonephritis, nephrotic syndrome, destructive lesions like tuberculosis of the kidney, neoplasms, calculi, nephrosclerosis etc. Those due to post-renal causes are pyelitis, cystitis, urethrits, prostatitis etc. Technique: Tests for the presence of protein are carried out on a clear specimen of urine which should be filtered or centrifuged to remove any turbidity. Boiling and Acetic Acid Test: Three quarters (3/4) of a test tube is filled with clear urine and the upper one third (1/3) of the column is boiled for about two minutes. The development of turbidity may be due to albumin is present. In phosphaturia turbidity will disappear on adding dilute acetic acid. Heller’s Test: Pour some nitric acid carefully over some urine in a test tube through a pipette. Albumin gives a white ring at the junction which persists on heating. Salicyl-Sulphonic (Sulphosalicylic) Acid Test: To a few ml. of urine 0.5 ml. of a 20% aqueous solution of Salicyl-sulphonic Acid is added. Turbidity indicates the presence of protein, but occasionally a false positive result may be due to presence of uric acid. QUANTITATIVE TESTS FOR ALBUMIN ESBACH’S ALBUMINOMETER: (Esbach – 1874) It is used to estimate albumin in urine. a) Identification: It is shaped like a test tube and is graduated in grams of dried albumin per 100 ml of urine. Graduations in the tube range from 1 to 20. It has a mark ‘U’ in the middle up to which filtered acidified urine is added. It has another mark ‘R’ up to which Esbach’s reagent is added. b) Method: Esbach’s Reagent: It consists of picric acid to precipitate proteins and citric acid to dissolve phosphates in urine. Esbach’s albuminometer is filled up to mark ‘U’ with urine. The reagent is then poured upto the mark ‘R’. The tube is corked and stood vertically for 24 hrs. The proteins settle down and the amount of precipitate is read as g’ltr. If the urine was diluted, the figure must be multiplied by the dilution factor. Picric Acid – 1 gm. *Esbach’s Reagent: Citric Acid – 2 gm Distilled water - 100 ml.
  93. 93. Note: If the urine is alkaline, it is rendered acid by acetic acid. It is subsequently filtered. The urine is then diluted to make specific gravity 1.008. c) Advantages: It is simple and easy to perform. It gives fairly reliable results. It is inexpensive d) Disadvantages: It is less precise and accurate, than currently available methods of estimating proteins. Aufrecht’s Albuminometer It is used to estimate albumin in urine. a) Identification: It consists of a small centrifuge tube with graduation in percentage of albumin. The graduations are in percentage from 0.02-1.70%. It has a ‘U’ mark upto which acidic urine is filed and an ‘R’ mark up to which Aufrecht’s reagent is filled. Aufrecht’s Method: Fill the Aufrecht’s albuminometer with filtered urine to the mark ‘U’. Add the Aufrecht’s reagent to the mark ‘R’. Close the tube with a rubber bung and invert several times. Centrifuge the tube for five minutes at 2500 R.P.M. Albumin is precipitated. Read the percentage of albumin directly from the graduations on the tube. Aufrecht’s Reagent: Picric Acid – 1.5 G. Citric Acid – 3.0 G. Distilled Water to 100 mts. Bence - Jones protein: Bence – Jones described a protein in urine which precipitates between 50 – 600C, redissolves on boiling and precipitates again on cooling at 580C. The presence of Bence-Jones protein is almost pathognomic of multiple myeloma. It has also been observed in leukaemias and carcinomatous metastases in bones. GYLYCOSURIA Various carbohydrates may be found in the urine. Glucose in by far the most common and is the one of much clinical importance. Techniques - Qualitative Test: If albumin is present in any considerable quantity, it interferes with reduction of copper sulphate tests and should be removed by acidifying with acetic acid or boiling and filtering. a) Benedicts Test: To 5 ml. of Benedict’s reagent* (qualitative) add 8 drops of protein free urine. The mixture is boiled for two minutes and allowed to cool. A yellow to red precipitate indicates the presence of reducing sugar. This is a quite sensitive test as it will detect 0.15 to 0.2 percent of glucose in the urine. b) Fehling’s Test: Take 1 c.c. Fehling’s solutions No.1* and 1.2. c.c of Fehling’s solution No.2** in a test tube and add about 3 c.c. distilled water. Boil the mixture. Then add
  94. 94. urine to be tested in fractions to the hot copper solution. The quantity of urine to be added should not exceed that of the reagent. A greenish yellow to red precipitate will appear depending upon the amount of sugar present. A positive Benedict’s test, in most cases implies glycosuria. Benedict’s solution may however be reduced by substances other than glucose. Homogenetistic acid (Alkaptonuria, and excess of mucin oxalates, uric acid, creatinine or urine may reduce copper. Many drug such as chloroform, formalin (used as preservation for urine) and ascorbic also may cause confusing colour changes. QUANTITATIVE ESTIMATION OF GLUCOSE IN URINE: The method of choice in most laboratories is the Benedict’s Method. Technique: Take 25 c.c. of Benedict’s Quantitative Reagent *** in small flask or porcelain dish and add 5 – 10 grams (a good pinch) of Anhydrous Sodium Carbonate. Heat to boiling and add the urine little by little from a burette until a chalk-white precipitate forms and the blue colour of the reagent begins to fade. Now add urine, a drop at a time, until last trace of blue just disappears which indicates the end point. Note the quantity of urine required to discharge the blue colour; this contains exactly 0.5 gm. of glucose. From this percentage of glucose can be calculated easily. The commonest condition of glycosuria is diabetes mellitus. But every case of glycosuria cannot be branded as diabetes, since there are conditions like renal glycosuria in which glucose appears in the urine due to the low threshold of the kidneys for glucose. Gycosuria is one of the manifestations of Cushing’s syndrome, phaeochromocytoma, some liver disorders etc. Hence to confirm the diagnosis of diabetes mellitus, tolerance test is essential.  Benedict’s Reagent (Qualitative) : Copper sulphate (pure crystallized) - 17.3 gms Sodium or potassium citrate - 173.0 gms Sodium carbonate (Crystalised) - 200.0 gms (or 100 gms of anhydrous sodium carbonate Distilled water to make - 1000 ml. * Fehlings Solutions No.1 Copper sulphate (Pire crystalline) - 34.64 gms Distilled water - 500.00 ml ** Fehling’s Solutions No.2 Potassium sodium tartarate - 163 gms Potassium hydroxide - 100 gms Distilled water - 500 ml. *** Benedict’s Quantitative Reagent Copper sulphate (pure crystallized) - 18 gms Anhydrous sodium carbonate - 100 gms Sodium or potassium citrate AR. Or CP - 200 gms Potassium sulfocynate AR or CP - 125 gms Potassium ferrocyanide solution 5% - 5 ml Distilled water to make - 100 ml
  95. 95. KETONURIA Ketone bodies are the three metabolically related compounds comprising of aceto-acetic acid, B-hydroxybutyric acid and acetone. In certain conditions where metabolisms of fat and carbohydrate are disturbed, the rate of ketogenesis in the liver is too great. Ketone bodies accumulate in the blood and are excreted in the urine. The most important condition associated with ketonuria is diabetes mellitus but ketonuria may occur in prolonged vomiting, starvation, high-fat, low carbohydrate diet, prolonged febrile illness, after either anesthesia and some cases of severe toxemia of pregnancy. TECHNIQUES: Rothra’s Nitroprusside Test for Acetone and Aceto Acetic Acid: About 2-5 ml of urine is saturated with ammonium sulphate in a test tube and a few crystals of sodium nitroprusside are added and dissolved. Now overlay with liquor ammonia. A gradually depending purple ring because of the chemical ferro pentacyanide shows the presence of acetone. A brown or red color is of no significance. Ferric Chloride (Gerhart’s) Test for Acetic Acid: To about 5 ml of urine add drop by drop a 10% solution of ferric chloride. A precipitate of ferric phosphate appears and then dissolves in an excess of the reagent. A red bordeaux colour indicates the presence of aceto-acetic acid. This is not sensitive test and the urine must be fresh because on standing, aceto-acetic acid is converted into acetone which does not give a positive reaction by this test. Salicylates and phenol when present in the urine strike a dark violet colour with this reagent. BILIRUBINURIA (Bile pigment) Billirubinuria is found in cases of hepatogenous and obstructive jaundice but not in pure haemolytic jaundice. Techniques Iodine ring test (Smith’s): Overlay a column of urine with tincture iodine diluted with nine times its volume of alcohol. A green ring at the zone of contact shows presence of bile pigment. Nitric Acid (Gmelin’s) test: Soak a filter paper in urine and put a drop of commercial nitric acid over it or pour-down the wall of test tube over a column of urine. A play colours, of which green and violet are most distinctive indicates bile pigment. Fouchet’s Test (Harrison’s): To 5 ml of urine 5 ml of 10% barium chloride is added and mixture filtered. Put one drop Fouchet’s reagent on the precipitate in the filter paper. A green or blue colour indicates the presence of Bilirubin. Fouchet’s Reagent: Trichloracetic acid ……. 25 ml 10% Ferric Chloride ……. 10 ml Distilled water ……. 100 ml
  96. 96. UROBILINURIA AND UROBILINOGENURIA The trace of urobilinogen present in normal urine is insufficient to cause a significant positive reaction with the tests described below: In pathological conditions, the rate of excretion of urobilinogen may be variable and unless a positive reaction is given by a casual sample, the following test should be carried out in a given sample of urine voided between 2 and 4 p.m. Technique: Schlesinger Test: To 5 ml. of urine add 3 drops of tincture iodine and then mix 5 ml. of a 10% suspension of zinc acetate in alcohol. The mixture is allowed to settle and the clear supernatant fluid shows green florescence in transmitted light due to urobilin or urobilinogen. Ehrlich’s Aldehyde test: Dilute Urine 1:2 with distilled water 4.5 ml of diluted urine + 0.5 ml. of Ehrlich’s Aldehyde reagent. Mix well. Run a control using undiluted urine in the similar order. Allow it to stand for 5minutes. Observe the cherry red colour from the top down through the test tube for positive reaction. Ehrich’s Aldehyde reagent: 4 dimethyl aminobenzaldehyde - 4.5 g Conc. HCI - 40 ml. Distilled water - 160 ml. Test for Bile Acid Excretion Hay’s Sulphur test: Sprinkle flowers of sulphur on the surface of a column of urine in a wide bore test tube (2 cm. diameter or more). If the sulphur granules sink immediately or on very gently shaking the test tube, bile acids are present the urine. HAEMOGLOBINURIA AND HAEMATURIA Chemical tests can be used to detect the presence of red corpuscles or haemoglobin in urine, but haematuria is best recognized by microscopy and haemoglobinuria by spectroscopy. TECHNIQUE (Chemical test for detecting blood) Benzidine TEST: Mix a good pinch of benzidine powder in 2ml. of glacial acetic acid in a test tube and heat, cool it and add 5 ml. of urine. Add 2 ml. of H2O2. A green colour changing to blue is indicative of blood or haemoglobin. Haemoglobinuria: means presence of free haemoglobin in urine and is seen in conditions where there is break down of R.B.C.s and liberation of free haemoglobin in blood, viz., black water fever, kala azar, paroxysmal nocturnal haemoglobinuria, mis-matched blood transfusion etc. Haematuira: means presence of erythrocytes in urine and is seen in the following conditions; bleeding diathesis, drug sensitivity, subacute bacterial encdocarditis, acute glomerulonephritis, destructive lesions of the urinary tract like carcinoma, tuberculosis and trauma.
  97. 97. MICROSCOPIC EXAMINATION OF URINE Whenever proteinuria is discovered or diseases of the urinary tract are suspected, the urinary deposits should be examined under a microscope. Since the nature of the sediment soon changes, the urine must be examined while fresh, preferably within 6 hours after it is voided. Technique: Fresh urine, about 15 ml, is centrifuged for 5 -10 minutes at 1500 – 2000 r.p.m. The supernatant fluid is decanted and sediment re-suspended in a small amount of urine (2-3 drops) remaining in the tube. A small drop of the sediment is transferred to a clean slide and covered with a cover slip. The preparation is best examined with the condenser of the microscope is lowered and the illumination reduced by almost closing the diaphragm. The high power is used to identify the constituents. The deposits may be considered under two main groups: (1) Organized deposits and (2) Unorganized deposits Organized Deposits: The elements which commonly form the organized deposits are red blood cells, leukocytes (pus cells), epithelial cells, casts, bacteria and occasionally spermatozoa, prostatic threads and foreign bodies. In acid urine, leukocytes usually retain their normal appearance; in alkaline urine they are swollen, granular and opaque. An occasional leukocyte may be present in normal urine, but more than one per high power filed indicates pyuria. The causative organism may be identified by bacteriological examination. A few epithelial cells from the bladder may be present in the normal urine, and cell from the vulva and vagina usually contaminate a routine specimen from women. The presence of more than a few epithelial cells in a clean or catheter urine specimen is abnormal and indicates disease of the urinary tract. Casts are formed by coagulation of albuminous material and cells in the renal tubules. They rarely occur in urine which does not contain, or has not recently contained albumin and in a general way, they have the same clinical significance as renal albuminuria. Hyaline Casts: They are the most common variety and in small numbers may be found in urine from healthy people. In large numbers they are found in all forms of renal disease.
  98. 98. Granular casts: When found in large numbers indicate tubular degeneration and occur in nephritis (Type II nephritis) and chronic glomerulo-nephritis. Fatty Casts: Consists of fat droplets often mixed with granular of epithelial cells. Waxy casts: These are never found in normal urine and indicate tubular degeneration. Casts containing organized structure: a) Epithelial Casts: Are composed of cells from the renal tubules and show nuclei in various stages of degeneration. Epithelial casts are usually found in acute renal disease such as acute glomerulo-nephritis. b) Blood and Leukocyte casts: These may consists of solid plugs of cells (Red or white cells) which have become adherent to a hyaline or granular cast. They usually imply acute disease and are seen in acute glomerulo-nephritis. Unorganized Sediments: In general these sediments have little diagnostic or prognostic significance. Most of them are substances normally present in solution which have precipitated either because they are present in excessive amounts, or more frequently, because of some alteration in the urine such as in reaction or in concentration. Unorganized sediments consist of crystalline or amorphous material, the exact nature of which varies according to whether the reaction of the urine is acid or alkaline. Crystals Formation and appearance of crystals in urine depends upon pH of the urine i.e. acidic or alkaline. Crystals in acidic Urine: These are as under: i) Calcium oxalate ii) Uric acid iii) Amorphous urate iv) Tyrosine v) Cystine i) Calcium Oxalate These are colourless refractile and have octahedral envelop like structure. They can also be dumb-bell shaped. ii) Uric Acid They are yellow or brown rhomboid shaped seen singly or in rosettes. They can also be in the form of prism, plates and sheaves.
  99. 99. iii) Amorphous Urate They appear as yellowish brown granules in the form of clumps. They dissolve on heating .When they are made of sodium urate, they are needle like in the form of thorn-apple. They are passed more often in patients having gout. iv) Tyrosine They are yellowish in the form of silky needles or sheaves. They are passed in urine in jaundice. v) Cystine They are colourless, hexagonal plates which are highly refractile. They are passed in urine in inborn error of metabolism, cystinuria. Crystals in Alkaline Urine these are as under i. Amorphous phosphate ii. Triple phosphate. iii. Calcium carbonate iv. Ammonium biurate v. Sulphonamide i) Amorphous Phosphate They are seen as colourless granules in the form of clumps or irregular aggregates. They dissolve when urine is made acidic. ii) Triple Phosphate They are in the form of prisms and sometime in fern leaf pattern. They dissolve when urine is made acidic. iii) Calcium Carbonate They are in the form of granules, spheres or rarely dumbbell-shaped. They again dissolve in acidic urine. iv) Ammonium Biurate They are round or oval,yellowish brown spheres with thorns on their surface giving ‘thorn apple’ appearance. They dissolve on heating the urine or by making it acidic. v) Sulphonamide They appear in yellowish sheaves, rosettes, or rounded with radial striations. They appear in urine after administration of sulphonamide drugs.
  100. 100. EXAMINATION OF URINE Physical Examination: Sp: Gr Colour: Odour: Turbidity: Deposits Chemical Examination: Test Observation Inference Reaction Blue Litmus Red/No Change Red Litmus Blue /No Change
  103. 103. EXAMINATION OF URINE Physical Examination: Sp. Gr Colour: Odour: Turbidity: Deposits Chemical Examination: Test Observation Inference Reaction Blue Litmus RED/No Change Red Litmus Blue/ No Change
  106. 106. GEORGE N PAPANICOLAOU (1883-1962) ‘FATHER OF EXFOLIATIVE CYTOLOGY’ George N Papanicolaou , American pathologist was born in Seaport town of Kymi, on May 13, 1883. George graduated from medical school in 1904 and earned his Ph D., in Zoology 1910, and called in to military service in 1912. Dr. Papanicolaou began using vaginal cytology of human in 1920 and continued to study for next 21 years and developed Pap test for detection and early diagnosis of uterine cervical cancer. In 1954 he published the Atlas of Exfoliative cytology
  107. 107. INTRODUCTION- Cytology is the study of body cells that are either exfoliated spontaneously from the epithelial surfaces or are obtained from various body tissues and organs by different techniques. Types EXFOLIATIVE CYTOLOGY -This is the study of cells which are spontaneously shed off from the epithelial surfaces into body cavities or fluid. ASPIRATION CYTOLOGY -In this study, samples are obtained from diseased tissue by fine needle aspiration (FNA) or aspiration biopsy cytology. IMPRINT CYTOLOGY -In imprint cytology touch preparations from cut surfaces of fresh unfixed surgically excised tissue are prepared on clean glass slides. These are fixed, stained and examined immediately. PAP SMEAR The Papanicolaou test (also called Pap smear, Pap test, cervical smear, or smear test) is a screening test used to detect pre-cancerous and cancerous processes in the endocervical canal (transformation zone) of the female reproductive system.
  108. 108. HARMONAL CYTOLOGY Superficial squamous cells Superficial Squamous cells 1) Scrapings from lateral wall of vagina and upper 1/3 rd of vaginal mucosa. 2) Estrogen have keratinizing effect (superficial squamous cells), where as progesterone tends to arrest maturation at intermediate stage (intermediate squamous cells) In par smear- superficial cells appear as mature polygonal cells with eosinophilic cytoplasm and pyknotic nucleus MATURATION INDEX-Ratio of parabasal cells, intermediate cells and superficial cells. MI-0/40/60-estrogen phase -seen at ovulation USES 1) Helps in investigating case of ammenorhoea. 2) Estrogen producing tumour 3) Investigating case of infertility
  109. 109. INTERMEDIATE CELLS Intermediate squamous cells - In Pap smear intermediate cells appear as polygonal or boot shaped with cyanophilic cytoplasm and have vesicular centrally placed nucleus MATURATION INDEX-0/70/30- progesterone phase - seen just before menstruation. - For hormonal studies exfolative cells should be free from inflammatory cells,glandular cells and anucleated squamous.
  110. 110. PARABASAL CELLS parabasal cells - In Pap smear parabasal cells appear as small round to oval cells, less mature than superficial and intermediate cells, with cyanophilic cytoplasm, large round to oval vesicular centrally placed nucleus. MATURATION INDEX-100/0/0- - seen in post menopausal period, post partum period
  111. 111. PARASITE IN PAP SMEAR Trichomonas vaginalis Trichomonas vaginalis Intermediate cell In Pap smear trichomonas vafinalis (Tv) appears as Pear shaped form with flagella along with superficial, intermediate cells and against abundant polymorphs background. Pap smear me also show BB shot or cannonball – aggregates of leukocytes covering the surface of isolated, mature squamous epithelial cell which is attached to T vaginalis organism. Most common parasitic infection occurring in lower female genital tract. Clinical symptoms – white discharge per vagina
  112. 112. DYSPLASTIC CELLS IN PAP SMEAR Dysplastic cells Pap smear - show cells in various size and shape Nuclei are round to oval and irregular in shape Nuclear chromatin is coarsely granular and hyperchromatic Increased nucleocytoplasmic (NC) ratio
  113. 113. MALIGNANT CELLS IN PAP SMEAR Keratinized squamous cell Malignant cell Pap smear – Show elongated and bizarre shaped cells and nuclei Cytoplasm show fibrillary keratin Nuclei are irregularly shaped and have coarsely granular hyperchromitic chromatin
  114. 114. EXFOLIATIVE CYTOLOGY OF BODY FLUIDS-PLEURAL FLUID Metastatic Deposit Malignant cells Mitosis Cytoplasmic vacuoles Moulded cluster a) Cells are seen in acini and single. b) Cells are variable size. c) Nuclei are irregular shaped with irregular border, hyperchromatic, nuclear overlapping and nuclear border touches the cell border. d) Chromatin is clumped with prominent nucleoli. e) Cytoplasma abundant deep purple coloured. f) Frequent mitosis.