THE GENERAL CONCEPTS — CBC (SAMPLING) EXERCISE 2 MAKING FILMS (SMEARS) FROM SAMPLES I. Clinical Significance: Examination of the sample film or aspiration smear is an important part of the hematologic evaluation, bone marrow evaluation, evaluation of blood parasites, as well as for cytological examination. The reliability of the information obtained depends heavily on well-obtained and well-made smears. Complimented with proper staining, the smear can then be systematically examined. Films and smears should be prepared immediately as soon as the specimen is obtained. II. Instruments & Material 1) Clean glass slides 2) Clean coverglasses = 2 3) Gloves III, There are 4 methods of making films, producing 2 types of smears: 1) Thick smears = preferred for blood parasite screening (e.g. Malaria) because a greater quantity of blood is examined in a shorter time. = Blood specimens ideally collected just prior to the next anticipated fever spike or at onset of fever.Technique: a) Place a drop of blood at the center of a clean glass slide. b) With the comer of another slide, or applicator stick, spread the blood immediately in a circular motion, making a 1cm diameter circle. ©) Allow the smear to dry, before submitting it for staining to the lab. 2)Thin smears = used for peripheral blood smear examination, bone marrow evaluation and tissue aspirates for cytological examination a. Wedge Method: 1)Place a drop of blood or specimen 2-3mm in diameter, about Lem from the end of a clean, dust- free slide that is on a flat surface. 2)Get a clean, dry, slightly narrower second slide to be used as “spreader”, with smooth edges. 3)With the thumb and forefinger of the right hand, hold the end of the “spreader” against the surface of the first slide at an angle of 30-45 degrees, and draw it back to come in contact with the specimen. 4)Allow the blood or specimen to spread along the angle between the 2 slides.5) With a steady position of the 2 slides, PUSH the "spreader slide” at a moderate, constant speed forward, until all the blood has been spread into a moderately thin film. 6) Blood Smear & Bone Marrow Smears should be RAPIDLY AIR DRIED, and submitted for staining. 7) Label the head of the smear with a pencil. ~ Slow drying results in contraction artifacts. 8) Smears prepared from Cytological aspirates should be immediately soaked in a Fixative Solution (e.g. Formol Alcohol) > The thickness of the film can be adjusted by: a) Using a smaller or larger drop of blood or specimen ) changing the speed of spreading c) Changing the angle of the “spreader slide” = Ata constant angle, increasing the speed will increase the thickness of the film. - Whereas, at a constant speed, increasing the angle of the "spreader slide” will also increase the thickness of the film. > A good film is characterized by the following: The film or smear does not cover the whole slide For blood films, there should be thick and thin portions, from one end to the other, & a gradual transition between these 2 portions. Blood smears should have smooth and even appearance, free from ridges, waves, or holes.3. Coverglass Method 1) No. 1 or 1 Y2 coverglasses 22mm square are recommended 2)Touch a coverglass to the top of a small drop of blood without touching the skin of the patient. 3)Place it , blood side down, crosswise on another clean coverglass, so that the corners appear as eight- pointed star, allowing the blood to spread evenly. 4)Then pull the coverglasses quickly but firmly apart, on an even horizontal position. 5)With the coverglass placed on a ‘film side up” position, allow the blood film to air dry immediately. 4, Spinner Method: > Blood films that combine the advantages of easy handling of the wedge slide and uniform distribution of cells of the coverglass preparation may be made with special types of centrifuges known as “spinners”. Cytospins are currently used for uniform cell distribution of aspirates.Blood Smear human LeishmanEXERCISE 3 HEMOGLOBIN DETERMINATION (Hb) I. Clinical Significance: Determination of the hemoglobin concentration of the whole blood is one of the most useful and frequently run tests in the clinical laboratory. Hb is the oxygen-carrying compound contained in red cells. Hb can be measured chemically, and the amount of Hb per 100 ml. of blood can be used as an index of the oxygen-carrying capacity of the blood. Total blood Hb depends mostly on the number of RBC and to a much lesser extent on the amount of Hb in each RBC. A low Hb level thus indicates anemia. It also provides essential information for calculations of the called Red Cell Indices. IL Instruments & Materials: 1) Lancet 2) Alcohol Swab 3) Test Tube with Sml Copper Sulfate III. Procedure: 1) Make a deep skin puncture. 2) Wipe the first drop of blood. 3) Apply 1 big drop of blood directly into the copper sulfate solution. 4) Watch for the bloods floatation characteristics.> Interpretation: (Copper Sulfate Specific Gravity = 1.053) If blood sinks = Normal or Polycythemic If blood floats = Anemia > Normal Values Birth oso . 19.5 + 5.0 Gm/100 ml. ADULTS: Females. 12.3 - 15.3g/dL MaleS....ccccnsesene 14.0 - 17.5 g/dL. READING ASSIGNMENT (make a copy) > DIFFERENTIATE the Hemoglobinopathies, with their pathophysiologic description for each.THE GENERAL CONCEPTS — CBC EXERCISE 4 PACKED CELL VOLUME (PCV) OR HEMATOCRIT (Hct) I. Clinical Significance: ‘The hematocrit value may be defined as the volume or red cell per 100 ml of whole blood. The percentage of the red cell mass to the original blood volume is the Hematocrit. Hematocrit determination is used as a simple screening test for anemia. In conjunction with accurate estimation of Hb and red cell count, knowledge of the hematocrit enables the calculation of “absolute” values - the BLOOD INDICES: the MCV, MCH anf MCHC. The hematocrit is usually about three times the Hb values (assuming there is no marked hypochromia) There are two methods of measurements in current use (1) a macro-method which uses the wintrobe tube and (2) a micro-method which uses the capillary tubes. The latter method is becoming the more popular because it has the advantages of shortened time of centrifugation and better packing of the cells.I. Instruments & Materials: 1) Heparinized Capillary Tube 2) Modeling Clay Sealer 3) Hematocrit Centrifuge 4) Ruler I, Procedure : 1) Fill a microhematocrit tube with blood directly from a skin puncture 2) Seal one end of the tube with a clay sealer. 3) Place in the Hematocrit centrifuge and spin for Smin. 4) After centrifugation, the height of the packed red cell column is measured and compared with the height of the original whole blood. I. Calculation: PCV = Red Cell column / Whole Blood column — x 100% ‘expressed as percentage (conventional) or as decimal fraction (SI units) y>Normal Values: Males isaac ie seer 41.5 - 50.4% Females... . 35.9 -44.6% NeWDOM rrsserrsensen iw 54+ 10% READING ASSIGNMENT (make a copy) What condition/s could lead to a false (relative) increase in the Hematocrit? and a false (relative) decrease?HEMATOCRIT LINKS https://www.youtube.com/watch?v=1SN_cmvsMto HEMOGLOBIN LINKS- TYPES OF HEMOGLOBIN DETERMINATION https://www.youtube.com/watch?v=9_old-bHDYs https://www.youtube.com/watch?v=mbpMq- dq9xohttps://www.youtube.com/watch?v=mbpMq-dq9xo0 https://www.youtube.com/watch?v=tOa2TB96KRM