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THE BASIS OF HEMATOLOGICAL DIAGNOSTICS Marustchak M.I. Overview of heme degradation. Heme is degraded to bilirubin, carried in the blood by albumin, conjugated to form the diglucuronide in the liver, and excreted in the bile. The iron is returned to the body’s iron stores. RES = reticuloendothelial system: RBC = red blood cells. RED CELL COUNT - Count the five(5) small squares indicated by "R". Each of those squares contain 16 smaller squares. Use high power magnification (400X). WHITE CELL COUNT - Count the four(4) large corner squares indicated by "W". Each of those squares contains 16 smaller squares the same size as one of the red cell squares. Use low power magnification (100X). HEMOGLOBIN DETERMINATION • Draw 20 mm3 of blood into a Sahli pipette. • Expel this blood into a test tube containing 5.0 ml of Drabkin's reagent. • Rinse out the pipette twice with the Drabkin's reagent. • Mix the contents using the vortex mixer. • Determine the absorbance for this solution using the • Spectronic 20 at 540 nm. Using the standard Hb curve, • determine the Hb concentration of your blood. WBC DIFFERENTIAL CELL COUNT • The WBC differential begins with preparation and staining of a blood smear. To prepare the smear place a small drop of blood on the surface of a clean microscope slide near the end; using a second microscope slide as a spreader held at 30-40 degrees, touch its end to the edge of the blood drop and push it toward the opposite end of the slide - the blood will be drawn into the acute intersection of the slides and will be "pulled" across the slide (see illustration). Allow the smear to air dry for about 5 minutes. • To stain the smears place the smears in Wright's stain for 3 minutes, transfer to water or phosphate buffer (pH 6.2) for 3 minutes and then briefly rinse with water. Place the slide in a vertical support and allow to air dry. < Simple Wright's stain setup for staining blood smears Note the unstained and stained blood smears in the photo's foreground. • Mount the stained blood smear on the microscope stage and examine at 400X magnification. Locate a region in the "feather" end of the smear where the RBCs are neither too difuse nor overlapping. < Binocular microscope for blood smear exams Feather regions: too thin, just right, too thick Calculation of Erythrocyte Parameters • The quality of erythrocytes is characterized by their MCV, their mean cell hemoglobin content (MCH), and the mean cellular hemoglobin concentration (MCHC). • MCV is measured directly using an automated hemoglobin analyzer, or is calculated as follows: • MCH (in picograms per erythrocyte) is calculated using the following formula: • MCHC is determined using this formula: Red Cell Distribution Width (RDW) Modern analyzers also record the red cell distribution width (cell volume distribution). In normal erythrocyte morphology, this correlates with the Price-Jones curve for the cell diameter distribution. Discrepancies are used diagnostically and indicate the presence of microspherocytes (smaller cells with lighter central pallor). Reticulocyte Count Reticulocytes are young erythrocytes immediately after they have extruded their nuclei: they contain, as a remainder of aggregated cell organelles, a net-like structure (hence the name “reticulocyte”) that is not discernible after the usual staining procedures for leukocytes, but can be observed after vital staining of cells with brilliant cresyl blue or new methylene blue. The staining solution is mixed in an Eppendorf tube with an equal volume of EDTA blood and incubated for 30 minutes. After repeated mixing, a blood smear is prepared and allowed to dry. The sample is viewed using a microscope equipped with an oil immersion lens. The ratio of reticulocytes to erythrocytes is determined and plotted as reticulocytes per 1000 erythrocytes (per mill).