AHT 1210 Lab Test Review Module D PDF

AHT 1210 Lab Test Review Module D Staining Polychrome Stain - Romanowsky Stain = Diff Quik - Polychrome stain impart different colors to certain parts of the cell - Have basic and acidic components - Acid dye components are eosins - they combine with the basic (eosinophilic) of the cel...

AHT 1210 Lab Test Review Module D Staining Polychrome Stain - Romanowsky Stain = Diff Quik - Polychrome stain impart different colors to certain parts of the cell - Have basic and acidic components - Acid dye components are eosins - they combine with the basic (eosinophilic) of the cell components = cytoplasm and granules = they stain red/orange - Basic dye components are methylene blue - they combine to the acidic cell components = nucleus and some granules = Stain blue/purple 3 Steps 1. Fixation - kills and dehydrates blood cells, changes refractive index, makes cell more permeable to stain, causes cells to adhere to slide 2. Staining - contains basic and acidic components which stain different parts of the cells 3. Washing - stops the staining process Fixative = Methanol - 5x for 1 second Eosin dye - 5x for 1 second Methylene Blue - 5x for 1 second Rinse with distilled water WBC Differential - Count minimum 100 WBC on a stained smear - if animal has a high WBC count then count 200 - Relative Values: expressed as a percent and merely reflects the number you see of each cell out of a total of 100 cells counted (%) - Absolute Values: indicate the ACTUAL number of those cells in a LITRE of the animals blood - multiplying the percentage value of individual cells in decimal form by the total number of white blood cells (individual patients WBC count) Reticulocytes and RBC Indices Reticulocyte - Non nucleated RBC - Still contains some RNA which decreases with maturity - Remains 2 days in bone marrow - Remains 1 day in circulation (peripheral blood) - Must contain 2 or more discrete blue granules (when stained with NMB) that are not refractive - usually due to poor drying or unfiltered stain - Refractile artifacts or stain precipitate must not be confused with reticulocytes - Heinz Bodies or Howell Jolly bodies which are erythrocyte inclusions may be confused with reticulocytes - Heinz bodies = stain blue/green and are found around the periphery of the RBC - Howell Jolly bodies will have one tow two deep purple dots in the RBC when using NMB stain Romanowsky Stained Smears (Diff Quik) - Polychromatophils (Reticulocytes) - Macrocytic Cells - Lack of central pallor - Stain a blue basophilia Supravital Stain (New Methylene Blue) - Reticulocytes will appear with bluish granules or a diffuse network of fibrils in the RBC - Cats have two types of reticulocytes: punctate and aggregate - Aggregate reticulocytes mature into punctate ones - Only the aggregate (red arrow) ones are counted as the punctate (black arrow) do not reflect the most recent bone marrow response Relative Reticulocyte % = Reticulocyte count/RBC count x 100 = % Example: Smear 1 500 RBC count 9 Reticulocyte count Smear 2 500 RBC count 8 Reticulocyte count Total RBC = 1000 Total = 17 17/1000 x 100 = 1.7% Corrected Reticulocyte Count = Observed reticulocyte % X (Patients PCV/Normal Mean PCV) = Corrected reticulocyte Example: Dogs PCV = 0.45 L/L 1.7 reticulocytes, PCV is 35% 1.7 X 35/45 = 1.3% Performing a Reticulocyte Count 1. Palace a few drops of blood in a test tube 2. Add equal amounts of NMB to the test tube which contains the blood  3. Place parafilm on top of tube 4. Mix gently 5. Let sit at RT for at least 20 minutes, mix gently every 5 min 6. Make 2 blood smears - let dry, don't forget to label slides - Count 500 RBCs on each slide keeping a tally for the number of retics you observe - Are you within 20% - Calculate the relative and corrected values - Report your findings on a lab requisition Mean Cell Volume - The result gives the average RBC volume indicating the size of the RBC - If the MCV is within the reference range, the RBCs are termed normocytic - If the MCV is below the reference range it indicates smaller than normal RBCs = microcytic - If the MCV is above the reference range it indicates larger than normal RBCs = Macrocyctic - If the MCV must be interpreted along with examination of a PBS as it is possible to have a variation in cell size = Anisocytosis MCV is calculated by: Hematocrit (L/L) = MCV in femtolitres (fL) 10^-15 RBC count (x 10^12/L) Example: RBC count 4.00 x 10^12/L, Hct 0.36 L/L 0.36/4.00 X 10^12/L = 0.090 need to multiply by 1000 = 90 fL Mean Corpuscular Hemoglobin (MCH) - The MCH is the average weight of hemoglobin in the RBC - If MCH is within the reference range the RBC will appear normochromic - If MCH is below the reference range the RBC will appear hypochromic - If MCH is above the reference range the RBC will appear hyperchromic MCH is calculated by: Hemoglobin (g/L) = picograms (pg) 10^-12 RBC count (X 10^12) Example: Hb 120 g/L, RBC count 4.00 X 10^12/L 120/4.00 X 10^12/L = 30 pg MCHC = Mean Cell Hemoglobin Concentration - MCHC is the ratio of the weight of the hemoglobin to the volume of the RBC - Values that fall within the reference range are termed normochromic - Red cell populations with values below the reference range can be termed hypochromic and microcytic - Values greater than the reference range are termed hyperchromic and macrocytic [Hb] and the PCV using the following formula = MCHC = Hb (g/L) / PCV Example: Hb = 120 g/L, Hct 0.36 L/L 120/0.36 = 333 g/L change to 33.0 g/dL due to units SNAP Tests - ELISA = enzyme linked immunosorbent assay - If antigen is present it binds to the antibody - Color development in the indicator spot is proportional to the antigen concentration in the sample - No color = results are negative - Some test for the presence of antibodies VS antigen - Heartworm Test Kit - Detects antigen for adult heartworm - FIV and Feleuk - detects for antigen Baermann Technique - Demonstrates lungworm larvae - The principle takers advantage of the inability of the lungworm larvae to swim against gravity - 5g of feces in gauze, place in funnel filled with warm water to level just above the feces - Let sit overnight (12-24 hrs) - Next AM gently release one large drop of fluid onto microscope slide - Coverslip and examine for lungworm larvae Blood Smears What contributes to a poor smear: - Blood drop size is too small or too large, or drop is partially dried - Tails may be caused by lifting or dropping the spreader at the end of the spreading motion - Tails are also the result of nicks in the spreader slide - Dirty or greasy film/slide - Holes may occur if there are fat particles in the blood, small lumps appear if the slide is covered with lint - Less than 30 degree angle pushes the blood out too much and makes the smear too long and thin - Greater than 30 degree angle does not spread out the blood enough and makes the smear too short and thick - If the spreader is pushed too fast = smear will appear thicker - If spreader slide is pushed too slow = the result will be too thin - A half bullet shape is the result of uneven pressure on each side of the edge - Waves are caused when the spreader is not pushed with an even motion - A long narrow bullet shaped smear is the result of pushing the blood drop too soon before the blood has spread along the edge of the spreader smear Criteria for a Good Smear - Thumb print or bullet shaped - Minimal visible indication as to where the drop of blood was applied - Entire film should cover more than ½ to ⅔ the slide area - Keep the portion of the smear away from the edge of the slide - Holes, ridges or line in smear are absent p - Progress evenly from thick to thin - Have adequate size of monolayer - Have an even EBC distribution when viewed microscopically - Thin edge free from nicks Blood Transfusion Medicine Blood typing: defined by the inherited antigens of the surface of the RBCs, species specific. Immunogenicity and clinical significance vary.  - Principle of typing methods is a visible hemagglutination reaction between patient RBC surface antigens and known reagent antisera - Naturally occuring antibodies: occur in most species, vary in their pathological significance - Acquired Antibodies: produced after exposure to an incompatible blood type, exposure s from blood or blood products containing erythrocytes (or their antigens) Blood Typing Cards - RapidVet-H QuickTest for Canine and Feline Major and Minor Cross Matching - Blood typing identifies certain known antigens in a patient and donor BUT does not identify antibodies in the patient or donor - A crossmatch procedure determines whether there is an antibody reaction to antigens in the donor and is performed to determine compatibility or incompatibility between donor and recipient. - Major = detect antibodies in the recipients serum that may agglutinate or lyse the donors RBCs - Minor = detects antibodies in the donors plasma directed against recipient RBCs Blood Types: Canine - DEA 1-8 - DEA 1 system significance = No pre-existing alloantibodies to DEA 1 - Dogs can be positive to either DEA 1.1 or DEA 1.2 but not both but can be negative for both Feline - AB system - Blood group antigens are defined by specific carbohydrates on the erythrocyte membranes  - Cats have naturally occurring antibodies Equine - 8 different red blood cell groups - A, C and Q most likely to stimulate an antibody response when given to a horse that is negative to them - Each system corresponds to a particular gene for which two or more alleles exist - The blood group genes produce surface molecules that contain antigenic sites which are known as factors Ruminant - 11 major blood group systems - J antigen is a lipid that is found in body fluids and is absorbed onto erythrocytes so it is not a “true” antigen - Sheep - 7 blood groups - M-L system is involved in active red cell potassium transport - R system is similar to the J system in cattle, in that antigen is soluble Fibrinogen Heat/Precipitation fibrinogen test: - The fibrinogen is denatured by heat and precipitates out of the plasma when heated - Fibrinogen is a plasma protein that is manufactured in the liver - Increased fibrinogen indicates inflammation - If decreased the blood will not rapidly clot Calculating fibrinogen: Plasma protein reading (prior to heat) - plasma protein reading (after heat) = fibrinogen value (g/dL)