Fall 2024 Labs 1 - 3.pdf

VETERINARY CLINICAL PATHOLOGY Laboratory Manual Fall 2024 Term 3 Richard Kabuusu Melinda Wilkerson India Paharsingh dawn Seddon Ruth Alexander Sharine Harris Shane Richards Pathobiology Ac...

VETERINARY CLINICAL PATHOLOGY Laboratory Manual Fall 2024 Term 3 Richard Kabuusu Melinda Wilkerson India Paharsingh dawn Seddon Ruth Alexander Sharine Harris Shane Richards Pathobiology Academic Program 1  Veterinary Clinical Pathology Laboratory Important laboratory safety precautions Full Name___________________________ 1. All bags and items not required for the laboratory MUST be kept in student lockers located just outside the laboratory. Bags on the floor of laboratory space WILL NOT be tolerated. 2. A clean laboratory coat MUST be worn during the practical class. It is to be worn over your scrubs or appropriate clothing. Lab coats should be removed before leaving the laboratory. 3. Closed shoes MUST be worn during all laboratory sessions 4. Long hair MUST be tied back and tucked under the collar of your lab coat 5. You are STRONGLY encouraged to wear gloves during all wet labs 6. Any cuts and abrasions MUST be covered with a waterproof plaster before entering the laboratory 7. Whilst in the laboratory you MUST NOT eat, drink, chew gum, make a phone call or wear a cap. Only water allowed. 8. Phone calls should be taken outside 9. Any spillages of samples MUST be reported immediately to the laboratory instructor present 10. All used consumable equipment such as slides, pipette tips, capillary tubes, Kim wipes,etc... MUST be discarded into Red biohazard containers 11. Do NOT use oil on the 40x objective lens – any oil on this lens should be cleaned off immediately with alcohol and a Kim wipe. 12. Clean oil off the 100X objective with lens paper / Alcohol and Kim wipe and cover microscope 13. Wipe down the bench with disinfectant provided 14. Remove your lab coat, then wash and dry your hands before leaving the laboratory 15. No animals are allowed in the lab space (this includes service animals) 2  16. No sitting on benchtops. 17. Avoid Horseplay: Maintain professional behaviour and avoid distracting behaviours that could lead to accidents. 18. Know Emergency Procedures: Familiarize yourself with the locations and proper use of safety equipment like fire extinguishers, eye wash stations, and first aid kits. 19. Know the Location of Exits: Be aware of the nearest exits and evacuation routes in case of an emergency. 20. Report Accidents: Immediately report any accidents, injuries, or unsafe conditions to the lab supervisor / lecturer in charge at the time. https://mycampus.sgu.edu/ehs/Accident-Injury-and-Illness-Reporting 21. Students are not allowed to enter the main diagnostic laboratories without permission. 22. You MUST attend all laboratory sessions and be present from the start to the end Failure to fulfill ALL of the above laboratory safety rules will result in a deduction of the points. Print your name and sign here that you have read and understood the safety precautions NAME (print): _______________________ SIGNATURE: _______________________ 3  Laboratory 1: Manual CBC Methods Instructional videos | eClinpath (watch video first for PCV, total protein and blood smear preparation) Ensure blood is on the roller (well mixed before) attempting any of the tests listed below. These techniques will be demonstrated to you by a laboratory instructor Spun Hematocrit [HCTs] or Packed Cell Volume [PCV] Fill a capillary tube 3/4 full blood from a purple top tube containing EDTA, and seal one end of each capillary tube with plasticine – holding the tube in a horizontal position to prevent blood spillage. Wipe outside of capillary tubes with Kim wipe Balance the microcentrifuge by placing capillary tubes (one directly across from the other) into the slots with the sealed end facing out Record the positions of your capillary tubes in the microcentrifuge Note that there is a double lid - Replace lid and spin at about 15,000 rpm for 5 minutes for all species (10 min for ruminants) Use a standard microhematocrit reader below to read off the percentage of the total blood volume occupied by packed red blood cells. Read and record the length (percentage) of blood column occupied by packed RBCs (PCV) Place the spun capillary tube in front of a white background and record the color of the plasma. Place capillary tube on a microscope and check microfilariae if small animal blood. 4  Spun Capillary Tube Example of determination of PCV or spun. Practice reading the tubes 5 Total Plasma Protein Clean a refractometer using Kim wipe and water Use a glass slide to cut and break the spun capillary tube just above the buffy coat Expel plasma from the capillary tube onto the refractometer by tapping on the prism using the unbroken end of the capillary tube. Read the protein concentration in g/dL that is the demarcation between the upper dark zone and lower pale zone (middle scale in the refractometer, g/100 mL = g/dL). Record results on page 6. Clean refractometer with Kim wipe / tissue and distilled water 6 Patient: ___________________________________________________________________ Partial CBC Results Parameter Result Reference Interval Bovine: 25 - 45 Equine: 35 - 50 PCV (spun HCT) % Canine: 37 - 62 Feline: 30 - 52 Plasma Color Clear – Straw Bovine: 6.0 - 8.0 Equine: 6.0 - 8.5 Plasma protein g/dL Canine: 4.6 - 7.2 Feline 6.0 - 7.5 DVM (SOON): __________________________ Date: _________________________ Making a blood smear (blood film) Smears should be prepared as soon as possible after collecting blood. If a sample is being forwarded to a commercial laboratory, it is a good idea to make, air-dry and a few forward smears along with the blood sample, as the blood sample may have deteriorated by the time it gets to the laboratory. To reduce artifacts, the smear should be air-dried and stained ASAP following the manufacturer's instructions. Smears may not stain properly because of inadequate staining time, degraded (old) stains and excessive washing/ rinsing or proximity to formalin. Place a drop of blood with a capillary tube onto a glass slide Place a spreader slide in front of the drop of blood at an angle Follow the steps shown in the diagram below to make a blood smear Dry the blood film by slanting it (in practice a hairdrier would be useful) Place smear in a tray 7 Stain Air-dry the smear quickly. Stain the blood smear with Diff-Quik stain through rapid action Diff-Quik is a modification of Wrights stain (Romanowski stains) First, 10 dips of the smear into fixative (Methanol, light blue) Then, 12 dips of the smear into Solution I (acidophilic or eosinophilic stain; orange – stains cytoplasm) Finally, 12 dips of the smear into Solution II (basophilic stain; dark blue – stains nuclear detail) Rinse the smear under gentle running tap water to remove excess stain. Shake excess water off the slide. Using a pencil, identify your slide with your name and group, species and date _______________________________________________________________ 8  Laboratory 2: Identification of Blood Cells Please read the information below before lab and look at this 5-minute video on how to operate a microscope using a 100X oil objective. Operation of a microscope Before placing the glass slide on the microscope stage, please check the condenser and make sure it is all the way up to the stage (see image below). You will start at low power (10X and scan the feather edge of the slide first for platelet clumps, then move to 100X. The objective needs a drop of oil. Please be careful focusing and ask for help if you have difficulties. The quality of the smear and how it is examined are critical 9 First, scan the entire slide at 10X to get an overview of the thickness of the smear and the erythrocyte, leukocyte, and platelet cell distribution. Leukocytes (neutrophils) are often pulled out to the feather edge. Parasites and platelet clumps can be seen in the feather edge. For routine smear examination, use the 10X lens to find a monolayer of cells, also referred to as the counting window (B), which is about midway between the base of the smear (A) and the end or feather edge of the smear (C). In the monolayer, the cells are evenly spread, not crowded on top of each other, and not in clumps, or too distant from each other. Platelet clumps and organisms can be found towards the feathered edge (C) of the smear. All stained smears should be examined under bright light, so the microscope condenser should be raised, and the aperture of the diaphragm open. Always scan the blood film using the lowest objective and particularly look for platelet clumps and organisms at the feather edge before using higher objectives. To view leukocytes, platelets, and to assess the morphologic changes in erythrocytes, apply a small drop of immersion oil and use the 100X lens. (All images below are Wright-Giemsa. A B C A. Too dense to evaluate, B. Monolayer (counting window), C. Feathered edge for screening of platelet clumps and organisms 10  Can you see? Platelet clumps Microfilaria Neutrophils 10X objective view of the feather edge of a blood film from a dog 10X and 20X view of feather edge of blood film from a dog with many platelet clumps present 50X objective view of the microfilaria 11 Examine and comment on RBC morphology, estimate platelet concentration (10-20 per oil immersion field), and lastly examine and comment on WBC morphology. To estimate WBC concentration, counting the number of WBC in 10 high-power fields (10X) and working out the average per field, and multiplying it by 100, to give the result ___ x103/µL. Carry out WBC differential concentration at 100X oil and calculate the absolute WBC concentration (add in your findings to the Table on page 15). Use the images below and in your lecture notes to properly identify leukocyte types. Please ask us if you have any questions. For glass or digital slides, they are labeled as K9 for dog, Bovine for cow, Eq or H32 for equine, H31 for cat observe the following (1 - 5 below): To access the digital slides, use this link: http://motic.sgu.edu/dsserver Log in Username: PTHB532 Password: MOTICF2024 Click the arrow down on the Teaching Folder to see the Hematology folder. Arrow down on the hematology folder to see the Healthy blood films folder. Select Healthy blood films to view the blood films from dogs (20104, 20- 8), cat (2096, 24-236), horse and nonmammalian species (123456). Clink on the blood film to open it, then you can move the slide around and zoom in on the fields. 1. Scan the feather edge for parasites and platelet clumps. 2. Note discocytes in the dog film have central pallor whereas equine, cat and ruminant erythrocytes lack central pallor. 3. Which species has abundant crenated erythrocytes and which has prominent rouleaux? 4. Note platelets in each species. 5. Be able to identify neutrophils, lymphocytes, monocytes, and eosinophils in each species. Note the cow has more lymphocytes than other species. Lymphocytes and neutrophils are nearly 1:1 in the cow. Video for Manual WBC Differential Count Try counting leukocytes (at least 25 leukocytes and keep track of each cell type (i.e. neutrophils, lymphocytes, monocytes, eosinophils, basophils). Then multiply the counts by 4 to get 100. Each percentage, i.e. 80% neutrophils, is 12 multiplied by the absolute WBC concentration to get the absolute neutrophil concentration. We will discuss this further in the leukocyte lectures/labs. Please note there is a WBC counter that you can download on your phone to count leukocyte subsets. Use either pattern to count leukocytes in the blood film WBC Counter App for iPhone or Android 13 Canine blood film Neutrophils Eosinophil Monocyte Monocyte Lymphocyte Basophil Normal platelet (L) Macroplatelets (R) Feline blood film 14 Neutrophil Eosinophil (rod shaped granules) Monocyte Lymphocyte, (Arrowhead = Howell Jolly body) 15  Equine eosinophil Bovine neutrophil Tortoise blood Nucleated erythrocytes Bovine eosinophil clump of thromobocytes, heterophil (arrowhead), Tortoise Eosinop hil, monocy te, heterop hil Eosinophil Monocyte Heterophil Lymphocyte Heterophils For the nonmammalian digital slide (view the slide, 123456, and identify the following a. Describe the morphology of the platelets (thrombocytes) ___________________________________________________________________________ b. Briefly describe the morphology of the following leukocytes. Lymphocyte: ______________________________________________________________ Heterophil (Neutrophil): _______________________________________________________ Eosinophil: ________________________________________________________________ Monocyte/Azurophil: _________________________________________________________ What differentiates Tortoise or Frog erythrocytes from mammalian erythrocytes. ___________________________________________________________________________ 16 Lab 3 Anemic blood film cases Access digital MOTIC SLIDES for case 1, case 2, and case 4 Link: http://motic.sgu.edu/dsserver Username: PTHB532 Password: MOTICF2024 Select Abnormal Blood Films found in Hematology folder under Teaching. You need to click the arrow down on the Teaching folder and the Hematology folder. Highlight Health Blood films to see the digital slides. Click on the blood film (i.e., Wilk969-A dog blood) and then you can use the mouse or track pad of your computer to move around and zoom in on the image. For the Anemic Blood Film cases, the following digital films will be evaluated. Case 1: Wilk969-A (Far right slide) Case 2: W60724b (Far left slide) Case 3: nondigital case (see pdf) Case 4: W68587a (Middle slide) CASE 2 CASE 4 CASE 1 17 Case 1 (slide # Wilk969-A) (provided by Dr. dawn Seddon, scanned by Dr. Steve Stockham). Signalment: 9-year-old male castrated mixed breed dog. History: Very weak. Physical Exam: Pale mucous membranes, barely able to stand Examine blood film and list RBC morphological size and shape changes that contribute to anisocytosis and poikilocytosis. (i.e. macrocytes, hypochromic microcytes, codocytes, schistocytes, and keratocytes). Also, identify polychromatophils (reticulocytes) Examine the partial CBC data and do the following: Total protein 5.0 g/dL 6-8 g/dl 1. Examine blood film and list RBC morphological changes. (Identify macrocytes, hypochromic microcytes, schizocytes, keratocytes, others?) Q2. Calculate the absolute reticulocyte concentration, determine if it is regenerative, and classify the anemia. The absolute reticulocyte concentration is: /µL Ref. Interval = 0 – 80,000 /µL Q3. Classify the anemia using morphologic and reticulocyte concentration. 18 Q4. Estimate platelet count; Average number of platelets in 5 fields using 100x magnification_____ , then multiple this average number by 15,000) to calculate the total platelet concentration/ µL). _ __________ The hematology analyzer reported out 600,000 platelets/ µL (Ref. Int. = 150-300,000/ µL = 150 - 300 x10^3/uL) Q5. Discussion about platelet concentration Based on your review of the erythrocyte morphology and platelets, do you think the platelet concentration from the analyzer is accurate? Please explain your answer Leukogram Results Analyte Result Ref. Interval Units WBC 42.5 6 - 17 X10^3/ μL Seg. Neutrophils 34.0 5 – 11.5 X10^3/ μL Bands 0.0 0 – 0.3 X10^3/ μL Lymphocytes 5.0 1.0 – 4.8 X10^3/ μL Monocytes 2.0 0.2 – 1.4 X10^3/ μL Eosinophils 1.5 0.1 – 1.3 X10^3/ μL Basophils 0 – 0.1 X10^3/ μL Q6. The most likely leukogram pattern is Choose from 1. Acute Inflammatory 2. Chronic inflammatory, 3. Steroid and 4. Physiologic Q7. What is the most likely cause of the anemia, leukogram and plasma protein findings? What type of blood loss is this? (Acute or Chronic) Please explain your answer. Case 2 (slide # W60724) (provided by Dr. dawn Seddon, scanned by Dr. Steve Stockham) 19 Examine blood film at low power first (scan feather edge) & describe all RBC morphological changes using high power. Use proper terminology (i.e. macrocytes, spherocytes, polychromatophilic erythrocytes). Q1. Classify anemia and determine if it is regenerative Q2. What contributes to anisocytosis and elevated MCV (2 possible answers) 20 Q3. Why is the manual PCV or spun HCT included rather than the calculated HCT from the analyzer? Review the relationship of MCV to calculated HCT, review formula Q4. Provide an estimate of platelet concentration. Q5. Classify the leukogram pattern (two words needed). Q6. Based on all findings, what is the most likely cause of anemia? A. Immune mediated B. Acute blood loss C. Iron deficiency D. Oxidant damage E. Fragmentation Q7. What blood film findings support the cause of the anemia in this case? A. Polychromatic erythrocytes B. Hypochromic microcytes C. Schistocytes D. Spherocytes E. Codocytes 21 Case 3 (24-248): Trudy is a 3-year-old intact female mixed breed dog with anorexia, lethargy, and rapid breathing. Below is the HMV Abaxis hematology analyzer report. (See attached images that represent the leukocyte findings) Manual CBC Compare the absolute concentrations of leukocyte subsets with that determined by the HMV above with the manual leukocyte subset concentrations below. What leukocytes did the HMV NOT identify? 22 Morphologic findings: Band neutrophils have 3+ toxic changes consisting of Dohle bodies and foamy cytoplasm. Reactive lymphocytes are present (see second image below). 23 Classify the anemia and provide the most likely cause of the anemia. Describe the pathogenesis (cause) of the anemia List the terms for the leukogram subset changes (i.e. leukocytosis due to…….). Classify the leukogram (use the manual results). Note there could be a mixture of patterns. Describe significance of toxic neutrophils and reactive lymphocytes. Veterinary Clinical Pathology Laboratory Full Name___________________________ Case 4 (W68587) (provided by Dr. dawn Seddon, scanned by Dr. Steve Stockham) Refer to Lecture 11 Bone Marrow Signalment: Kelsey Dog 7-year-old, breed unknown History: Rapid breathing, painful abdomen, losing weight. *NOTE: MCV, MCHC and HCT are all inaccurate due to the numerous lysed leukocytes interfering in the measurement of MCV and MCHC/HCT calculations Examine the blood film and evaluate platelets, erythrocytes, and leukocytes. Describe morphology of leukocytes, erythrocytes, and platelets. Answer the following questions. Q1. Describe the anemia (note, morphologic classification cannot be used due to inaccurate Wintrobe Indices, use reticulocyte concentration) Q2. Evaluate the leukogram and the morphology of the leukocytes (note numerous blast cells). What is the most likely condition in this dog, based on the leukocyte findings? Q3. Provide an explanation as to why this dog has anemia considering the underlying condition. 25