Clinical Pathology Learning Objectives for Exam 1 PDF

VM 7589 Clinical Pathology Learning Objectives for Exam 1 Lecture 1: Introduction 1. Provide a definition of Clinical P...

VM 7589 Clinical Pathology Learning Objectives for Exam 1 Lecture 1: Introduction 1. Provide a definition of Clinical Pathology Clinical pathology is a field of medicine that uses the laboratory to study changes in organs/tissues/cells secondary to disease and/or other factors. 2. Define the term “reference interval” a. Describe how they are obtained A reference interval indicates a “normal” range for a specific laboratory value. Reference intervals are obtained with a Gaussian/normal distribution. 40(minimum)-120(recommended) “normal” (fasted and healthy adults) patients are sampled; the mean is determined; and then the interval is set at 2 standard deviations from the mean value. 95% of “normal” patients fall within the interval at 2 SD. 99.7% of patients fall within 3 SD. Reference intervals (RIs) are set at 2 SD from the mean so that we don’t miss the sick patients. RIs are affected by age, breed, sex, time of year, etc. A low number of healthy patients will fall slightly outside of the RI.  3. Describe the difference between serum and plasma Serum: liquid portion of blood WITHOUT coagulation factors - Placed in a tube w/out anticoagulant - Blood is allowed to clot → then centrifuged to remove solids - Fibrinogen and coagulation factors are tied up in the clot (so can’t test) Plasma: liquid portion of blood WITH coagulation factors - Obtained by centrifugation of anticoagulated blood - Remove cells ** Plasma → more letters, more stuff in it (aka coagulation factors and fibrinogen) Serum → less letters, less stuff  4. Be able to list several different anticoagulants a. Describe how they might be used Lithium heparin - In green top tube - Binds antithrombin - Product → plasma EDTA - In purple top tube - Irreversibly binds calcium (Factor IV) - Product → plasma Sodium citrate - In blue top tube - Reversibly binds calcium (Factor IV) - Product → plasma Lecture 2: Quality Assurance  1. Define and calculate diagnostic sensitivity and specificity a. Explain how they influence our interpretation of assay results Sensitivity: a measure of frequency of the test being positive in animals that have the disease How well does this test detect the disease? What is the certainty that, if the animal has the disease, the test will detect it? It affects our interpretation of results as it gives an approximation of how many truly afflicted animals will be missed by a test. Ex: a test with 90% sensitivity will detect 9/10 animals with the disease Specificity: a measure of the frequency of the test being negative (or normal) in animals that do not have the disease Ex: a test with 90% specificity will have normal (negative) test results in 9/10 non-diseased animals **See slide, “Using sensitivity and specificity” 2. Define and calculate positive and negative predictive values and prevalence a. Explain how they influence our interpretation of assay results Predictive values include prevalence of the disease and the likelihood of the disease in the animals tested (prevalence = % of the population at one time with a certain disease) Positive predictive value: tells you what % of animals with a positive (abnormal) test result actually have the disease A PPV of 95% means that if you obtain a positive/abnormal result, you are 95% confident that the animal has the disease Negative predictive value: tells you what % of animals with a negative (normal) test don’t have the disease If a test has a NPV of 95%, if you obtain a negative/normal test result, you are 95% confident that you can rule out the disease Predictive values help you decide if a test is worthwhile to use in your practice; in what instances it is appropriate to use a test; how to interpret your results; whether or not further testing is needed. 3. Know the phases of laboratory testing Start - A decision is made to run a specific laboratory test/set of tests Pre-analytical phase - Patient is approached - Sample obtained - Sample placed in collection tube - Sample transported to the instrument - Sample prepared for evaluation Analytical phase - Sample is evaluated Post-analytical phase - Result generated - Numbers are recorded and evaluated  - Result is reported Finish - Decision is made on patient care 4. List what the veterinarian’s responsibilities are in a quality control program - Ensure that a quality control program is in place and routinely re-evaluated - Properly train ALL instrument users - Systemic monitoring of proper orientation to equipment use - A system for monitoring reagent inventory - Controls with known ranges of acceptable results for each test run - KEEP RECORDS Lecture 3: Erythrocytes (Erythron 1) 1. Explain key features of red blood cell production. Where are erythrocytes produced? Erythropoiesis is regulated by erythropoietin (EPO) and other growth factors (IL-3, stem cell factor for early erythropoiesis) EPO is a glycoprotein hormone produced mainly in the kidney - Secreted in response to cellular hypoxia, via hypoxia inducible factor (HIF) - Stimulates RBC production in the bone marrow Erythrocytes are produced in the bone marrow. a. What growth factor is primarily responsible for red cell production and what triggers its production/release? Erythropoietin (EPO). Secreted in response to cellular hypoxia, via hypoxia inducible factor (HIF). b. What are reticulocytes, and under what conditions are they seen in peripheral blood? Reticulocytes are a late-stage immature RBC, making up about 1% of peripheral blood RBC. - An increased # is seen with regenerative response - Look “blue” (polychromatophilic) on regular Wright’s stain - New methylene blue (NMB) stains mRNA (used to identify reticulocytes) - 2-3 types in cats (ex: aggregate; punctate) - NOT seen in horses in blood! Reticulocytes are seen in peripheral blood when there is regenerative anemia. c. What is a Howell Jolly body? A Howell Jolly body is a fragment of nucleus seen with a Wright’s stain in an RBC. - May indicate reduced splenic function  d. What are nRBCs, and under what conditions are they seen in peripheral blood? nRBCs are nucleated RBCs, aka Metarubricytes. They are seen when there is a regenerative response, which may occur w/ - Splenic disorder (decreased clearance) - Marrow injury - Hematopoietic neoplasia (nRBCs w/out reticulocytes in peripheral blood = inappropriate response!) 2. Describe the two types of red blood cell destruction. (1) Extravascular RBC destruction (a) Occurs in the spleen – macrophages break down old erythrocytes into bilirubin, iron, amino acids (2) Intravascular RBC destruction (a) RBC lysis in circulation → free Hb → Hb dimers go to liver macrophages → excreted in urine (10%) or excreted in feces (90%) a. Which is more common? Extravascular RBC destruction. b. How can they be differentiated in disease states? Extravascular RBC destruction – spleen or liver dysfunction Intravascular RBC destruction – pink hue to blood serum (hemoglobinemia) or in urine (hemoglobinuria)  3. Apply what you have learned about red cell metabolism to see how pathology can occur a. What happens when an enzyme in the glycolytic pathway or other pathways is inhibited? Decreased 2,3-DPG or 2,3-BPG (intermediate of glycolysis) results in a left shift of the Hb-oxygen dissociation curve → more affinity of hemoglobin to oxygen, so oxygen is bound at a lower PO2 → more difficult to offload oxygen → decreases oxygenation. b. How would this manifest in the animal? Decreased oxygen transport → hypoxia, dyspnea, cyanosis, tachycardia, exercise intolerance 4. Discuss why changes in the temperature or blood pH of an animal might affect oxygenation Increased temperature → shifts the dissociation curve to the right (decreases Hb binding capability, which increases Hb saturation in the blood, thus increasing oxygenation) Increased CO2 → decreases blood pH → shifts dissociation curve to the right (decreases Hb binding capability, which increases Hb saturation in the blood, thus increasing oxygenation) 5. Describe the basics of iron metabolism a. Where is it absorbed? Iron is absorbed in the small intestine. b. How is it regulated? Hepcidin is a key negative regulator (decreases serum iron) which is regulated by inflammatory cytokines (ex: IL-6, which increases hepcidin).  c. What is hepcidin? Hepcidin is produced in the liver. It is a key negative regulator of iron (increased hepcidin means decreased serum iron). It is increased with inflammation, and is the main cause of anemia seen with inflammation (more on this later). Increased IL-6 → increased hepcidin → decreased iron d. Where is it stored? Iron is stored as serum ferritin (can be measured) or as hemosiderin (found in tissues). e. What is its role in the body? Iron is used for the production of hemoglobin. f. List the tests used clinically to assess iron Serum iron (SI) = iron bound to the carrier protein transferrin - Usually about ⅓ of iron-binding sites of transferrin are occupied by Fe3+ Total iron binding capacity (TIBC) = amount of iron transferrin can bind when totally saturated with iron - Indirect measurement of serum transferrin % saturation of transferrin = percentage of transferrin iron binding sites that are occupied - Saturation (%) = 100 x (SI / TIBC) Serum ferritin (a form of iron storage) can be measured. g. What test results might be found with iron deficiency? True iron deficiency: - Serum iron decreased  - Serum ferritin decreased - TIBC normal or increased - % saturation of transferrin is decreased - Small RBC (microcytosis) Lecture 4: Evaluation of the Erythron 1. Identify the components of blood when centrifuged in a hematocrit tube When centrifuged in a hematocrit tube, it contains: - At the bottom: RBCs - Next: buffy coat (WBCs, platelets, microfilaria, nRBCs) - At top: plasma 2. Calculate RBC indices and describe how they are used RBC indices are used to evaluate the erythron. Mean Corpuscular Volume (MCV) → cell size MCV (fL) = PCV x 10/RBC in millions fL = femtoliter, 1 x 10^(-15) L Mean Corpuscular Hemoglobin Concentration (MCHC) → ratio of Hgb weight to RBC volume MCHC (g/dL) = (Hgb (g/dL) x 100) / PCV How mature the blood cells are Mean Corpuscular Hemoglobin (MCH) → ratio of Hgb weight per RBC; less useful than MCHC MCH (pg) = (Hgb (g/dL) x 10) / RBC in millions Red Cell Distribution Width (RDW) → variation in MCV RDW = (Standard deviation of MCV/MCV) x 100 (machine derived) Variation in size example: a young reticulocyte – large and less Hb → pale and larger, increased RDW, decreased MCHC, increased MCV 3. Identify changes in RBC shape and offer possible explanations for their shape change **eClinpath: https://eclinpath.com/hematology/morphologic-features/red-blood-cells/quick-guide/ Echinocyte: regularly spiculated,”burr” cells or crenated cells Expansion of the outer leaflet of RBC membrane, ATP depletion Acanthocyte: irregularly spiculated RBC or “spur” cell Could indicate splenic hemangiosarcoma Speculated to be due to alterations in lipid composition of RBC membrane or fragmentation injury to RBCs Spherocyte: sphered RBC Could indicate IMHA Lack central pallor → could be bc coated by Ab or mac partially phagocytosed Removal of membrane by macrophages (trogocytosis) Keratocyte: “bite”, “helmet” or “blister” cells Oxidant or fragmentation injury. Low numbers may be seen in non-anemic cats. Target cell: RBC with a bullseye; AKA codocyte Only recognized in dogs, which have central pallor Expansion of the inner leaflet of the RBC membrane from alterations in phospholipid:cholesterol content in the membrane, cells that spread in a smear than normal (leptocytes) because they are larger than normal (polychromatophils) or thinner than normal (iron deficient cells). Schistocyte: RBC fragments or ”schizocyte” Shearing of RBC in the circulation due to abnormalities in the vasculature (endothelial cell, fibrin strands, blood flow) or mechanical RBC fragility (iron deficiency). Could indicate splenic hemangiosarcoma Dacryocyte: tear-drop RBC Unknown. Physiologic?: Low numbers in non-anemic camelids. Stomatocyte: RBC with a slit or mouth-like central pallor (“stoma”) Expansion of the inner leaflet of the RBC membrane 4. Discuss the appearance of reticulocytes in different species, their importance, and techniques for calculating reticulocyte numbers Reticulocytes look “blue” or polychromatophilic on a regular Wright’s stain (pale, slightly basophilic). They can officially be identified with an NMB stain, which stains fragments of RNA blue. In cats, there are 2-3 types seen (including particulate, aggregate types). Reticulocytes are NOT seen in horses! Reticulocytes are immature RBCs and are important in discerning what the bone marrow is doing and identifying a regenerative anemia. Reticulocyte counts: - Reticulocyte percentage (#retic/1000 RBC x 100) - Enumeration of reticulocytes - % reticulocytes = # counted per 1000 RBC on blood smear - Can be very inaccurate! - Corrected reticulocyte percentage - Observed reticulocyte % on NMB smear x patient PCV/normal PCV = corrected reticulocyte percentage - >1% in dogs and >0.4% in cats indicates bone marrow response - Reticulocyte production index - Corrected reticulocyte % / maturation factor - Absolute reticulocyte count (preferred technique) - Absolute reticulocyte count = (% retic / 100) x RBC count  5. Identify what structures and features can be detected by a New Methylene Blue stain Reticulocytes (verified because we can see RNA fragments) Heinz bodies (denatured Hgb) 6. List some of the immunologic or immunohematologic tests commonly used in the veterinary lab and understand what these tests measure a. Define the following terms. If it is a structure, describe its appearance on a blood smear. Anisocytosis, basophilic stippling, Coombs’ test, erythron, Heinz body, hemoglobinuria, Howell Jolly body, hypochromic, normochromic, LE cell, macrocytic, microcytic, normocytic, poikilocytosis, reticulocyte, rouleaux, spherocyte, target cell Immunohematologic tests: - PCV/Hematocrit (Hct): the percent of blood composed of RBCs - Used synonymously but technically PCV is the manual test and Hct is the calculated value - Hemoglobin - RBC indices - MCV → cell size  - MCHC → ratio of Hgb weight to RBC volume (maturity of cells) - MCH → ratio of Hgb weight per RBC - RDW → variation in MCV - RBC morphology - Anisocytosis, poikilocytosis, specific shapes/sizes - Reticulocyte count - Bone marrow examination - Used more in cases of nonregenerative anemia and other cytopenias - Evaluated concurrently with a CBC, clinical history - Immunologic tests - Coomb’s test: antibody against RBC - Used as ⅓ criteria for IMHA Dx - Antinuclear antibody test (ANA): antibody against nucleoprotein - Used for Lupus Dx - Rheumatoid Factors (RF): IgM against patient IgG - Rheumatoid arthritis Dx presumably Anisocytosis: variation in RBC size Basophilic stippling: seen on Wright’s stain; presence of numerous basophilic granules that are dispersed through the cytoplasm of RBCs; can be demonstrated to be RNA; indicative of disturbed erythropoiesis Coomb’s test: tests for anti-RBC antibodies; used as 1 out of 3 criteria for IMHA diagnosis Erythron: mass of erythroid cells in the body, including circulating cells and precursor cells Heinz body: seen on Wright’s stain (same color) or NMB (basophilic); inclusions within RBCs composed of denatured hemoglobin Hemoglobinuria: free hemoglobin in the urine; usually due to intravascular destruction – possible hemolysis or hemolytic anemia Howell Jolly body: when the nucleus is extruded from the metarubricyte during the last developmental stage of the RBC (formation of retic as ER remains and Hgb synthesis continues), occasionally a small fragment of the nucleus remains, called a Howell-Jolly body Hypochromic: MCHC is less than the reference interval values Mean Corpuscular Hemoglobin Concentration Used to classify anemias Normochromic: MCHC is within the reference interval values Mean Corpuscular Hemoglobin Concentration Used to classify anemias **Increased MCHCs are falsely increased due to interferences in the Hgb assay (lipemia or Heinz bodies) or due to in vitro hemolysis; RBCs cannot carry too much Hgb LE cell: a lupus erythematosus (LE) cell (AKA Hargraves cell) is a neutrophil or macrophage that has phagocytized the denatured nuclear material of another cell Macrocytic: MCV is greater than reference value range Mean Corpuscular Volume; cell size indicator Used to classify anemias Microcytic: MCV is less than reference value range Mean Corpuscular Volume; cell size indicator Used to classify anemias Normocytic: MCV is within the reference values Mean Corpuscular Volume; cell size indicator Used to classify anemias Poikilocytosis: variation in RBC shape Reticulocyte: immature RBC; polychromatophilic on Wright’s stain; NMB will precipitate remnant mRNA, forming a reticulated pattern Increased number seen in the peripheral blood with regenerative anemias Rouleaux: cells stacking on top of each other Horses and cats can show this in health Canines typically show little rouleaux unless inflam Dz present Differentiate from agglutination with a saline dilution/dispersion test **Rouleaux disperses with the addition of saline, whereas agglutination will persist Spherocyte: dense, spherical RBC associated with IMHA and fragmentary changes Target cell: (AKA codocyte); RBC containing dense, central areas of hemoglobin Associated with liver disease, regenerative anemia, and in vitro (artifactual) changes https://eclinpath.com/hematology/morphologic-features/red-blood-cells/quick-guide/ Lecture 5: Anemia and Polycythemia 1. Discuss the classification of anemia and give examples of different techniques for classification Anemia: a decrease in the PCV, hemoglobin concentration, and RBC count Absolute: a decrease in the total body red cell mass Relative: normal red cell mass but increased plasma volume Classify anemia by: - RBC indices - Is the MCV microcytic/normocytic/macrocytic? - Is the MCHC hypochromic/normochromic/hyperchromic? - Is the RDW increased? - Marrow response (reticulocytes)  - Pathophysiologic mechanism RBC indices: Macrocytic, hypochromic? (increased MCV, decreased MCHC) - Think regenerative Microcytic, hypochromic? (decreased MCV, decreased MCHC) - Think iron deficiency - Too small (microcytic) because too little iron Normocytic, normochromic? Think everything! Marrow response: Regenerative → increased reticulocytes, nRBC - Usually caused by hemorrhage or hemolysis - Remember it takes time for regen to occur (3-4 days) - The most severe anemias have the highest regen response - Regen is difficult to detect in the horse – sometimes the MCV and RDW and changes in PCV help - **An increase in nRBC without reticulocytosis does NOT indicate a regenerative response! Nonregenerative → little to no increase in reticulocytes Pathophysiologic mechanism: Regenerative - Hemorrhagic anemia → both PCV and TP decreased - Hemolytic anemia → PCV decrease, TP normal, icterus, hemoglobinemia may be present Nonregenerative - Anemia due to reduced or ineffective erythropoiesis 2. When given a case, classify the anemia and give a possible cause Classification of anemia by pathophysiologic mechanism: - Regenerative (bone marrow still functioning well) - Hemorrhagic anemia – both PCV and TP decreased  - Note: chronic hemorrhage is a little different - Hemolytic anemia – PCV decreased, TP normal, icterus, hemoglobinemia may be present - Nonregenerative (problem w/ bone marrow) - Anemia due to reduced or ineffective erythropoiesis 3. Discuss the laboratory characteristics of acute and chronic blood loss and give possible causes Hemorrhagic anemia: acute vs chronic Anemia from acute blood loss: - Initially, PCV is normal as cells and plasma lost in similar proportions - Depending on the severity, hemorrhagic shock may occur - Loss of ~40% → hemorrhagic shock - After 2-3 hours, blood volume restored by interstitial fluid - RBC regeneration apparent in peripheral blood by 3-5 days after blood loss - Must recheck to confirm Anemia from chronic blood loss: - Animals are normovolemic - Iron stores may become depleted, causing a microcytic, hypochromic anemia - Anemia may be only poorly regenerative - May or may not be hypoproteinemic **(From lecture book notes): A. Characteristics of acute blood loss a. PCV (plus hemoglobin and RBCs) are decreased, protein is often decreased b. Evidence of hemorrhage may be readily apparent in these cases i. Remember that blood can also be lost within body cavities or within the GI tract and not be visible 1. Protein in these cases may only be mildly decreased, as it is resorbed c. Hemorrhage typically results in loss of both plasma protein and RBCs in equal proportions with the vascular network collapsing around the reduced blood volume (hypovolemia)  i. The body and dietary water can flow into the vascular space and partially correct the hypovolemia, decreasing HCT and plasma protein 1. This is in contrast to hemolytic or anemias due to decreased erythrocyte production, where the RBCs are lost or not produced and plasma protein remains at its pre-anemia value 2. The fluid that replaces the fluid lost in hemorrhage comes from the interstitial space and GI tract at least 2 hours after the loss d. Signs of regeneration (reticulocytosis, increased MCV) will not be apparent until 3-5 days after the blood loss B. Characteristics of chronic blood loss a. Anemia typically develops slowly i. Can become more severe as the animal has had time to adapt to the anemia b. Iron stores often become depleted i. Leading to a microcytic, hypochromic anemia related to iron deficiency c. Check for evidence of bleeding into the GI tract (tarry stool/melena), as this is often the cause of the blood loss d. This type of anemia can be difficult to classify i. Will vary with time and amount of blood loss ii. Early on, there should be evidence of regeneration 1. May not be profound iii. As blood loss continues (weeks to months) there is less iron present and red blood cell regeneration will slow down iv. In very chronic cases, a microcytic, hypochromic anemia may develop 1. When there is less iron in the body, the cells cannot make enough hemoglobin 2. Instead of being the normal red color, these cells are hypochromic from less hemoglobin and pale with increased central pallor a. In contrast to the hypochromic immature cells seen with regeneration, which will be pale pink, not blue +/- increased central pallor e. Animals may only be mildly hypoproteinemic Causes of acute or chronic blood loss: - Trauma - Parasitism - Hemostasis disorders  - Ulcers - Vascular neoplasms 4. Discuss the laboratory characteristics and possible causes of hemolytic anemia Hemolytic anemia: - Damaged or antibody coated RBCs are rapidly removed or destroyed within the circulation - PCV is decreased, TP is often normal (everything still in vasculature) - Signs of icterus, hemoglobinemia, hemoglobinuria, bilirubinemia, bilirubinuria - Intravascular and/or extravascular hemolysis can occur - Intravascular → see hemoglobinuria, hemoglobinemia, as Hgb released directly into circulation - Extravascular → see icterus, as macrophages, spleen, and liver involved Causes of hemolysis: - Immune-mediated (most common) - Toxins/chemicals - RBC parasites - Mechanical injury - Inherited RBC defects - Oxidants - Hypophosphatemia (least common) 5. Discuss the classification of anemia from reduced or ineffective erythropoiesis, and give examples of each category Anemia from reduced/ineffective erythropoiesis (nonregen) – classification Normocytic, normochromic anemias with normal or increased Anemia due to lack of erythropoietin (EPO) neutrophils and platelets Anemia of inflammation (most common) Anemia of endocrine disease and neoplasia Infectious causes of anemia – FeLV  Immune-mediated anemia – pure red cell aplasia (PRCA), precursor targeted immune-mediated anemia (PIMA) – Ab targets precursor in bone marrow Normocytic, normochromic anemias with decreased neutrophils and Aplastic anemia platelets (pancytopenia) - Infectious - Drugs/toxins - Irradiation - Idiopathic Myelophthisic anemia Microcytic, hypochromic anemias with variable neutrophils and Iron deficiency – low serum iron and ferritin platelets **microcytic → think iron deficiency** Anemia of chronic inflammation (longstanding) Portosystemic shunts Pyridoxine (B6) deficiency (nutritional) Copper deficiency (nutritional) Macrocytic, normochromic anemias with variable neutrophil and Vitamin B12 and folic acid deficiencies IN HUMANS **don’t see in vet platelet numbers med!! FeLV infection Hematopoietic neoplasia (erythroid line) 6. Define erythrocytosis (AKA polycythemia), provide possible causes and discuss laboratory tests to determine the cause Polycythemia: increase in multiple cell lines (RBC, WBC, platelets) Erythrocytosis: increase in hematocrit, hemoglobin, and RBC count - Absolute - Primary: Polycythemia vera - Secondary  - Appropriate/hypoxic: cardiopulmonary - Inappropriate: EPO-secreting tumors - Relative (more common) - Dehydration - Splenic contraction How to determine the mechanism of erythrocytosis? - Check hydration and persistence to r/o relative erythrocytosis - Check for hypoxia – assess cardiopulmonary system, look at PaO2 - EPO secreting tumor? Look at kidneys (test EPO or ultrasound) - Polycythemia vera/primary erythrocytosis is Dx of exclusion; if you can measure EPO, it will be normal or low; can also test for JAK2 gene mutations Lecture 6: Evaluation of White Blood Cells 1 1. Morphology of the white blood cells of common domestic species Segmented vs Band Neutrophils - Segmented = mature - Band = immature  2. Normal production, function, and kinetics of different white blood cells a. Where are cells produced? Leukocytes are formed in the… Liver/spleen: important for fetus Bone marrow: important for adults b. What cells do we normally see in peripheral blood? Mononuclear leukocytes: monocytes and lymphocytes Granulocytes: neutrophils, eosinophils, and basophils c. What do these cells do in the body? Neutrophils – first line of defense - Can phagocytize bacteria directly - Contain granules with microbicidal elements and enzymes - Can produce reactive oxygen species (ROS) - Can kill bacteria by trapping them in a matrix of denatured DNA and cell debris with neutrophilic extracellular entrapment (NETs) Monocytes - Like neutrophils, can phagocytize and digest/destroy foreign material - Can produce reactive oxygen species (ROS) - Major source of cytokines - Antigen-presenting cells - Involved in wound healing and bone resorption Eosinophils - Primarily involved in killing helminthic parasites and allergic reactions  - Granules contain substances that can neutralize histamine and other components Basophils - Contain large, basophilic granules which have variable morphology by species (may be moderately lavender in color) - Often seen with eosinophils and mast cells in hypersensitivity/allergic reactions - Granules contain substances important for inflammation and coagulation (heparin and histamine) Mast cells - Mast cells are NOT normally found in the blood - In tissues, often involved with hypersensitivity response but also general inflammatory reactions - Like basophils, contain substances important for inflammation and coagulation (heparin and histamine) - What does it mean when you see mast cells in the blood? - In cats: usually indicates a visceral mast cell tumor – aspirate the spleen! - In dogs: most often seen with disease OTHER than MCTs – inflam disease, neoplasia, trauma, etc.; meh in the dog Lymphocytes - Play a major role in the adaptive immune response and chronic inflammation - B cells, T cells, NK cells, and more - Life span ranges from weeks to years - Most lymphocytes in the blood of domestic animals are small in size; use neutrophils as size comparison! - Too many intermediate to large lymphocytes? → concern for neoplasia - Cattle and other ruminants can have a higher number (up to 50%) of intermediate lymphocytes T cells - Primarily involved in cell mediated immunity (but crucial for antibody mediated immunity!) - Mature in the thymus - Defined by the expression of a TCR - Many subtypes  - Most common lymphocyte B cells - Primarily involved in humoral/antibody mediated immunity - Mature in the bone marrow - Differentiate into plasma cells and produce antibodies d. What are the different “pools” of white blood cells? Proliferation and maturation/storage pools (bone marrow) Circulating pool Marginating pool Tissue pool When we take a blood sample, we are only measuring the circulating pool. The marginating pool refers to cells stuck to the sides of blood vessels. Neutrophils are located… - Proliferation and maturation/storage pools (bone marrow) - Circulating and marginating pools (blood) - Tissue pool Monocytes are located… - Produced in bone marrow; NO storage pool - Circulation pool (for a few hours) - Tissue pool (known as macrophage) Eosinophils are located… - Produced in bone marrow; NO storage pool - Circulation pool (short, minutes to hours) - Migrate to skin, respiratory tract, and GI tract primarily Basophils are located…  - Produced in bone marrow; NO storage pool - Circulate for ~6 hours before going into tissues Mast cells are located… - NOT in the blood! Shouldn’t be there - Typically reside in tissues Lymphocytes are located… - After they are produced in the bone marrow, lymphocytes go to the secondary lymphoid organs (LN, spleen, tonsils, etc.); if T cell, matures in thymus - Key point: unlike other WBCs, lymphocytes can re-circulate from blood to tissue and back to blood - In the blood, they exist in a circulating pool and marginating pool Lecture 7: Evaluation of WBCs 2 1. How to describe and interpret changes seen in the leukogram “-osis” or “-philia”: refers to an increase in absolute counts of leukocytes compared to a reference interval - Leukocytosis, lymphocytosis, monocytosis - Neutrophilia, eosinophilia, basophilia (granulocytes use -philia) “-penia”: refers to a decrease in the absolute counts of leukocytes compared to a reference interval - Leukopenia, lymphopenia, neutropenia How to evaluate changes in the leukogram: - Typically starts with a complete blood count (CBC) - Total WBC count – from instrument - Differential (% of each cells) – from instrument and blood smear review - Morphology changes – from blood smear only - If we need to know more… - Serial CBCs - Bone marrow exam Look at the absolute counts when interpreting leukograms. 3 major mechanisms that can lead to -philias or -penias: - Production and release from bone marrow - Shifts in the blood in the circulating and marginating pools - Tissue utilization and/or destruction What determines cell concentration? - Reminder: all we can see on a CBC is the circulating pool - Shifts in the blood in the circulating and marginating pools can occur Key Point: to understand a patient’s leukogram, need to interpret all leukocytes and morphology changes (in combination with history, physical exam findings, and other lab work) **KNOW: When describing and interpreting a leukogram… Step #1 = Describe: a. Leukocytosis characterized by a neutrophilia with a left shift, monocytosis, and toxic neutrophils Step #2 = Interpret: b. Consistent with an inflammatory leukogram **Understand the mechanism and consider a cause for each change when applicable. 2. Identify whether neutrophilic “shifts” are present, and how that might affect patient outcome Neutrophilia - Inflammatory neutrophilia - Epinephrine induced (“physiologic” or “excitement”) neutrophilia - Corticosteroid-induced neutrophilia - Chronic myeloid leukemia (RARE) Inflammatory Neutrophilia Mechanism Characteristics Cause Additional Info Due to release of inflammatory Can be mild to marked mature Any infectious or non-infectious Neutrophil count in inflam cytokines released during tissue neutrophilia (dep on cause of inflammation (e.g. conditions is a balance between injury production/release and tissue abscess, tumor, IMHA, etc.) production and release by the consumption) with or without a bone marrow and consumption Release of segmented left shift Remember that inflammation of neutrophils in tissues (i.e., a neutrophils from the storage pool does NOT equal infection lack of neutrophilia does NOT (within hours) and can result in a Often have toxic change (refers exclude the presence of inflam) neutrophilia if it exceeds to toxic neutrophils) usage/destruction in tissue Can be seen with a Once storage pool is depleted, lymphocytosis, monocytosis, release of bands +/- eosinophilia, basophilia, etc. metamyelocytes from maturation pool → left shift With chronic inflam (usually >7 days), there will be hyperplasia in bone marrow Epinephrine-induced (“physiologic”) Neutrophilia Mechanism Causes Characteristics Additional info Marginated neutrophils distribute Excitement and release of Typically mild neutrophilia, can be Effects of catecholamines on to circulating pool epinephrine associated with accompanied by a lymphocytosis circulatory system – increase CO coming into the clinic - Especially in young cats Corticosteroid-induced (“stress”) Neutrophilia – common! Mechanism Characteristics Causes Additional info Neutrophils shift from Typically mild, mature neutrophilia Increased corticosteroid release Stress leukogram marginating → circulating pool (but can double neutrophil count from acute or chronic stress (downregulates adhesion in dogs, horses, and cattle and (often expected in sick patients), Most sick animals have stress! molecules) triple neutrophil count in cats) increased production of glucocorticoids in Decreased tissue migration Often accompanied by hyperadrenocorticism, iatrogenic lymphopenia**, monocytosis*, Mild increased release from and eosinopenia (AKA stress storage pool leukogram/corticosteroid-induc ed leukogram) Chronic Myeloid Leukemia (RARE) Mechanism Cause Characteristics Additional info Neoplastic proliferation of Neoplastic proliferation of Marked neutrophilia (often DIAGNOSIS OF EXCLUSION well-differentiated neutrophils well-differentiated neutrophils >50,000/uL), often have a left shift, often have increases in Do bone marrow to confirm other leukocytes Leukemoid Response vs Chronic Myeloid Leukemia Leukemoid Response Chronic Myeloid Leukemia MORE COMMON in veterinary medicine Similar features to leukemoid response Marked neutrophilia (>50,000/uL), often with a concurrent “ordered” RARE left shift Diagnosis of exclusion only! Can be due to infectious or noninfectious causes of severe inflammation - Pyometra, pyothorax, etc. - IMHA - Can be seen with neoplasia (either from inflam or paraneoplastic) - Necrosis Neutropenia - Inflammatory neutropenia - Endotoxin-associated neutropenia  - Peripheral destruction neutropenia - Granulocytic hypoplasia (of bone marrow) Inflammatory Neutropenia Mechanism Cause Characteristics Additional info Excessive tissue demand for Infectious or noninfectious causes Patients are often very sick! Indicates poor prognosis neutrophils and depletion of of inflammation - Often see left shift and storage pools toxic changes in - Can be seen with acute neutrophils bacterial/viral infections - Common in sick adult (often overwhelming) cattle as they have a relatively small storage pool of neutrophils Endotoxin Neutropenia Mechanism Cause Characteristics Endotoxins (from gram-negative bacteria) Endotoxemia Transient and may not be observed clinically; cause a rapid shift from the circulating often followed by a neutrophilia neutrophil pool to the marginal neutrophil pool - Lasts about 1-3 hours after initial exposure - Mild depletion of storage pools Peripheral Destruction Neutropenia Mechanism Cause Characteristics Additional info Antibodies form against Antibodies form against Respond to immunosuppressive Consider bone marrow to solidify neutrophils → destroyed by the neutrophils → destroyed by the dose of glucocorticoids diagnosis mononuclear phagocytic system mononuclear phagocytic system Can be very severe Peripheral = in the blood Often show granulocytic  hyperplasia in the bone marrow (trying to produce more) Granulocytic Hypoplasia Mechanism Causes Characteristics Additional info Can occur when stem cells Drugs (chemotherapy, estrogen, Neutropenia + evidence of Confirm with bone marrow exam (neutrophil precursors) become other), infectious agents granulocytic hypoplasia on bone damaged → results in hypoplasia (Parvovirus, chronic Ehrlichiosis, marrow exam → decreased production of FeLV), or myelophthisic effect neutrophils (neoplasia effaces bone marrow) May see decreases in other cell lines (RBCs and platelets) If associated with chemotherapy, the nadir typically occurs 7-10 days after dosing 3. Describe WBC changes seen in some common leukocyte profiles Abnormal neutrophil morphology - Toxic change - Associated with inflammation - Hyposegmentation (decreased segmentation of nucleus) - Pelger-Huet anomaly in Australian Shepherds: defect in proteins that aid segmentation - Hypersegmentation - Indicates increased lifespan - Seen with corticosteroids, dysplasia, or cobalamin/folate deficiency - Intracytoplasmic granules - Miscellaneous inclusions (ex: viral inclusions) - Associated with infection; Distemper viral inclusions, Anaplasma or Ehrlichia spp., Mycobacterium spp., Histoplasma capsulatum, rod and cocci bacteria, protozoa  4. Describe the appearance of toxic neutrophils and understand the mechanism Toxic neutrophils - Accelerated granulopoiesis associated with inflammation - Often accompany left shift (have same mechanism as band neutrophils) - Characteristics: - Dohle bodies** – retained endoplasmic reticulum - Cytoplasmic basophilia** – retained ribosomes and rough endoplasmic reticulum - Cytoplasmic vacuolation – indistinct foamy appearance → degranulated lysosomes - Toxic granulation – retained primary granules  5. Describe and recognize additional changes that can be present in leukocytes on a blood smear Lymphocytosis - Epinephrine induced lymphocytosis (COMMON) - Antigenic stimulation (inflammatory) lymphocytosis - Lymphoproliferative disease (AKA – neoplasia) - Other - Hypoadrenocorticism (lack cortisol) Epinephrine Induced Lymphocytosis Mechanism Cause Characteristics Epinephrine induces left shift of marginating Epinephrine release secondary to excitement Seen in young healthy animals most often lymphocytes to circulating pool of coming into clinics (cats!) Often accompanied by mature neutrophilia, no May be some release of lymphocytes from the left shift spleen or lymph node Usually not more than 10,000/uL but can be higher Usually short-lasting  Antigenic Stimulation (Inflammatory) Lymphocytosis Mechanism Causes Characteristics Increased lymphopoiesis (production of Can be seen with chronic ehrlichiosis, chronic May see reactive lymphocytes lymphs) in response to chronic antigenic inflammation Usually mild/moderate lymphocytosis stimulation (can be infectious or May see mature neutrophilia, +/- left shift, +/- noninfectious) monocytosis, +/- eosinophilia, +/- basophilia Neoplastic Lymphocytosis Mechanism Characteristics Proliferation of neoplastic lymphocytes Lymphocytosis is variable and can range from mild to severe Acute lymphoid leukemia (ALL), chronic lymphocytic leukemia (CLL), or lymphoma at the level of the bone marrow (stage V lymphoma) More in the hematopoietic neoplasia and lymphoma sections Other causes of lymphocytosis: - Hypoadrenocorticism (Addison’s / adrenal insufficiency) - Typically mild lymphocytosis - Can be accompanied by an eosinophilia - Pay attention to the absence of a stress/glucocorticoid leukogram in a sick animal!! **subtle cue!! Lymphopenia 1. Corticosteroid induced lymphopenia (COMMON) Less commonly: - Acute inflammatory lymphopenia (acute systemic inflammation) - Depletion lymphopenia (lymphangiectasia, thoracic duct trauma) - Lymphoid hypoplasia/aplasia (hereditary - SCID foals)  Corticosteroid Induced Lymphopenia Mechanism Causes Characteristics Immediate shift of lymphocytes from Increased corticosteroid release from acute or Hallmark of a “stress circulating pools to other pools chronic stress (often expected in sick leukogram/corticosteroid” (COMMON, the Lymphocytes get “stuck” in tissues patients), increased production of most consistent change) Glucocorticoids can cause lysis of glucocorticoids in hyperadrenocorticism, Can be seen with a concurrent neutrophilia, lymphocytes (lympholysis) iatrogenic (Pred, Dex) +/- monocytosis, +/- eosinophilia Changes in Monocyte Concentration Monocytosis - Glucocorticoid induced (common in dogs and cats) - Acute and chronic inflammation - Increased tissue demand for macs - Often seen with neutrophilia Monocytopenia - Not significant Changes in Eosinophil Concentrations Eosinophilia - Seen with parasites or Type I hypersensitivity - However, many dogs with eosinophilia do NOT have parasites - Inflammatory disorders of mast cell rich organs - Can be seen with hypoadrenocorticism - Can be paraneoplastic (IL-5 producing tumors can draw eosinophils to them) - Hypereosinophilic syndromes Eosinopenia - Occurs in response to glucocorticoids - Often not clinically significant, esp if other WBC counts are within range Changes in Basophil Concentrations Basophilia - UNCOMMON - Usually accompanies an eosinophilia - Can be associated with allergies, parasites, or neoplastic conditions Basopenia - Not clinically significant Lecture 8: Hematopoietic Neoplasia 1. Define the term leukemia Leukemia: presence of neoplastic hematopoietic cells are seen in the peripheral blood - Can be the result of lymphoid or myeloid neoplasia - Malignant disease of hematopoietic tissue characterized by replacement of bone marrow with an abnormal clone of proliferating blood cells (primarily bone marrow origin) 2. Describe basic types of leukemia Lymphoid or myeloid neoplasia 3. Discuss types of lymphoid neoplasia Lymphoma Acute lymphocytic leukemia Chronic lymphocytic leukemia Large granular lymphoma Plasma cell myeloma Primary macroglobulinemia Plasmacytoma  4. Describe the classification schemes used for lymphoma Anatomic classification - Multicentric - Thymic/mediastinal - Alimentary - Other (skin, kidney, etc.) Histologic classification - Architecture - Mitotic rate - Cellular morphology Cytologic classification - Small lymphocytes - Intermediate lymphocytes - Large lymphocytes - Large granular lymphocytes Immunophenotyping - Monoclonal antibodies - Flow cytometry Clonality - Lymphoid neoplasms arise from the clonal expansion of a single cell (monoclonal) 5. Describe the clinical laboratory features and tests available for diagnosis of lymphoma in the dog, cat, cow, and horse Laboratory features of canine lymphoma: - Mild non-regenerative anemia (anemia of inflam from neoplasm and other effects) - Variable WBC, lymphocyte, platelet counts - Possible leukemic blood profile - Hypercalcemia due to PTH-related proteins in some dogs - Poor prognostic indicator - More common in T-cell lymphoma but can happen in either - High yield problem!  - Hypercalcemia of malignancy 6. Describe the main types of plasma cell neoplasia Multiple myeloma: neoplasm of plasma cells where one type of immunoglobulin or immunoglobulin subunit (light chain) is secreted - Dx based on having at least 2 of the following features: - Radiographic evidence of osteolytic lesions - Increased numbers of plasma cells in bone marrow - Monoclonal gammopathy - Bence Jones proteinuria (light chains in urine) - Paraneoplastic syndromes associated: - CRAB signs - Calcium (increased) - Renal (dysfunction) - Anemia - Bone lesions Macroglobulinemia: AKA Waldenstrom’s macroglobulinemia / primary macroglobulinemia; neoplasia of lymphocytes which produce IgM (IgM has more binding sites than others, leads to hyperviscosity syndrome) - Rare! - Can cause a hyperviscosity syndrome - Mucous membrane bleeding - Retinopathy Plasmacytoma: focal tumors of bone or soft tissue; often as skin tumors - Vary in behavior from benign to aggressive (no way to tell histologically) - Rarely monoclonal gammopathies can occur 7. Describe the different types of myeloid neoplasia Myeloid neoplasia: neoplasia of non lymphoid hematopoietic cells - Includes acute disorders, chronic disorders, and myelodysplasia (some dysfunction in bone marrow)  - Leukemic blood profile with circulating neoplastic cells usually present - Less common than lymphoid neoplasia - Classify with evaluation of blood and bone marrow smears, cytomorphology, cytochemistry and immunophenotyping Acute myeloid leukemias Chronic myeloid leukemias Polycythemia vera (neoplasia of erythroid cells) Dysmyelopoiesis, myelodysplasia, myelodysplastic syndrome Characterized by severe nonregenerative anemia +/- other cytopenias due to ineffective hematopoiesis Evidence of abnormal cells in bone marrow Several types in dogs Lecture 9: Hemostasis *** Disease BMBT Platelet Count APTT OSPT FDP ITP Increased Decreased N N N (immune-mediated thrombocytopenia) Thrombopathy Increased Normal Normal Normal Normal Hemophilia A Normal Normal Increased Normal Normal Hemophilia B Normal Normal Increased Normal Normal FVII deficiency Normal Normal Normal Increased Normal Vit K antagonist Normal or increased Normal or decreased Increased Increased Normal or increased DIC Increased Decreased Increased Inreaces Increased vWD Increased Normal Normal or increased Normal Normal  1. List the key players in the hemostasis process and briefly describe their functions Platelets - Primary hemostasis is a result of platelet and vessel functions - Platelets are produced in the bone marrow by megakaryocytes - In dogs, platelets circulate for ~6 days - Mean platelet volume (MPV) = average vol of a platelet - Increased MPV = active/accelerated thrombopoiesis - Cavalier King Charles Spaniels have high MPV/large platelets normally - Platelets circulate in an inactive state - Damage to the endothelium → platelets adhere to subendothelial structures via platelet receptors - Platelet activation - Adhere to subendothelial vWF via platelet glycoprotein receptors  - Adherence and thrombin promote platelet activation and cause secretion of granule contents, formation of thromboxane - Leads to aggregation of platelets - Platelet aggregation occurs, producing a platelet plug (final step in primary hemostasis) - Thrombin generation occurs on the platelet surface - Platelet plug stabilized by a fibrin mesh - Contraction of platelet filaments helps to bring the edges of the wound together - Secondary hemostasis: platelet plug is stabilized by fibrin; occurs through action of coagulation factors Coagulation factors - Produced in the liver - Many factors circulate in blood as zymogens (inactive enzymes) → need to be activated - Identified by Roman numerals; “a” after indicates activated coagulation factor Fibrinolytic factors - Lysis of fibrin, breakdown of clots Endothelium/vessels - Normally nonthrombogenic - Vasoconstrict when stimulated (slows blood flow) - Endothelial damage → collagen exposure → primary hemostasis - Store / release vWF, which is necessary for platelet activation Inhibitors - Inhibit coagulation - Antithrombin (AT) - Thrombin activatable fibrinolysis inhibitor (TFPI) - Alpha2 antiplasmin - Plasminogen activator inhibitor (PAI) 2. Describe the correct procedure and anticoagulant for collecting a blood sample for platelet or coagulation assays EDTA for platelet counts Na Citrate for most coagulation tests **Never heparin!! Blood collection: - Minimal trauma - Correct anticoagulant to blood ratio (can affect cell and platelet count – be sure to add right amount of blood) - Plastic or siliconized containers Separate plasma within 30 minutes of collection Keep samples at 4C Test within 3 hours Plasma samples may be frozen in small aliquots for submission to outside labs 3. Describe the key steps used in the diagnosis of a bleeding disorder Patient Presentation - Evidence of bleeding? - Physical evidence - Pale mucous membranes - Blood in body cavities - Hematomas - Pinpoint hemorrhages (petechiae) - Bruises - Laboratory evidence - Decreased PCV - Decreased protein - Is the bleeding appropriate? - Can the bleeding be explained? - Is bleeding occurring from multiple sites?  - Is the bleeding proportionate to the injury? History - Previous bleeding - Age at first inappropriate bleeding episode - Drug history - Previous or present illness - Vaccination history - Travel history Physical Exam **Note the type of hemorrhage, and the site of hemorrhage** - Muscle, joint, body cavity, hematoma - Suggests coagulation factor deficiencies - Secondary hemostasis - Petechiae - Suggests platelet or vessel defects - Primary hemostasis - Ecchymosis - Primary and/or secondary hemostasis defect Putting it together: - History - Inherited or acquired - Acute or chronic - Physical exam - Defect in primary or secondary hemostasis - Laboratory tests - Defect of primary or secondary hemostasis - Evidence of fibrinolysis  4. Describe appropriate tests for the assessment of platelet numbers and function and discuss the possible causes of abnormal test results Platelet evaluation - Platelet counts - Bleeding time - Platelet antibodies - Clot retraction - Platelet adhesion - Platelet aggregation Platelet count - Smear estimate - Should see 8-10 platelets per 100X field in monolayer - Automated count - Interpretation: - Thrombocytopenia: