Clinical Pathology Kristin Canga, RVT Anemia Clinical Pathology Kristin Canga, RVT

Reading Assignment Page 68 – Lab Pro book ‘Clinical Application’ box (Iron Deficiency Anemia) on pg. 12 of A&P book Pages 55 – 57 Lab Pro book (about counting reticulocytes)

Anemia Literally means “no blood” but clinically means an ______________ ____________________below normal in any of the following values: ________________________________________ In other words, anemia is a condition of reduced oxygen carrying capacity of RBCs Rate of RBC ______________________ = decreased Rate of RBC ______________________ = increased

Classification of Anemia Classification is to aid in discovering the _______________ and to help guide __________________. Remember: Anemia is not a __________________, but a sign of an underlying health concern. Anemia may be considered _____________ or __________ and is generally classified in one of two different ways: Classification by RBC ________ and ____ concentration a. RBC ____________________ (MCV, MCHC) Classification according to bone marrow response ________________________________

Aiding in Classification of Anemia A thorough ___________ must be obtained. This helps the doctor know: what the patient has been ____________ /____________, where they have been, how long they have been suffering, and possibly _________ the anemia has occurred. A physical exam should be completed. Put your ____________ on the animal! Look for _____________, _____________, _____________, active bleeding, elevated heart/respiratory rates, etc… A complete _____________evaluation is a MUST. HBC, toxins, iron deficiency, post-operative, parasites, etc…. Lab tests:

PATIENT HISTORY 1. __________________________of clinical signs ______________ onset suggests acute _________________ or ______________ ______________ onset suggests chronic ______________ or bone marrow depression 2. Evidence of blood loss ______________ Blood in ______________ Melena = blood in stool Hemoptysis = blood originating in lungs being expelled through nose, or coughing it up.

PATIENT HISTORY 3. ____________________________ 4. Existence of an underlying condition or prior illness ____________________________ 5. Exposure to drugs - human ______________ , ______________ 6. Exposure to toxic ______________ in the ______________ - ______________ , poisonous _________, ______________

PHYSICAL EXAMINTION ______________ Character of ___________________: Suspect: infection, leukemia, hemorrhage, or hemolysis Character of ___________________: ______________ – liver disease or hemolysis ______________ + ______________ = hemolysis ______________ - hypoxia ______________ or ______________ = platelet or vascular defect Ecchymoses = larger than petechia.

Petechia

Ecchymosis

PHYSICAL EXAMINATION 3. Palpation ______________ 4. ______________ signs of underlying disease 5. External wounds

LABORATORY EVALUATION Initial laboratory tests to evaluate the anemic patient include (but not limited to): ______________ (and color of supernatent plasma) Total ______________ protein Examination of ______________ and ____________________________ Total ______________ count ______________ estimation ______________ concentration Total ______________ count ______________ ** ______________ evaluation ** ** are the two ways to classify anemia.

PCV: Test yourself  What is it measuring? Normal ranges for dogs? Normal ranges for cats Plasma (supernatent) colors?

Plasma Protein What is it measuring? How is it measured? What is normal range for dogs and cats?

How Many Cells should you have? As a rule, the following values should be considered: RBC total numbers should be in the ______________ . (106/μL) Plt total numbers should be in the ______________ of ______________ . (200,000 – 500,000/μL) WBC total numbers should be in the ______________ to ____________________________ . (6,000 – 17,000/μL) Neutrophils: 60 – 77% Lymphocytes: 12 – 30% Monocytes: 3 – 10% Eosinophils: 2 – 10% Basophils: rare (<2%) In dogs and cats

Blood Film Evaluation and WBC Differential What area are you evaluating? How are cells arranged? Are RBCs normal? How many WBCs are counted? How many fields are counted for plt. estimation? What is calculation for plt. estimation? CAN look at entire slide on 10x and 40x to see how cells are appear. Following that, move to 100x and monolayer

Total WBC Count Overall count should be in ______________ to ____________________________ . (6,000-17,000) Total count calculated by machine Manual hemacytometer is rare in clinic and diluent is no longer available. Increased WBCs = ______________ Decreased WBCs = ______________ Diluent would lyse the RBCs and allow only WBCs to remain.

Hemoglobin Calculation Done by machine. Aids us in calculating average ______________ of RBCs (_______) Aids us in calculating average ______ concentration within RBCs (_______) Can aid in calculating average ______________ of Hb within average RBC. (_______) *** MCH is LEAST accurate*** MCH is least accurate because it uses Hb and RBC count instead of PCV.

Classifying Anemia by RBC indices MCV: ____________________________ MCHC: ____________________________ MCH: ____________________________

Rules of Thumb (ROTs): Hb concentration is ~_______ of PCV (in g/dL) Total RBCs are ~_______ of PCV (in millions)

Classification by RBC Indicies Recall that MCV (mean corpuscular volume) describes the average volume of the individual RBC Normal MCV = _____________________ Increased MCV = _____________________ Decreased MCV = _____________________ FORMULA: (PCV / Total RBC) X 10 = MCV (femtoliters) Normal MCV = canine: 60 – 77 fl. feline: 40 – 55 fl.

Let’s do the math: The MCV of a patient with a PCV of 12% is: Step 1: Recall the formula: (_______/ ______________) X 10 = MCV (femtoliters) Step 2: Remember the ROT total RBC ≅ _______ PCV so: ______________ = ______________ Step 3: plug in the numbers ___________________________________ Is this normal for k9/fel? How would you classify this RBC? Normal for k9, not for feline. Canine = normocytic, Feline = Macrocytic

Possible Causes of Abnormal MCV Possible causes of Increased MCV: Increased _____________________activity = #1 Reticulocytosis Congenital (___________&_________________) Cats with _______ (+/- anemia) Possible causes of Decreased MCV: ______________ deficiency = #1 Congenital disorder (_______and ______________) Polycythemia can also cause

Classification by RBC Indicies MCHC (mean corpuscular hemoglobin concentration): Describes the ratio of the _______of hemoglobin to the ______________in which it is contained (concentration of hemoglobin in the avg. RBC) Normal MCHC = ______________ Decreased MCHC = ______________ High MCHCs = artifact WHY???

Formula (______ / ______) X ______= MCHC (g/dL) Normal MCHC = canine: ______________g/dL feline: ______________g/dL Remember the ROTs? If you calculate MCHC by estimating Hb, the values will always come out the same. Lets do the math!

Using the ROT The MCHC of a patient with a PCV of 33% is: Step 1: Recall the formula (_______ / _______) X _______= MCHC (g/dL) Step 2: Remember the ROT Hb ≅ _______ of PCV so: _______= _______ Step 3: Plug in the numbers __________________________________________

Using actual numbers The patient’s Hb is 9g/dL, and their PCV is 30% Formula: (_______/_______) x _______ SO: _______________________=_____g/dL Is this normal for k9? Fel? How would it classify the RBC? Not normal for either, both would be Hypochromic

Low MCHC usually results from: Severe _______deficiency Marked, regenerative anemia ____________________________RBCs that do not yet have their full complement of Hb. MCHC increase: Presence of ______________, ______________, and ___________ can interfere with tests and ______________increase MCHC True _____________________anemia cannot exist; the erythrocyte cannot be oversaturated with ______.

Morphologic Classification of Anemia by RBC Indicies MCHC normal MCHC decreased MCV normal Normocytic Normochromic Hypochromic MCV increased Macrocytic MCV decreased Microcytic Hyprochromic

Normocytic ; Normochromic Macrocytic Microcytic Hyperchromic Hypochromic

Calculating MCH You will need to know HOW to do this for VTNE, even though it is the _______accurate of the indices. Calculates the average _______of Hb contained in average RBC. (_______/_______) x _______= MCH in picograms (pg) Normal ranges: K9: _______pg Fel: _______pg

Let’s do the Math The MCH of a patient with a PCV of 54% is: Step 1: Remember the formula (_______/_______) x _______= MCH Step 2: Remember the ROT Hb ≅ _______ PCV and RBCs ≅_______ PCV _______ = _______ and _______ = _______ Step 3 Plug in the numbers: _____________________pg

Classification of Anemia According to Bone Marrow Response Most pertinent of anemia classification Distinguishes between _____________________and _____________________ anemia

Classification Based on Bone Marrow Response Regenerative anemia Characterized by evidence of increased ______________ and delivery of new erythrocytes into ______________ (usually within 2-4 days). Usually suggests bone marrow is responding appropriately to either: _____________________ (acute or chronic; internal or external) or _____________________ (intravascular or extravascular) Involves determining whether absolute _____________________ numbers are increased in the blood.

Classification According to Bone Marrow Response Nonregenerative anemia Lack of circulating ______________ RBCs in the face of _______ indicates a nonregenerative anemia and likely results from bone marrow ______________. Either reduced erythropoiesis or defective erythropoiesis No response evident in ______________blood. (usually ______________; ______________) _____________________examination may be helpful with the diagnosis.

Regenerative Anemia Blood Loss Anemia Acute _____________________– relatively large amount of blood lost in a brief period. (______________; ______________) PCV initially = ______________ Reticulocytes should appear ~_______ hrs (peak within ~ 1 week) Causes: ______________ Internal or external Accidental or surgical ______________disorders Bleeding ______________ or large ______________ Normal PCV initially because fluid and cells lost in proportion. Takes time for spleen to release immature cells.

Regenerative Anemia Chronic blood loss (_______Deficiency Anemia) – lost ______________and ______________for a period of time. a. Parasites ______________, _______, blood-sucking _______, coccidia spp. b. GI ulcers and neoplasms c. Inflammatory bowel disease d. Overuse of ______________donors Note: neonates can become iron deficient due to lack of adequate dietary _______ intake.

Iron Deficiency Anemia Body compensates for anemia by lowering _______-_______ affinity, preferential shunting of blood to vital _______, increased ______________output (tachycardia), and increased levels of _____________________. With decreasing _______ stores, erythropoiesis is limited and RBC’s become ______________and deficient in _______ (______________and _____________________). Hallmark of iron deficiency anemia is decreased _______. Keratocytes & schistocytes Clinical signs include: lethargy, weakness, decrease exercise tolerance, anorexia, lack of grooming, mild systolic murmur.

Regenerative Anemia a. Immune mediated ______________________ 2. _____________________– Increased rate of erythrocyte ______________________within the body a. Immune mediated ______________________ Incompatable ______________________ b. Blood parasites Hemotrophic Mycoplasmas ______________________spp.

Cytauxzoon felis inclusions Look like a ring inside the cell.

Regenerative Anemia c. Heinz body anemia Plants Drugs or Chemicals Onions*, garlic ______________________ Drugs or Chemicals (______________________, Propylene glycol, Zinc, Copper, Methylene blue, Naphthalene, ______________________, phenothiazine, benzocaine Diseases (in cats) Diabetes mellitus Hyperthyroidism Lymphoma Propylene glycol and Ethelene glycol are both ingredients in antifreeze. Propylene = green, Ethelene = blue

Regenerative Anemia d. ______________________ induced hemolysis RBC glycolysis is inhibited by hypophosphatemia; no glycolysis = no ATP (energy) for RBC = cell lysis Diabetic cats Enteral alimentation Phosphate = required for bone density. Also binds with ATP to create energy for the cell.

Regenerative Anemia e. Other Microorganisms ______________________ Clostridium spp. and Leptospirosis (cattle) f. ______________________intoxication (usually calves) can also occur as a result of inappropriate administration of ______________________ therapy. g. ______________________ RBC defects ______________________ (shortened RBC lifespan) RBC membrane transport defects Chronic intermittent hemolytic anemia (Abyssinian and Somali cats) H2O intoxication may occur in claves given ad lib access to H20 for the first time. Excessive intake without appropriate renal response causes intravascular hemolysis due to dilution of the blood. C/s on set within 1 hour and can include: neuro, resp distress, bloat, hemoglobinuria, and death.

Regenerative Anemia h. Miscellaneous Metabolic disorders (anything that interferes with synthesis of ______________________, RBC, etc. or anything that interferes with ______________________processes of RBC)

Nonregenerative Anemia Most nonregenerative anemias are ______________________ Further subclassified based on whether ______________________ (neutrophil production) and ______________________ (platelet production) are also affected. Animals with nonregenerative anemia in conjunction with ______________________ (neutropenia and thrombocytopenia) usually have ____________ cell injury. Possible causes: drugs, toxins, viruses (FeLV), radiation, and immune-mediated stem cell injury.

Nonregenerative Anemia Reduced ______________________ Chronic ______________________ disease Endocrine deficiencies Inflammation and neoplasia Cytotoxic damage to the ______________________ Estrogen toxicity Cytotoxic cancer drug therapy Chlormphenicol (cats) Radiation Other drugs

Nonregenerative Anemia e. Infectious agents FeLV ______________________ spp. ______________________ f. Immune-mediated Continued treatment with recombinant erythropoietin ______________________ aplastic anemia g. Congenital/inherited h. ______________________ and other ______________________ disorders

Nonregenerative Anemia 2. Defective ______________________ Disorders of ___________ synthesis Iron, copper, and pyridoxine deficiencies; lead toxicity; drugs Folate and ____________ deficiencies Abnormal ______________________ can be inherited, drug-induced or idiopathic Pyridoxine = Vit B6 Cobalimin = Vit B12

Reticulocyte Count Probably the most important diagnostic tool used in the evaluation of anemia. Fewer _____________ erythrocytes are present in anemic animal; more ______________________are present. Expressed as a _____ of the RBCs present. The lifespan of a normal RBC is about 110 days (dogs) and 68 days (cats). Bone marrow should replace ___ % of the RBCs daily so the reticulocyte count should be _____-______%.

Reticulocyte Count Gently mix 4 drops of blood with 4 drops of new methylene blue in a test tube. Let mixture stand for 15 minutes Use 1 drop of mixture to prepare a diagnostic blood film and observe under high-power, oil-immersion field. Count 1,000 RBCs while separately keeping track of the number of reticulocytes (only aggregate form) Divide the reticulocyte number by 1,000 and convert to a percentage. (Multiply by 100) We will be using two blood smears and counting 500 cells on each slide to tally the 1000 cells.

Example Reticulocyte count If you see 10 retics on slide 1 and 15 on slide 2, your total is 25 reticulocytes. 25/1000 = 0.025 x 100 = 2.5%

Corrected Reticulocyte Count Performed to take in account the reduced number of circulating RBC’s in the anemic animal. Called CRC or Corrected Reticulocyte Count FORMULA: Observed retics % x PCV / normal PCV (Normal PCV: use 45% for dogs and 35% for cats) Ex: 2.5% X 30% / 45% = 1.67% Note: This calculation is necessary because the reticulocyte count is misleading in anemic patients. The problem occurs because the reticulocyte count is not really a count but rather a percentage: it reports the number of reticulocytes as a percentage of the number of red blood cells. In anemia, the patient's red blood cells are reduced, creating an elevated reticulocyte count.