VM605 Clinical Pathology

Understand and explain the diagnostic tests used to evaluate the different branches of the coagulation cascade and the fibrinolytic pathway

PT (prothrombin time): tissue factor and calcium added as reagents, evaluates extrinsic and common pathways, limited sample stability and unsuitable for shipping

PTT (partial thromboplastin time): negatively charged glass beads added to stimulate surface of activated platelets, evaluates intrinsic and common pathways, limited sample stability, unsuitable for shipping

ACT (activated clotting time): in-clinic test to evaluate intrinsic and common pathways, need specialized tubes/cartridges, more sources of variability can affect results, does not evaluate extrinsic pathway

Fibrinogen:

Method 1: Heat Precipitation: two PCV tubes w/ EDTA blood, one tube gets placed in a water bath for 3 minutes, measure TP by refractometry; most commonly used in equine/large animal practice as marker for inflammation, insensitive to decreases in fibrinogen - less useful for evaluating coagulopathies

Method 2: Clauss Method: citrated sample needed, thrombin added as reagent, method of coice for detecting hypofibrinogenemia

FDPs (fibrin degradation products): latex beads coated with antibodies against FDPs are incubated with patient serum, used to support dx of excessive fibrinolysis (DIC and severe liver disease), cannot be interpreted alone, specialized test kits needed

D-Dimers: one specific type of FDP, direct measurement more specific for fibrinolysis than FDPs, performed only by a few specialized laboratories, validated assays for veterinary species are limited

Viscoelastic tests (TEG/ROTEM/VCMVet): global assessment of hemostasis, most commonly used to identify hypercoagulable patients, limited diagnostic utility for hypcoagulable states, high preanalytical variability

Specific factor tests: indicated when marketly prolonged PT or PTT is not explained by liver failure or rodenticide toxicosis

requires specific protocols: sodium citrate tube, fill exactly to line indicated on tube, clean draw, immediate gentle mixing

Be able to interpret secondary hemostasis results

Prolonged PT, Normal PTT: hereditary factor 7 deficiency, early liver failure or rodenticide toxicosis (factor 7 has shortest half-life of all factors

Normal PT, Prolonged PTT: hereditary factor deficiencies (intrinsic pathway factors only, 12, 11, 9, 8)

Prolonged PT, Prolonged PTT: rodenticide toxicosis (most classic pattern; Vitamin K antagonism, 2, 7, 9, 10), liver failure (all coagulation factors synthesized in liver), decreased fibrinogen, heparin treatment, DIC (also low PLT, high FDPs), hereditary factor deficiencies (common pathway factors - 10, 5, 2, 1)

Hypofibrinogenemia: liver failure, DIC (most common), congenital fibrinogen deficiency (rare)

Hyperfibrinogenemia: inflammation (especially in horses/cattle)

FDPs <5 microliter/mL is considered normal; above this value is considered excessive fibrinolysis

VCM = coagulability of patient

Recognize the differences in clinical signs between primary and secondary hemostatic disorders

severity usually worse with secondary dz; can overlap with primary dz

secondary: large accumulations of blood (hematoma, bleeding into cavities, hemarthrosis, epistaxis, melena, rebleeding from wounds, hematuria)

Understand and explain the specific disorders discussed in lecture including what abnormalities would be noted in coagulation testing

Hereditary disorders:

Hemophilia A and B

Hemophilia A: Factor 8 deficiency, most common inherited coagulopathy, German shepherd dogs, X-linked - typically only male patients affected; clinical signs depend on factor activity (<30% factor activity needed to detect on PTT, carriers have 40-60% factor activity, abnormal or spontaneous bleeding typically does not occur until 2-5% factor activity)

Hemophilia B: Factor 9 deficiency, X-linked - typically only male patients affected, clinical signs depend on factor activity (similar to H.A., carriers have 40-60% factor activity, abnormal or spontaneous bleeding typically does not occur until factor activity is very low, specific cutoffs not as well-defined)

Other factor deficiencies are less common

Acquired disorders:

Vitamin K deficiency or antagonism: anticoagulant rodenticide toxicosis, liver disease (infrequent), decreased absorption/production (rare)

Liver failure: most coagulation factors are produced in th elier, severe liver disease can result in a coagulopathy

Disseminated intravascular coagulation (DIC): inciting stimulus (severe inflammatory/neoplastic/traumatic disease (burns/vehicular trauma))

1. triggers widespread endothelial injury

2. systemic release and overexpression of tissue factor (Factor 3)

3. tissue factor systemically activates platelets and initiates the extrinsic coagulation cascade (result is widespread clot formation and consumption of platelets and coagulation factors)

4. healthy endothelium reacts by initiating fibrinolysis (result is massive clot breakdown and generation of FDPs)

5. net result is initially hypercoagulable state with microvascular thrombosis followed by widespread hypocoagulability affecting both primary and secondary hemostasis

Grave prognosis

Decreased PLT, Increased PT/PTT, Decreased fibrinogen, Increased FDPs; need three or more findings to support DIC dx

Understand how to evaluate platelets quantitatively and qualitatively using laboratory tests

platelet count (PLT): measured as platelets/microliter of blood

decreased platelet mass = thrombocytopenia; <20,000-30,000 can result in spontaneous hemorrhage; less severe magnitudes may result in prolonged hemorrhage due to trauma or surgery

increased platelet mass = thrombocytosis; >1,000,000 is theoretically associated with thrombotic disease in dogs/cats, hypercoagulable patient

measured on automated CBC via impedance or flow cytometry, common sources of error are platelet clumping (in all species, especially cats) and limited size variation between platelets and RBCs (in cats). both can lead to underestimation of platelet count (or pseudothrombocytopenia)

can be estimated on blood smear evaluation

confirm an automated count with manual estimate when:

if analyzer reports thrombocytopenia, especially if patient has no clinical signs

if your patient HAS clinical signs of primary hemostatic disease but analyzer reports normal platelet count

treating or monitoring a patient for primary hemostatic disease

always trust your eyes over your analyzer

species variation exists! mice have 2x what dogs/cats do, horses are lower than dogs/cats; evaluate platelet count using species-specific reference interval

mean platelet volume (MPV): measured in fL, only reliable if automated platelet count is accurate

plateletcrit (PCT): = ([PLT]*[MPV])/1,000, when available takes both number and size into account for best approximation of platelet mass

all require anticoagulated blood, typically EDTA

Be able to interpret primary hemostasis test results and use them to formulate or refine a differential diagnosis list

look at test results - high or low --> what would cause that --> DDX list

Understand and explain the different platelet disorders discussed including possible pathogenesis

Thrombocytopenia:

1. decreased production: drug-associated (chemo, estrogen compounds, phenobarbital, chloramphenicol, sulfa antibiotics), toxins (aflatoxin B, bracken ferm), infectous (FeLV, parvovirus, late-stage rickettsial disease (ehrlichiosis), EIA, BVD), primary bone marrow disease (neoplasia, myelofibrosis)

2. sequestration: entrapment of platelets within congested organs or neoplasms, uncommon and typically mild

3. increased destruction: immune-mediated thrombocytopenia (primary or secondary to infectious/neoplastic/other dz), rickettsial disease, typically results in marked thrombocytopenia

4. increased consumption/utilization: hemorrhage or trauma (typically mild, not commonly recognized), disseminated intravascular coagulation (DIC) (may be severe)

Thrombocytosis:

1. splenic contraction or post-splenectomy, in response to adrenaline; horses are very common

2. reactive thrombocytosis: response to cytokine release to stimulate thrombopoietin, neoplasia/chronic inflammatory disease/iron deficiency anemia/others, most common cause of thrombocytosis (dogs/cats are typically between ~700,000-1,000,000)

3. neoplasia: essential thrombocytosis - rare chronic myeloid leukemia affecting megakaryocytes, causes high platelet counts that are persistent and progressive over time without known cause for reactive thrombocytosis, may increase risk for thrombotic disease

Disorders of Platelet Volume:

1. congenital macrothrombocytopenia of CKCS: genetic defect in beta1-tubulin which is heavily involved in platelet shedding from megakaryocytes, platelets are lower in number but larger in size (double the normal range), plateletcrit is typically normal, not associated with abnormal clinical bleeding and does not require clinical intervention, is very common - 30-50% of CKCS dogs in US, genetic test available

Disorders of Platelet Function:

Thrombopathy: typically suspected in patients with clinical signs of primary hemostatic disorders when platelet count is adequate

Congenital causes: von Willebrand disease, uncommon genetic diseases (breed-specific) (Bernard-Soulier syndrome, Glanzmann's thrombasthenia, Basset Hound/Spitz thrombopathia, Kindlin-3/LAD III in German shepherds)

von Willebrand Disease: Type 1 (most common), all vWF multimers are present but at decreased concentrations, varying severity, very common in Doberman pinschers; Type 2 is a deficiency of vWF with disproportionate decrease in large multimers, severe but uncommon, German shorthaired pointers; Type 3 is an absence of all vWF multimers, is often fatal but is rare, Chesapeake Bay retrievers, Scottish terriers, Shelties

Acquired causes: Aspirin/other NSAIDS (due to inhibited thromboxane A2 production), uremia (chronic kidney failure, build-up of uremic acids in bloodstream impairs platelet adhesion), hyperglobulinemia (multiple myeloma, less commonly inflammatory/infectious disease; multiple myeloma is a syndrome of functional plasma cell neoplasia with excessive immunoglobulin production, interferes with platelet adhesion)

Be familiar with the concept of blood types, important blood types within veterinary species and influence of incompatibilities (need for crossmatching, occurrence of transfusion reactions, neonatal isoerythrolysis)

RBCs have surface molecules called "RBC antigens", antibodies against these antigens may occur

presence of anti-RBC antibodies can result in a Type 2 hypersensitivity response in which RBCs are destroyed (hemolysis)

alloantibodies = naturally occurring antibodies the patient was born with

cats and horses are the main veterinary species

sensitization = acquired antibodies after exposure to foreign RBC antigens, occurs when an animal is exposed to RBC antigens different than their own, following exposure the immune system develops antibodies against the foreign RBC surface antigen, re-exposure can elicit a transfusion reaction (now the blood antigens are incompatible), encounter this mostly in dogs

transfusion reactions: effects of anti-RBC antibodies, opsonization of RBCs and removal by splenic macrophages (extravascular hemolysis) and activation of MAC with subsequent RBC pore formation (intravascular hemolysis); can range from mild to life-threatening

agglutination: due to antibody binding to antigen on cell surface, antibody is called hemagglutinin, leads to premature removal of transfused RBCs or neonate RBCs (extravascular hemolysis)

hemolysis: rupture of erythrocyte within the vasculature, antibody is called hemolysin, leads to acute intravascular hemolysis

Feline AB System:

three blood types: A, B, AB (frequency of each depends on geography), naturally-occurring antibodies are common - cats are born sensitized against other blood types without receiving a previous transfusion (ALWAYS BE ON GUARD when giving transfusions)

Type A: most common (>90% of cats), present in most mixed breed cats in the US, have naturally-occurring alloantibodies against B antigen, antibodies are weak hemagglutinins and hemolysins (leads to early removal of Type B erythrocytes transfused into a Type A cat, relatively mild transfusion reaction but DO NOT ignore clinical signs)

Type B: usually purebred cats but can occur in mixed breed cats, have naturally-occurring alloantibodies against A antigen, causes SEVERE hemolysis of type A erythrocytes when transfused into a type B cat (antibodies are naturally present at high concentrations in the blood and are STRONG hemagglutinins and hemolysins --> much more life-threatening response)

Type AB: have both A and B antigens on RBC surface, NO naturally-occurring alloantibodies, rare (<1% of cats, ragdolls have relatively high incidence), give type A blood if type AB is not available (packed RBC units of type A would be safest option

less bad of two options and should get less transfusion reaction - the plasma is reduced/removed so there are less antibodies present; the donor cat antibodies are the biggest concern

Dog Erythrocyte Antigen (DEA) System:

DEA1 is highly immunogenic and considered most important

~60% of US dogs are DEA1+

Neonatal Isoerythrolysis (NI):

destruction of neonate's erythrocytes induced by antibodies ingested with dam's colostrum

cats and horses of greatest concern

low incidence but may result in neonatal death (they cannot handle the hemolytic reaction)

Understand the principles of blood crossmatching and blood typing, interpretation of results, and clinical application (accept/reject a potential donot for a needy recipient)

commercially available tests (cards, cartridges, gel columns), use antibodies against specific blood groups (antisera), can be done in-clinic

large animals/exotics: likely impractical to blood type individuals, safest to do a crossmatch prior to transfusion, may be impossible in the field

Crossmatching: diagnostic test that assesses for blood incompatibility between animals (some animals with same blood type will still be incompatible), most commonly used to identify a compatible donor (occasional animals with compatible crossmatch results will still develop a transfusion reaction due to other/unknown causes, may be used to predict neonatal isoerythrolysis in horse/mule foals (expose sire's RBCs to dam's serum), major and minor crossmatches are separate tests

Major crossmatch: ALWAYS want to do, test recippient's serum against donor's RBCs, attempts to simulate in vitro what is happening in vivo with the transfusion (looks for evidence that recipient has antibodies that recognize donor RBCs - agglutination and/or hemolysis of donor RBCs, want to avoid incompatibility because massive lysis of transfused RBCs in the patient is extremely dangerous)

Minor crossmatch: do depending on situation, tests donor's serum against recipient's RBCs (looks for evidence that the donor has antibodies that recognize recipient RBCs - clinically an incompatibility is considered much less dangerous, if using packed RBCs which contains very little plasma then a minor crossmatch is generally not necessary)

If using whole blood: both major and minor are recommended

If using packed RBCs: only major recommended, requires serum (or EDTA plasma) from recipient and aliquot of packed RBCs from donor

Results: a compatible result shows no damage to erythrocytes; an incompatible result will show agglutination and/or hemolysis of RBCs

Anemia from Destruction

aka hemolytic anemia, extravascular hemolysis (phagocytosis by macrophages) is much more common than intravascular hemolysis (within the blood vessels), with acute hemolysis clinical signs are commonly overt (body has no time for physiologic adaptation)

lab findings: regenerative effort is usually strong (polychromasia/reticulocytosis), normal to increased plasma proteins, look for evidence of hemoglobin release (hyperbilirubinemia), abnormal RBC morphology may be present to lend support for hemolysis as the cause (spherocytes, Heinz bodies, RBC parasites, etc.)

extravascular hemolysis: phagocytosis of RBCs by macrophages of the spleen, hyperbilirubinemia, mechanisms (antibody and/or complement-mediated, decreased RBC deformability, abnormally increased cellular phagocytic activity, inherited erythrocyte defects)

antibody and/or complement-mediated: antibodies and/or complement binds to RBCs --> recognizzed by macrophage receptors --> complete or partial phagocytosis of RBCs by macrophages (EX. IMHA --> usually IgG-mediated, if IgM-mediated then may also see agglutination and spherocytes; primary (idiopathic) vs. secondary IMHA)

Primary IMHA: body produces anti-RBC antibodies that cause early RBC removal, this is true autoimmune hemolytic anemia (AIHA), no stimulus or cause identified, diagnosis of exclusion, most common in dogs

Secondary IMHA: immune response has an inciting cause --> infectious agents/toxins/drugs/paraneoplastic syndrome (tumor somewhere in body with epitope like RBCs)/vaccines?; most common in everyting BUT dogs

Diagnosis of IMHA: expected bloodwork findings: anemia (often severe), spherocytes, regernative response, hyperbilirubinemia (important clue); may be present/helpful: agglutination, Coombs test positive (in research settings, several limitations)

Decreased RBC deformability: less deformable RBCs are sequestered by the spleen and rmeoved (RBC shapes formed from fragmentation injury, oxidant damage, parasitized erythrocytes, spherocytes from IMHA and other processes)

Increased cellular phagocytic avtivity: not very common, hemophagocytic neoplasms such as hemagiosarcoma and histiocytic sarcoma; hemophagocytic syndrome - abnormal behavior by tissue macrophages, generally secondary to neoplasia (lot of RBCs being removed by healthy macrophages)

intravascular hemolysis: lysis of RBCs within blood vessels releasing hemoglobin into plasma, usually peracute or acute onset, may see hemoglobinemia +/- hemoglobinuria, mechanisms are complement-mediated lysis, physical injury, oxidative damage (sometimes) osmotic lysis, other membrane alterations

complement-meidated lysis: membrane attack complex (MAC) - some transfusion reactions, some cases of neonatal isoerythrolysis, some cases of IMHA

physical injury: fragmentation injury (DIC, blood turbulence, abnormal vasculature), increased RBC fragility, oxidative damage, osmotic lysis, other membrane alterations (endotoxemia, snake envenomation)

Anemia from Decreased Production

most present as non-regenerative anemia, classic pattern is normocytic, normochromic anemia with lack of reticulocytosis, bone marrow can't keep up with normal body demand for blood cells, may be primary (intra-marrow causes) or secondary (extra-marrow causes), bone marrow evaluation may be necessary to delineate cause

extra-marrow causes of decreased production: inflammatory/chronic disease, chronic renal disease, nutrient deficiency, "hypo-"endocrinopathies (hypothyroidism, hypoadrenocorticism)

inflammatory disease: especially chronic inflammation (anemia of inflammatory disease (AID) or anemia of chronic disease (ACD), most common non-regenerative anemia of domestic species, mild to moderate, often normocytic, normochromic anemia, mechanisms are hepcidin production (reduces iron availability), blunted EPO release and the erythroid response to EPO, shortened RBC lifespan (oxidative damage)

chronic renal disease: mild to severe, normocytic,normohromic anemia, must correlate with biochemical findings indicative of renal disease/insufficiency, main mechanism is decreased erythropoietin (EPO) due to loss of renal cells (uremia)

nutrient deficiency: classic pattern is a microcytic, hypochromic anemia, iron deficiency anemia (chronic external blood loss), copper deficiency (copper needed in proteins essential for iron transport, results in functional iron deficiency)

intra-marrow causes of decreased production: aplastic anemia, myelophthisis, FeLV-induced erythroid hypoplasia, precursor-directed immune-mediated anemia (PIMA) (pure red cell aplasia (PRCA)

general information: damage to one or more of the following: hematopoietic stem cells, blood vessels/sinusoids, bone marrow stromal cells; may be reversible or irreversible, may or may not affect multiple cell lines

aplastic anemia: thought to be due to stem cell injury, decreases in RBCs/WBCs/platelets expected, numerous causes: infectious/drugs/toxins/therapeutic or environmental irradiation/idiopathic (immune-mediated?)

myelophthisis: replacement of normal marrow cells with non-hematopoietic tissue/neoplastic tissue/inflammation, often presents as pancytopenia, can occur in lymphomas, leukemias, and other cancers (carcinomas)

FeLV-induced erythroid hypoplasia: FeLV targets and damages erythroid stem cells and progenitor cells, may be acrocytic,normochromic or normocytic/normochromic anemia, numerous abnormalities of erythroid cells

PIMA: may be primary or secondary immune-mediated disease (directed against precursor cells), bone marrow reveals increased numbers of immature RBC forms which may not progress past a certain level of maturation, some cases respond to immunosuppressive therapy

PRCA: considered a severe form of PIMA, non-regenerative anemia with markedly decreased (<5%) or absent erythroid precursors in the marrow, no growth of RBC population

Understand the types of erythrocytosis and common underlying causes

Erythrocytosis: an increase in RBC count and/or hematocrit, may interchangeably use "polycythemia" but "erythrocytosis" is preferred

relative: secondary to something and not a true increase in circulating RBC mass (proportionate to something else, like fluid change), most commonly seen

dehydration (hemoconcentration), splenic contraction (physiologic erythrocytosis), endotoxemia (due to fluid shifts)

absolute: a true increase in the circulating RBC mass (normal sighthounds often have higher counts than most non-sighthounds)

primary: polycythemia vera, rare (the most rare of all erythrocytoses), autonomous (EPO-independent) proliferation of erythroid precursors resulting in elevated numbers fo mature erythrocytes in peripheral blood, considered a neoplastic condition, diagnosis of exclusion (must rule out causes of secondary erythrocytosis)

secondary: due to appropriate or inappropriate increased secretion of EPO

appropriate: hypoxia of normal tissues is present that drives release, causes: cardiac disease with left-to-right shunting of blood, diffuse lung disease, moving from low to high altitude, some cats with hyperthyroidism (increased metabolic rate), persistent methemoglobinemia

inappropriate: hypoxia is NOT present yet EPO is being released (the tissue feels like it's being starved of oxygen), causes: EPO-secreting renal tumor or renal lesions (rare), non-renal lesions (case reports)

differentiation of appropriate/inappropriate: check arterial blood PaO2 (should be low in appropriate case), diagnostic imaging to assess for cardiac and/or pulmonary disease and renal lesions/tumors and other masses

Plasma EPO concentration measurement: expect elevation with secondary erythrocytosis, should be normal or low with primary erythrocytosis, lots of overlap limits diagnostic usefulness, the bigger problem is testing is not typically available

Assess clinical information (history/clinical signs) and bloodwork data to classify the type of erythrocytosis and identify the most likely differential diagnosis/es

consider breed to see if elevation is normal (sighthounds it is)

look at previous bloodwork (especially healthy bloodwork) to compare values when sick

if there is diarrhea, consider that the patient is dehydrated (so don't assume an elevation is a bad thing)

recheck bloodwork later to compare how bloodwork is changing (look for evidence of regeneration/values returning more to baseline)

Understand the different classification schemes for anemia

Anemia: decrease in RBC count/Hct/and/orHgb

two types of "faux" anemias: pseudoanemia (collecting of whole blood samples through IV fluid lines/catheters) and relative anemia (excess plasma volume, ex is anemia of the newborn)

history and clinical signs are important to help differentiate true from relative, patients with chronic anemia (which develops slowly over time) may have few outward clinical signs (due to adaptation), acute anemia usually presents with outward clinical signs (sudden large-volume blood loss)

classification: based on RBC indices, based on bone marrow response, based on pathologic mechanism

By RBC Indices: earlies mechanism to identify

use MCV and MCHC, by convention characterization of cell size is stated first followed by characterization of hemoglobin concentration

some common types of anemias observed: normal MCV/MCHC (normocytic, normochromic anemia), increased MCV/decreased MCHC (macrocytic, hypochromic anemia), and decreased MCV/MCHC (microcytic, hypochromoic anemia); hyperchromia (increased MCHC) is ALWAYS a false increase (hemolysis, lipemia, Heinz bodies)

By Bone Marrow Response:

regenerative: bone marrow is working appropriately to resolve anemia, evidence of bone marrow response in peripheral blood

expect to see increased polychromasia on peripheral blood smear and an elevated reticulocyte count (reticulocytosis) with other possible findings, classic pattern is macrocytic/hypochromic anemia based on MCV/MCHC, presence of bone marrow response suggests either loss or destruction of the RBCs (hemorrhage or hemolysis respectively), needs 3-5 days to get from bone marrow to peripheral blood

non-regenerative: bone marrow is NOT working appropriately to resolve anemia, NO evidence of bone marrow response in peripheral blood

classic pattern is normocytic, normochromic anemia based on MCV/MCHC, multiple potential causes (either something outside the marrow is affecting marrow function or the marrow is damaged/diseased in some way), also consider if enough time has passed for the marros to have responded to the anemia

By Pathologic Mechanism:

Loss (hemorrhage): hemorrhagic anemia, can be acute or chronic in nature, source may be obvious or hidden, broad causes include damage to vasculature (trauma/sx/ulceration/neoplasia/etc.)/coagulation abnormalities (congenital or acquired)/severe thrombocytopenia (can transition to iron deficiency anemia)/parasitism (endo- or ectoparasites)

acute hemorrhage: clinical signs depend on amount of blood lost, period during which bleeding occurred, and site of hemorrhage; if over 1/3 of blood volume lost rapidly hypovolemic shock can develop (but hard to get a handle on severity of anemia with PCV)

lab findings: Hct is initially within RI, splenic contraction may temporarily compensate Hct by delivery of high-Hct blood into circulation; blood volume is subsequently restored via shifting of interstitial fluid into vasculature, decreased RBC count/Hct/Hgb will become evident, regeneration evidence becomes evident 3-5 days after event, plasma protein should start to normalize within 2-3 days after loss, values should return to RI within 1-2 weeks following acute single-episode of blood loss

acute internal loss: autotransfusion may occur (some intact RBCs and plasma proteins are resorbed via lymphatics), RBCs that don't autotransfuse will lyse or be phagocytosed by macrophages to allow for recycling of iron/amino acids/plasma proteins; regenerative response to internal bleeding is more robust than with external hemorrhage (see stronger response)

chronic hemorrhage: anemia occurs slowly so patients may not show clinical signs, blood loss associated with parasitism or GI ulceration is often chronic

lab findings: likely to have decreased plasma proteins, non-regenerative anemia common at time of diagnosis

Destruction (hemolysis): See in lecture 6

Decreased production (reduced or defective erythropoiesis)

Understand principles of automated erythron evaluation

takes measure of an animal's ability to oxygenate tissues, may give clues to underlying disease(s), helps assess need for care (supplemental oxygen, IV fluids, blood transfusion, etc.), expands findings of peripheral blood smear (get most info from taking both together)

lavender/purple top tube: used in most mammals, uses (potassium) EDTA anticoagulant

green top tube: consider for horses and exotics (pocket pets/avians/reptiles/etc.), uses lithium/sodium heparin anticoagulant, can use for serum analysis also

Limitations of hematology analyzers:

unable to detect: blood parasites, most RBC shape changes, toxic changes within neutrophils (can help ID inflammation)

imperfect detection of: nucleated RBCs, immature or neoplastic white blood cells, specific leukocyte types for differential count, platelet count of samples with platelet clumps

Interpret data from automated methods (RBC, Hct, Hgb, MCV, MCH, MCHC) to evaluate the circulating erythron

RBC = RBC count or concentration; machine counts individual RBCs, not yet able to accurately do automated counts on species with nucleated RBCs (use spun PCV and manual count with hemacytometer)

Hgb = hemoglobin concentration; total hemoglobin content within an aliquot of blood, false increases caused by Heinz bodies/hemolysis/lipemia

Hgb x 3 = Hct (+/-3%), used to double check the machine Hgb and Hct values, hemoglobin should be ~1/3 of Hct when RBCs are of normal size (if they do not match run a PCV)

Hct = hematocrit (typically equivalent to PCV (packed cell volume), PCV is a direct measurement and gold standard); 55% plasma & 45% cells (most of which are RBCs), calculated value on many automated analyzers (an indirect measurement)

RBC count/Hgb/Hct all collectively reflect the erythrocyte mass of the patient, generally increase/decrease together, sometimes only one or two analytes will be outside their respective reference intervals (caused by: hemoglobin impacted by hemolysis/lipemia/Heinz bodies, RBC population is in flux (borderline anemia), concurrent changes in RBC size and Hgb content of cells (macrocytes), miscounting of RBCs by analyzer (agglutination, platelet clumps, large platelets))

decreases in these typically indicate anemia (decrease in erythrocyte mass within an animal) but consider hydration status (true severity of anemia can be masked by dehydration); can also be caused by sample dilution/miscounting of RBCs, RBC clumping leading to miscounting

increases frequently due to dehydration, referred to as "erythrocytosis"

RBC Indices: characteristics of the average RBC, use MCV/MCH/MCHC collectively

MCV = mean cell/corpuscular volume; measured directly by most analyzers but can be calculated, normocytic is the descriptive term to mean MCV is within the reference interval (significant numbers of unusually small or large RBCs are not present), mean and reference interval will vary by species

increased: macrocytic/macrocytosis, most commonly due to increased numbers of circulating reticulocytes/polychromatophils, typically observed in patients with active erythropoiesis, often an anemic patient with a bone marrow that IS responding to the problem, may be only hint of regeneration in an anemic horse; other causes are in vitro RBC swelling due to sample age (fairly common), RBC agglutination, FeLV+ cats (typically without polychromasia), sighthounds, uncommon breed abnormalities

decreased: microcytic/microcytosis, iron deficiency is the most common cause (often due to chronic hemorrhage such as bleeding gastric ulcers), significant liver disease or vascular anomalies (portosystemic shunts), several east Asian dog breeds, may be seen in neonates of most species (because reference intervals are based on adult values)

Hemoglobin content of erythrocytes: uses MCH and MCHC

MCH = mean cell/corpuscular hemoglobin; mean amount of hemoglobin in an average RBC that is a calculated value using the RBC count

MCHC = mean cell/corpuscular hemoglobin concentration; mean amount of hemoglobin in an average RBC that is a calculated value using the Hct (can sub in the PCV)

MCHC considered more reliable than MCH, can readily verify the Hct using a PCV when values don't match (cannot easily verify the RBC count)

increased: hyperchromic/hyperchromasia, RBCs do not make too much hemoglobin so increases in MCH/MCHC should get your attention, the analyzer assumes all hemoglobin present is within the cell (Heinz bodies are read as a higher concentration of Hgb, hemolysis and lipid molecules from lipemia are also read as intracellular Hgb)

decreased: hypochromic/hypochromasia, caused by many circulating immature RBCs (polychromasia/reticulocytes, lower concentration of Hgb in immature RBCs), often seen in patients with regenerative anemia, iron deficiency anemia (not enough iron in body to fully stock RBCs with hemoglobin, hypochromasia withOUT reticulocyte change)

Use the absolute reticulocyte count to assess for a regenerative response to anemia

Last stage of erythropoiesis, RBCs that contain residual RNA and have not fully completed hemoglobin synthesis --> not fully mature though may be found in circulation

2 methods of quantification:

1. automated count: via flow cytometry with certain hematology analyzers

2. manual count: New Methylene Blue stain and count number of reticulocytes

counts using either method are important for an accurate assessment of the bone marrow's response to anemia for most species

in most species we expect increased numbers of circulating reticulocytes in regenerative anemia

Identify erythrocyte hemoparasites and understand clinical relevance and findings

Hemoparasites:

Epicellular: hemotropic mycoplasmas

HM: associated with RBC membrane surface, may be found individually or in chains, can fall off the RBCs during transport in vitro, can be confused for stain precipitate, PCR testing is recommended for definitive diagnosis if infection is suspected

HM: virulence varies between organisms, when they cause anemia expect hemolytic anemia which is often extravascular in nature, treated animals may become carriers (recurrence possible)

Mycoplasma haemofelis: can cause disease in normal animals, concurrent retroviral infection (FeLV/FeIV) can increase susceptibility; infection can result in severe hemolytic anemia, cyclic parasitemia, survivors often remain carriers; transmission: presumptive vectors (fleas/ticks/mosquitoes), blood (including transfusions), vertical (queen --> kittens)

Most domesticated species are affected by at least one Mycoplasma spp.

Extracellular: microfilaria, Trypanosoma spp.

Microfilaria:

dog and cat heartworm (US) = Dirofilaria immitis

dog and cat heartworm (Europe) = Dirofilaria repens

dogs = Acanthocheilonema reconditum

cattle and horses = Setaria sp. (intestinal roundworm that has a microfilarial stage

Intracellular: Theileria spp., Babesia spp., Anaplasma spp., Cytauxzoon felis, Hemoproteus spp., Plasmodium spp., Leucocytozoon spp.

Theileriosis: protozoal organism affecting ruminants (including cervids), species in US are often non-pathogenic but can cause hemolytic anemia, multi-stage life cycle (piroplasms in RBCs, schizonts in lymphocytes)

Cytauxzoon felis: protozoa related to Theileria, feline pathogen of southern/south-central/midwest/mid-Atlantic US, piroplasms in RBCs, Bobcat is the wild reservoir, tick vectors, macrophages cause most of clinical signs (cells get large and impact function) - agent itself causes mild anemia; can cause severe disease and fatality - fatal for symptomatic (have gotten better with this, survivors are lifelong carriers --> reservoirs)

Babesiosis: protozoan, sporozoites penetrate and multiply in RBCs (can rupture RBCs and subsequently invade other RBCs, intravascular hemolytic anemia +/- thrombocytopenia, do not infect other cell types), large and small Babesia spp.

RBC Anaplasmosis (cattle): Anaplasma marginale: widespread, tick vectors (many species), mechanical transmission, increasing parasitemia occurs until anemia develops, must be differentiated from Howell-Jolly bodies (usually Anaplasma organisms are not perfectly round and may be smaller than typical H-J bodies; survivors are chronic carriers --> reservoirs

Avian and Reptile Hemoparasites:

Hemoproteus (avian)

Plasmodium (avian and reptile) (questionable pathogenicity)

Hemogregarines (reptile) (catch-all term that encompasses 4 genera, including Hemogregarine spp., pathogenicity pretty low)

Leucocytozoon/Leukocytozoon spp. (pathogenicity pretty low)

Understand the steps of and be able to perform a basic blood film review

MIX THE BLOOD via invertion several times before making the smear!

Must start with a good smear - has a monolayer of cells

Evaluation should utilize 10x, 40x, and 100x examination

10x: orient yourself to the feathered edge, monolayer and body; evaluate the BODY of the smear (typically this area is of limited use but may be able to identify platelet clumps or large organisms, do NOT evaluate RBC agglutination/clumping in the body - this often occurs as an artifact in this area), evaluate the MONOLAYER of the smear (central pallor of RBCs in dogs will be most prominent here, determine if RBC density is normal/increased/decreased), evaluate the FEATHERED EDGE of the smear (also usually of limited use but should always be assessed, larger organisms/large cells/clumps of cells/platelets are often pulled out to this area because of their size), continue higher power evaluations primarily in the monolayer

40x: perform 100 cell differential count of leukocytes, differentiate Rouleaux formation from agglutination (Rouleaux = linear association of RBCs and should disperse with the addition of physiologic saline to an unstained wet mount of whole blood; Agglutination does not disperse and has a cluter of grapes appearance), assess for nucleated RBCs (these are quantified during the leukocyte differential and reported as the number of nRBCs per 100 WBCs, typically seen in response to anemia, when increased nRBCs are observed in non-anemic patients then bone marrow or splenic disease should be considered, do not mistake for lymphocytes, automated analyzer may count them as WBCs and categorize them under lymphocytes so can falsely elevate your count)

100x: remember to stay within the monolayer, closest assessment of cellular detail (WBC morphology/inclusions/etc., platelet count/morphology/inclusions, RBC morphology), identify and quantify RBC characteristics (anisocytosis, presence of polychromasia, poikilocytosis, presence of hemoparasites or other inclusions), quantification is somewhat subjective but should be as consistent as possible

Be able to identify erythrocyte shape changes and inclusions

Macrocytes: larger-than-normal RBCs, if they stain more blue/grey to purple, then they are polychromatophils; if mature red-orange (normochromic) macrocytes are present consider reasons (sample age, possible regneration in anemic horses, FeLV+ cats, hereditary macrocytosis in poodles)

Microcytes: smaller-than-normal erythrocytes; causes are iron deficiency anemia (main), vascular anomalies, severe liver disease, Heinz body remnants, fragmentation of RBCs, breed-related

Anisocytosis: variation in apparent size across a population of erythrocytes, may be due to the observation of many large or small RBCs or both

Hypochromasia: decreased cytoplasmic staining of RBCs with increased area of central pallor (less hemoglobin present), causes are many immature erythrocytes produced in regenerative anemias, iron deficiency, in birds from lead toxicity and inflammation

Know which erythrocyte shape changes and inclusions are associated with clinically relevant conditions

Poikilocytosis: umbrella term for RBCs that deviate from normal morphology for the species (regardless of the specific shape change)

Acanthocytes: unevenly spiky erythrocytes with irregular membrane projections, presumptive mechanisms are RBC trauma/fragmentation and increased cholesterol:lipid ratio in the RBC membrane

reported with: some types of cancer, liver disease, iron deficiency anemia, trauma within the vascular system, altered lipid metabolism, normal in young cattle and young goats

Codocytes: central area of Hgb separated from the peripheral Hgb by a clear area, target appearance, have more membrane than usual relative to cytoplasm (from increased lipid/cholesterol and/or decreased cytoplasmic volume)

causes: young erythrocytes (polychromatophils), liver disease, iron deficiency anemia

Echinocytes: spiculated erythrocytes with evenly-spaced uniform membrane projections, common in healthy cats and pigs, most commonly this is an ex vivo artifact from changes in temperature/pH/drying - drying most common

causes: snake envenomation, uremia, electrolyte depletion, lymphoma, doxorubicin toxicity, glomerulonephritis

Keratocytes: have one or two cell membrane projections created from a ruptured vesicle, blister cells - erythrocytes where the vesicle is still intact

reported causes: trauma within the vascular system (fragmentation injury), oxidative damage, iron deficiency anemia, liver disease (haptic lipidosis), doxorubicin toxicity (cats), myelodysplastic syndrome (dogs)

Ovalocytes: oval erythrocytes, normal in camelids and non-mammals (avians/reptiles/amphibians)

causes:

dogs: myelofibrosis, myelodysplastic syndrome, rare hereditary/congenital diseases of dogs

cats: liver disease (hepatic lipidosis), portosystemic shunt, doxorubicin administration

may be observed in iron deficiency

Schistocytes: irregular erythrocyte fragments, due to trauma (including tortuous vasculature, intravascular fibrin strands (DIC), turbulent blood flow)

reported with: disseminated intravascular coagulopathy (DIC), hemangiosarcoma, congestive heart failure (CHF), vasculitis, glomerulonephritis, chronic doxorubicin toxicity, myelofibrosis

Spherocytes: CLINICALLY IMPORTANT, small dark red erythrocytes that completely lack central pallor, decreased membrane with normal cytoplasmic volume, can only be reliably identified in monolayer of blood smears in dogs (DONT TRY in other species)

causes: IMHA is most common, after transfusion with stored RBCs, removal of Heinz bodies/damaged membrane/parasite/etc by splenic macrophages, rattlesnake/coral snake/viper envenomation, bee stings

Know clinical and artifactual causes of erythrocyte shape changes and inclusions

RBC Inclusions:

Basophilic stippling: diffuse basophilic punctate inclusions, due to residual aggregates of ribosomes, see of Diff-Quik

observed in: regenerative anemias (especially ruminants), lead toxicity in any species

Siderotic inclusions: focal blue or brown rounded or irregular structures within RBC cytoplasm (just a few dots), inclusions represent accumulated iron

reported in: hemolytic anemias, lead toxicity, dyserythropoiesis, myeloproliferative diseases, chloramphenicol treatment, idiopathic

Howell-Jolly bodies: small (2-3 microns) spherical dark blue/purple nuclear remnants, nuclear material that is left behind when nucleus is expelled (must differentiate from parasitic organisms), often removed by the spleen

seen in: low numbers not uncommon in healthy animals, regenerative anemia, splenectomized animals

Heinz bodies: aggregates of denatured hemoglobin caused by oxidative damage, appear as round protruding structures from the side of RBC membrane (looks like a "nose") and is small pale or slightly refractile cytoplasmic spots when sitting on top of the RBC, up to 5% Heinz bodies can be seen in healthy cats

Nucleated erythrocytes (nRBCs): erythrocytes should be nucleared in birds/reptiles/fish/etc; metarubricytes (most common), rubricytes, or earlier precursors in mammals, when observed in higher-than-normal numbers in mammalian peripheral blood must determine if their presence is appropriate or inappropriate

Appropriate: premature release in patients with regenerative anemia, hypoxia that is not due to anemia (significant cardiopulmonary disease), splenectomized animals, healthy piglets less than 3 weeks old)

Inappropriate: lead toxicity, iron or copper deficiency, splenic disease (such as hemangiosarcoma), bone marrow disease

Viral Inclusions:

canine distemper

inclusion body disease (IBD)

Rouleaux Formation: linear association of RBCs, "stack of coins" appearance, common finding in healthy animals of certain species (especially horses and cats), can be secondary to increased proteins in plasma, should disperse with addition of physiologic saline to an unstained wet mount of whole (anticoagulated) blood

Agglutination: irregular clumping or aggregates of RBCs, typically want 3-5 RBCs in an aggregate before counting as agglutination, does not disperse with addition of physiologic saline, if present concerning for antibody or complement coating the RBC surface allowing for cross-linking of nearby RBCs, agglutination often signifies immune-mediated disease (specifically immune-mediated hemolytic anemia - IMHA)

Understand the components (circulating erythrocytes and erythroid precursors within the marrow) and purpose (carry oxygen to tissues) of the erythron

Components: 55% fluid (plasma) and 45% cells

erythrocytes, leukocytes, and platelets (buffy coat is leukocytes and platelets)

circulating erythrocytes and erythropoietic cells in bone marrow

Purpose: transportation of oxygen - through hemoglobin (tetrameric metalloprotein)

millions of hemoglobin molecules in a single erythrocyte

if hemoglobin is oxidized (Fe2+-->Fe3+) it is turned into methemoglobin which severely reduces/negates the oxygen carrying capacity

Know normal erythrocyte morphology (biconcave disc, oval) of discussed species

mature mammalian RBCs lack nuclei and mitochondria (produce energy through anaerobic glycolysis) --> glucose is the major energy source for RBCs

normal mammal: biconcave disc with a thin central area that may display central pallor (most consistent in dogs)

Feline: weird shapes normally

Equine: normal mammalian shape

Bovine: uniconcave appearance

Ovine: smaller indentation, appear uniconcave

Caprine: spherical

Camelids: flat and oval RBCs

Avian/reptile/amphibian/fish: oval nucleated erythrocytes

Know how to calculate the volume of blood that can be safely collected from a patient based on the patient's body weight in kg/grams and total blood volume in mL

Blood volume as a percentage of body weight varies by species/disease condition/hydration status

Some routine values:

2-3mL from cats/dogs

<10mL for production animals/horses

~100-500microliters from pocket pets/reptiles/birds/etc

extrapolate these guidelines to similar wildlife/zoo species

20-30% blood volume loss = hypovolemic shock

up to 20% total blood volume can be lost without complication in many animals

in healthy small mammals and birds typically draw 10% or less of total blood volume

estimate that 10% of BW (body weight) is blood volume in all patients, and we can collect 10% of the 10%BW --> this is 1% of body weight (use 0.8% for reptiles)

Draw minimum volume needed

Example: 8lb cat - how much blood can be collected?

8lb --> 3.6kg

3.6kg x (1000g/kg) x (1mL/g) = 3600mL body weight equivalence

2 ways to calculate total blood volume: specific and general

specific: uses 6% of body weight as total blood volume

3600mL x 0.06 = 216mL

216mL x 0.2 = ~43mL total blood to be collected

generic uses 10% of body weight as total blood volume; overestimates total blood volume but safe blood volume is underestimated (technically can draw 20% of total blood volume without complication

3600mL x 0.1 = 360mL

360mL x 0.1 = 36mL total blood to be collected

Identify the five types of leukocytes visually and recognize differences in morphology between species

Granulocytes: neutrophils, eosinophils, basophils

Mononuclear cells: lymphocytes, monocytes

Understand and explain the different leukocyte pools in the body and how cells shift between them (kinetics)

Bone Marrow:

Mitotic Pool: undergo mitosis, houses ~20% of total population

Maturation Pool: where metamyelocytes go to become mature segmented neutrophils

Storage Pool: where segmented neutrophils are housed until needed in circulation

Blood:

Marginating Pool: sits along periphery of vessels, slow-moving pools in tissues

Circulating Pool: in laminar flow, what is measured with blood work

Understand and explain the function of each leukocyte type

Neutrophils: in tissue - phagocytosis/microbicidal activity/immunoregulation

Eosinophils: hypersensitivity responses, defense against helminthic parasites

Basophils: histamine release, parasitic responses

Lymphocytes: antibody production, regulate immune responses, cytotoxicity

Monocytes: provide macrophages to tissues, phagocytosis, immune modulation

Recognize the morphologic features of neutrophil toxic change, understand the underlying mechanisms, and explain the clinical significance

Mechanism: immature neutrophils are released into circulation before they are ready due to demand

Clinical significance: occur at the level of the bone marrow, indicate inflammation (accelerated release from bone marrow, direct effects of endotoxins on precursors), cytoplasmic changes to neutrophils

Changes:

Dohle bodies: aggregates of rough ER, low numbers may be present in normal cats

Basophilic cytoplasm: rough ER and polyribosomes, see a busier cytoplasm

Vacuolated cytoplasm: toxic vacuoles are foamy with indistinct margins

Less Common Changes:

toxic granulation (primary granules), see in birds and reptiles

giant neutrophils - skipped a division, see in cats

Identify and understand the significance of reactive lymphocytes and blast cells

Reactive lymphocytes: have increased cytoplasmic basophilia (+/- volume & +/- Golgi apparatus) and no nucleolus

significance: increased antigenic stimulation, non-specific, may be seen in health

Blast cells: concern for neoplastic population (acute lymphoid/myeloid leukemia vs circulating lymphoma), low numbers may be seen transiently with severe inflammation

Differentiate infectious organisms within leukocytes

Monocytes: Cytauxzoon felis major; minor include: Ehrlichia canic, Ehrlichia chaffensis, Neorickettsia risticii, Mycobacterium spp., Histoplasma capsulatum, Leishmania spp., Hepatozoon spp.

Neutrophils: Distemper viral inclusions, Ehrlichia ewingii, Anaplasma phagocytophilum, Histoplasma capsulatum, Leishmania spp., Hepatozoon spp.

Explain how total WBC count and individual leukocyte concentrations are determined

WBC concentration is a measurement of nucleated cells per volume of blood (typically cells per microliter)

Can use manual counts (hemocytometer), automated count, or estimation from blood smear to get value

Manual differential count is performed on a blood smear to calculate how many leukocyte types are present (and in what percentages later)

Recognize abnormal WBC scatterplot features and choose when to reject automated leukocyte counts

Normal features: each color should be a distinct cloud separate from other colors, no clusters should extend beyond the upper end of the y-axis, the uRBC or debris cluster should not be continuous with any other clusters

if these conditions are met then automated differential counts by flow cytometry are typically reliable

Abnormal features:

inflammation - red/blue/purple bleed into each other, little green

blasts - reds and blues bleed into each other, red maxes out top line, very little purple, no green

platelet clumps - banana shape

Use appropriate medical terminology to describe leukogram abnormalities

leukocytosis: increased WBC count

leukopenia: decreased WBC count

left shift: presence of band neutrophils (hallmark of severe inflammation or infection)

neutrophilia: increased absolute count

neutropenia: decreased absolute count

lymphocytosis: increased absolute count

lymphopenia: decreased absolute count

monocytosis/monocytopenia

eosinophilia/eosinopenia

basophilia/basopenia

Recognize and differentiate the common leukogram patterns seen in domestic animals

Glucocorticoid/stress pattern: cortisol-mediated; mild-moderate leukocytosis, mild-moderate neutrophilia, lymphopenia (major hallmark), +/- monocytosis, +/- eosinopenia, no left shift, common finding in clinically ill animals

Adrenaline/excitement pattern: epinephrine-mediated; mild-moderate leukocytosis, neutrophilia, lymphocytosis, +/- monocytosis

Antigenic stimulation pattern: degree is mild (up to 2x URL), marked in dogs with tickborne disease, +/- reactive and/or atypical lymphocytes

Inflammation pattern: left shift, increased immature neutrophils (usually bands), toxic change to neutrophils, concentration varies

Acute: neutrophilia, left shift, increase in bands, toxic change, +/- monocytosis

Severe: neutrophilia >50.100K/microliter, regenerative or degenerative

Chronic: rebound neutrophilia +/- mild left shift, +/- monocytosis, need serial data to identify

Granulocytic hypoplasia: neutropenia (persistent and progressive), no left shift

Hemic cell neoplasia (leukemia):

acute: immature/blast cells present in large numbers, marked leukocytosis

chronic: marked leukocytosis, some other cell is elevated (no blast cells)

Define left shift and correlate associated leukogram changes with the severity of inflammatory disease

Left shift: seeing presence of immature neutrophils in peripheral blood (usually bands)

Regenerative left shift: orderly presence of immature neutrophils in the presence of neutrophilia, segmented outnumber bands, bone marrow is responding

Degenerative left shift: inflammatory neutrophilia with bands outnumbering segmented OR left shift with neutropenia, associated with increased mortality, bone marrow is overwhelmed

Be familiar with major species differences when interpreting leukogram abnormalities

exotic mammals: heterophils, Foa-Kurloff cells

birds/reptiles: heterophils, azurophils (reptiles), nucleated erythrocytes, nucleated thrombocytes, leukocyte morphology is highly variable between species