Clinical Pathology and Focused Ultrasound

Haemoglobin - HB

• Most accurate and reliable measure of RBC mass

• approximately 1/3 of HCT

HCT - haematocrit

calculated number, accuracy depends on RBCC and MCV values

PCV

• approximately 2-3% higher than HCT

MCV

Average volume of a single erythrocyte in a population of RBC

MCHC - mean corpuscular haemoglobin concentration

• average concentration of haemoglobin in a RBC

• elevated MCHC not generally physiologically possible - haemolysis most likely

  • iatrogenic - hyponatraemic samples

  • lipaemia

  • secondary to haemolytic disorders

MCH - mean corpuscular haemoglobin

• important value as MCHC and MCV are both affected by storage time and temp

• reticulocytes will cause an increased MCV and decrease MCHC

  • under these circumstances MCH is more accurate than MCHC

RDW - Red Blood Cell Distribution Width

• most sensitive marker for variability of RBC size

• increased in regenerative anaemia and iron deficiency anaemia

Erythrocytosis - Causes

1. Relative erythrocytosis

   • Splenic contraction

   • isotonic dehydration

2. Absolute erythrocytosis

   • appropriate

     • chronic hypoxia → increased EPO → increased RBC production

       • Right-to-left shunts

       • chronic pulmonary disease

   • Inappropriate

     • increased EPO without systemic hypoxia

       • renal neoplasia

       • Hyperthyroidism or hyperadrenocorticism → mild, usually not enough for clinical signs of erythrocytosis

Anaemia - HCT, Hb and RBCC below normal reference interval

1. Regenerative anaemia

   • Haemolysis or haemorrhage

   • absolute reticulocytes (/uL) = reticulocyte % x RBC count (10^6/L)

2. Non-regenerative anaemia

   • Primary bone marrow disease

   • anaemia of chronic inflammation/chronic disease

   • decreased production of EPO

   • pre-regenerative

Poikilocytes

General term that describes an abnormally shaped erythrocyte

Acanthocytes

• irregularly spaced, asymmetrical blunt-tipped spicules (2+), projects from the surface of a round RBC

• causes:

  • fragmentation injury

    • shearing injury in DIC

    • fragile RBC in iron deficiency anaemia

    • usually accompanied by schistocytes and keratocytes

    • haemangiosarcoma in dogs - important blood biomarker

  • altered lipid metabolism

  • hepatic disease (dogs and cats)

  • others:

    • neoplasia - osteosarcoma, lymphoma

    • renal, AKI, CRF, glomerulonephritis

    • GIT - GE, GDV

    • musculoskeletal - trauma

    • internal haemorrhage

    • cardiac disease - degenerative valvular disease

Eccentrocytes

• RBC’s with a transparent crescent-shaped area on one side of the RBC

• causes:

  • oxidant-induced haemolytic anaemia (e.g., onion poisoning; Zinc toxicity)

  • endogenous oxidant

    • can occur in sick dogs without concurrent anaemia

Echinocytes

• Small, evenly spaced, uniform, sharp/blunt spicules

• causes:

  • artefact - common

  • drugs:

    • frusemide

    • doxorubicin

    • salicylates

    • phenylbutazone

  • Disorders

    • renal disesae (glomerulonephritis; uremic toxins) (most commonly in dogs)

    • inherited RBC disorders

    • snake envenomation

    • neoplasia

Elliptocytes:

• Elongated RBC’s

  • type 1: slightly oval-shaped

  • type 2: round to oval shaped

  • type 3: elongated elliptical cell

  • causes:

    • liver disease:

      • lipidosis (cats) - acanthocytes + type 3 elliptocytes

    • myelofibrosis

      • type I elliptocytes (dogs)

      • non-regenerative immune mediated anaemia

      • pre-cursor directed immune mediated anaemia

      • pure red cell aplasia

    • inherited/congenital RBC abnormality (rare)

    • neoplasia

    • artefact

Keratocytes (helmet cells)

• Bite shaped defect - two horn like projections due to rupturing of a vesicle

• causes:

  • fragmentation injury (DIC; HAS; Vasculitis)

  • oxidant injury

    • possibly accompanied by Heinz bodies, eccentrocytes

  • liver disease

    • hepatic lipidosis (cats)

Schistocytes

• Irregular fragments of RBCs

• causes:

  • roughing or irregulatiry of the blood vessel lining (e.g., fibrin presence)

  • turbulent flow

  • iron deficiency anaemia

  • drug toxicity - doxorubicin

  • disease processes

    • DIC

    • glomerular disease

    • vasculitis

    • PSS

    • vascular neoplasma

    • CHF

    • splenic torsion

  • rarely seen in cats - liver disease if present

Spherocytes

• globoid RBCs that appear smaller than regular RBCs and lack central pallor

• causes:

  • immune mediated anaemias

    • moderate to marked spherocytosis

    • strong regenerative response

  • others:

    • oxidative injury to RBCs

    • abnormal macrophage function

    • snake envenomation, bee stings

    • transfused or stored RBC’s

  • normal if only few are seen

Stomatocytes

Elongated central pallor (mouth like)

• causes:

  • non-specific finding - regenerative anaemia, liver disease, lead poisoning

  • artefact

  • inherited RBC defect (dogs)

Codocytes (target cells)

• resembles a bullseye due to redistribution of Hb within the cell

• causes:

  • regenerative anaemia

  • iron deficiency anaemia

  • lipid disorders:

    • increase in cholesterol and phospholipid in RBC membrane

    • hypoT (dogs ) - high systemic cholesterol concentrations

    • hepatic disease

    • renal disease

Neutrophilia

Neutrophilia

• Left shift: increased number of immature or precursor neutrophils in the circulation

  • non-degenerative: mature/segmented neutrophil count exceeds immature/band neutrophil count

  • degenerative: immature/band neutrophil count exceeds mature/segemented neutrophil count: it indicates severe inflammation

• Corticosteroid response (stress leukogram)

  • endogenous or exogenous corticosteroid increase

  • Includes

    • neutrophilia - generally not more than 1x above reference limnit in dogs; but can be 2-3 times the upper reference limit in cats

    • lymphopenia

    • eosinopenia

    • monocytosis - common in dogs and rare in cats

  • not every patient has all of these changes - lymphopenia and neutrophilia are the 1st and 2nd most common seen respectively

  • biochemical changes that might be seen

    • hyperglycaemia - requires more chronic increase in corticosteroids

    • increase in ALP - in dogs with chronic corticosteroids (both endogenous or exogenous)

• Adrenaline response (physiological leukocytosis)

  • FAS

  • most common cats and puppies!

  • immediate neutrophilia without left shift

  • usually accompanied with lymphocytosis, can also cause temporary hyperglycaemia

  • eosinophilia and basophilia may also be present in cats

• Acute Inflammatory Respose

  • Neutrophilia in response to disease process

    • may feature a left shift

  • ± toxic changes due to rapid transit time from bone marrow to circulation

  • concurrent lymphopenia (with or without eosinopenia) are common

  • causes:

    • infectious agents - bacteria, viruses, protozoa, fungi

    • immune mediated disease

    • neoplasia

    • hypoxic injury and necrosis

    • foreign body, e.g., migrating grass seed

• Leukemoid response

  • marked leukocytosis secondary to strong inflammatory stimulus

    • >70×10^9/L dogs

    • >50×10^9/L for cats

  • associated with high mortality rate

  • reduced risk of mortality if infectious aetiology vs neoplasia

Toxic Changes in Neutrophils

• Caused by dysplasia from accelerated granulopoiesis

• most frequently associated with inflammation

• characterised by:

  • dohle bodies - normal in low numbers in healthy cats

  • increased cytoplasmic basophilia

  • cytoplasmic vacuolation

  • nuclear immaturity

  • toxic granulation

• help differentiate inflammation (present) from corticosteroid/adrenaline responses (absent)

• can only be assessed via blood smear assessment

Hypersegmentation

• normal aging process of neutrophils

• more than 5 lobes within nucleus

• increased hypersegmentation

  • increased lifespan - corticosteroid therapy

  • abnormal neutrophil production - FIV, myelodysplastic syndrome, vitamin B12/folate/cobalt deficiency

  • inherited condition - toy or miniature poodle with macrocytosis

Leukopenia and neutropenia

Causes:

• Overwhelming demand for cells with increased migration from circulation into tissues or third spaces

  • pyometra, pyothorax, peritonitis, abscess

• Ineffective granulopoiesis (and erythropoiesis)

  • myeloproliferative disorders

  • dysplasia of bone marrow

• decreased cell survival time

• viral infections:

  • FeLv, FIV, parvovirus

• infectious agents

  • cryptococcus, histoplasmosis

• protozoan parasites

  • leishmania, rickettsial infections (ehrlichia canis, rocky mountain spotted fever etc.,)

• endotoxic shock or anaphylactic shock

• vitamin B12 and folic acid deficiency

• inherited neutropenic syndrome of collies - e.g., cyclic haemopoiesis

• drug reactions

  • chloramphenicol

  • oestrogens

  • phenylbutazone

Lymphocytosis

Causes:

• adrenaline response in the cat and puppy

• protozoan infections

• canine ehrlichiosis

• some viral diseases

• chronic antigenic stimulation or tarnsiently following vaccinations

• hypoadrenocorticism

• lymphoid leukaemia

  • monoclonal proliferation

  • lymphoid count >20×10^9/L

Lymphopenia

Causes:

• stress/steroid response

• decreased production - FIV/FeLV

• loss of cells: chylothorax, lymphangiectasia

• acute inflammatory response/acute phase of infection: some viral diseases, sepsis/endotoxaemia

• CRF

• immune mediated disorders

• immunosuppressive drugs

• lymphoproliferative disease

• dogs with CHF

Eosinophils

Eosinophilia

• IgE related hypersensitivies and migrating parasites

• paraneoplastic - lymphoma cats: disseminated/intestinal mast-cell tumours

• hypoadrenocorticism

• idiopathic hypereosinophilic disease

• Chronic eosinophilic leukemia

• Cats: CRF and cardiac disease

Eosinopenia

• characteristic finding in stress/glucocorticoid leukogram

Basophils:

• appears in conditions associated with eosinophilia - allergies or inflammation

• endocrinopathies - hypoT, DM

• oestrogen administration

• mastocytosis, neoplasia, myeloproliferative diseases

• may also appear after fatty meal

Mast cells

• mast cell neoplasia - especially if in circulation

• acute inflammation - in low numbers

Thrombocytosis

An increased number of platelets

Causes:

• most common - iron-deficiency anaemia/chronic GI bleeding

  • due to cross reactivity between EPO and TPO

• Rebound following acute haemorrhage or thrombocytopenia

• Reactive thrombocytosis

  • secondary to inflammatory state

    • IL-6 most important inflammatory cytokine contributing to thrombocytosis

  • stress/corticosteroids (common finding in Hyperadrenocorticism)

  • Post splenectomy

Thrombocytopenia

Four major mechanisms of acquired thrombocytopenia

• increased consumption

• decreased production

• destruction

• splenic sequestration (least common)

Can be normal or inherited in some breeds - generally asymptomatic

• macrothrombocytopenia in CKCS and norfolk terriers (30-150×10^9/L)

• greyhounds 80-295×10^9/L

MPV - mean platelet volume

Highly susceptible to platelet clumping

Increased:

• increased thrombopoiesis

• CKCS - macrothrombocytopenia:

  • high MPV, slightly lower than normal platelet count with normal coagulation

Hyperproteinaemia - Albumin and Globulin

1. Albumin and globulins both increased

   • Dehydration - only way this occurs

   • TP higher in neonates by 10% due to colostrum intake (normalises by 8 weeks)

2. Increased albumin, normal globulins (increased A:G)

   • overproduction of albumin is not possible

   • any increase should be interpreted as dehydration or laboratory error

   • most common cause is haemolysis or lipaemia

3. Increased globulins only

   • acute phase reaction

     • in chronic inflammation there is a concurrent compensatory decrease in albumin (negative acute phase protein)

     • microbial invasion, neoplasia, injury or inflammation

   • polyclonal gammopathy

     • antibodies produced in response to any inflammatory/infectious conditions

     • chronic bacterial infection, parasitic disease, fungal, viral infections, autoimmune disease and neoplasia. ANYTHING THAT CAUSES PERSISTANT STIMULATION OF IMMUNE SYSTEM

   • Monoclonal gammopathy

     • neoplasia of immunoglobulin producing cells - plasma cell neoplasm, lymphoma, lymphosarcoma, multiple myeloma etc.,

Hypoproteinemia

1. both albumin and globulin decreased!

   • Hemodilution

   • haemorrhage

   • excessive loss of protein

     • outside the body - parasitic, dermal burns

     • inside body cavities - 3rd space effusions/peritonitis

     • protein losing enteropathy - diarrhoea

     • protein losing nephropathy/nephrotic syndrome - glomerular disease

2. Decreased globulins and normal albumin

   • Neonatal pre-colostrum serum

     • less important in dogs and cats compared to large animal species

   • Primary immunodeficiencies

     • primary severe combined immunodeficiency - basset hounds, cardigan corgis, dachshunds

     • IgA deficiency - sharpei, beagle, airedale terriers, GSD

   • Breed variations - greyhounds have a lower reference range in comparison to other breeds

   • infectious diseases - viruses (FeLV and FIV in cats, canine distemper in dogs,) parasites (toxoplasmosis)

3. Decreased albumin +/- decreased globulin

   • Inadequate dietary intake

   • failure to synthesise albumin

     • severe hepatic disease

4. Decreased albumin with increased globulins

   • acute phase response

Lipids

• Broken down into triglycerides and cholesterol

• cholesterol is most readily tested in house

  • Increased - failure of cholesterol excretion (target cells and acanthocytes)

  • Decreased: reduced cholesterol production (more severe hepatic disease)

Hyperlipidaemia

1. post-prandial

2. primary hyperlipidaemia

   • not common in animals

   • idiopathic in miniature schnauzers

   • hypercholesterolaemia in briards, doberman, rottweilers, rough collies and shetland sheepdogs

3. secondary hyperlipidaemia

   • hypoT

   • DM

   • pancreatitis

   • hyperadrenocorticism

   • hepatic disease/cholestasis

   • pregnancy

   • obesity

   • nephrotic syndrome/protein-losing nephropathy

   • gram-negative sepsis

   • drug induced: corticosteroids; phenobarbitone, oestrogens, methimazole

Hypolipidaemia

• no significance in most cases

• significant causes:

  • impaired hepatic function - PSS or cirrhosis

  • malabsorption - EPI

  • protein-losing enteropathies

  • hypoadrenocorticism

Blood urea Nitrogen (BUN)

• End product of protei metabolism

• small quantities ingested

• processes which induce protein catabolism

• Causes of an increased BUN

  • Increased protein catabolism - fever, burns, corticosteroids, starvation, infection

  • increased protein digestion - haemorrhage into GIT, high protein meals

  • decreased filtration - pre-renal, renal or post-renal cause

• Causes of a decreased BUN

  • reduced synthesis

    • reduced protein intake

    • PSS

    • severe hepatic insufficiency

  • increased loss

    • chronic polyuria → medullary washout

  • haemodilution

    • polydipsia

    • fluid therapy

Creatinine

• Small amounts ingested

• mostly derived from non-enzymatic breakdown of muscle phosphocreatinine

• greyhounds normally have more than other species

• crude estimate of GFR

Causes of increased creatinine?

1. pre-renal azotaemia

   • more common than renal azotaemia

   • USG >1.030 (dog) or USG >1.350 (cat) -indicates renal tubular function is most likely adquate

   • decreased renal perfusion → decreased GFR

     • shock, dehydration, cardiovascular insufficiency

     • maybe accompanied by increased PCV and TP

   • Increased protein catabolism → only mild azotaemia in that case

     • e.g., SI haemorrhage, infection, fever, corticosteroids

     • BUN will increse without a concurrent increase in creatinine

2. Renal azotamia

   • 75% of nephrons must stop functioning before GFR sufficiently decreased for values to increase to above reference range

   • dog USG 1.008-1.029

   • cat USG 1.008-1.034

3. Post-renal azotaemia

   • Most commonly associated with obstruction or leakage distal to the kidneys

   • clinical signs: oliguria/anuria

   • USG - anything

   • Repture of LUT without significant obstruction

     • >1.4x increase in potassium in peritoneal fluid compared with serum

     • >2x increase in creatinine in peritoneal fluid compared to serum

   • hyperkalaemia common - reduced ability to excrete

Amylase

Causes of increased Amylase

• non-specific enzyme

• leakage when damage to pancreatic acinar cells - absence of which does not rule out pancreatitis

• elevations >3-5 times normal can be suggestive of pancreatitis

• also secondary to non-pancreatic disorders

  • especially intestestinal but also kidneys and uterus

• hyperamylasaemia (2-3 x upper reference range) occurs in canine renal failure due to reduced excretion

• not a feature of feline pancreatitis

Lipase

• non-specific test

• leakage enzyme when there is damage to pancreatic acinar cells

• found in mainly the pancreas but also the gastric mucosa

  • elevations >=3-5 times normal can be suggestive of pancreatitis

• absence does not rule out pancreatitis

• in renal failure (3-5x upper reference range)

• other causes

  • chronic gastritis, duodenal obstruction and other enteric disease

  • hepatic disease

  • post-abdominal

  • pancreatic and hepatic neoplasia

Canine pancreatitis lipase - Snap

negative test results generally rules out pancreatitis, a positive test result requires further testing to confirm the diagnosis

Alanine Aminotransferase (ALT)

Increased ALT

• reflects either leakage from damaged or necrotic hepatic cells

• or increases in volume of secreting tissue

• and/or increased intracellular enzyme concentration

• also present in heart, kidneys and muscle

  • mild to moderate increases in moderate to severe muscle injury (CK and AST will also be significantly elevated)

  • mild increases can occur with cholestasis and inflammatory gastrointestinal disease

• ALT also present in RBCs - mild increases in haemolysed and lipaemic samples

• hepatotoxic drugs

  • anti-convulsant medications (<4x increase)

  • phenobarbitone

  • glucocorticoids

  • doxycyline and methimazole (cats)

  • paracetamol

• persistant increase is evidence of ongoing innury

• rate of clearance is more important than magnitude of elevation

  • half-life is reported to range from 3hours to 4 days

    • hepatic derived ALT is most prolonged as it is also secreted by regenerating hepatocytes

  • approximately 50% reduction every 3-4 days in acute hepatopathies can be suggestive of a good prognosis - normal in 3-4 weeks

Aspartate aminotransferase - AST

• found in skeletal muscle, cardiac muscle, liver and erythrocytes

• used to investigate muscle damage (all species) and liver damage in some

  • must look at with CK

• increased

  • elevated AST with parallel increase of ALT - hepatic

  • elevated AST with normal ALT - extrahepatic

ALKALINE PHOSPHATASE (ALP)

• Mainly found in liver and bone (osteoblasts)

• Also found in intestinal wall, kidneys and placenta - insignificant

• Hepatic isoenzyme of ALP

  • in dogs:

    • sensitive indicator of cholestasis (precedes hyperbilirubinaemia and often seen with elevated GGT also)

    • increases in a variety of causes:

      • severe hepatic necrosis

      • anything that causes hepatocellular swelling

      • phenobarbital

      • hyperadrenocorticism

      • DM

      • hypoT

  • Interpretation in the cat:

    • short half life only 6 hours

    • not sensitive for cholestasis but very specific!

    • chronic neutrophlic cholangitis

    • idiopathic hepatic lipidosis

    • hyperthyroidism - resolves with treatment\

• Bone-isoenzyme of ALP

  • increased in young animals (increase in osteoblastic activity)

    • kittens <15 weeks

    • puppies <6-8months

  • normal adult ragnes expected by 12 months of age at the latest

  • older animals

    • osteomyelitis, osteosarcoma, primary and secondary hyperPTH

• Steroid induced isoenzyme of ALP

  • in the dog but NOT THE CAT

  • endogenous and exogenous glucocorticoid

  • prednisolone

  • phenobarbitone

Bilirubin

Hyperbilirubinaemia

• retention hyperbilirubinaemia

  • primarily unconjugated

  • increased production (haemolysis) or hepatocellular lesion proximal to conjungation - e.g., intravascular haemolysis

• regurgitation hyperbilirubinaemia

  • primarily conjugated

  • due to hepatocellular lesions distal to the conjugation step or intra- or extra-hepatic cholestasis

    • e.g., liver failure, obstructive biliary disease (intra-hepatic or extra-hepatic)

• mixed patterns

  • reflects saturation of the system

  • most cases have mixed patterns by the time they present

Creatinine Kinase (CK)

• Present primarily in skeletal muscle, myocardium and brain

• Increases:

  • any event that causes muscle damage

  • myopathies

  • short half-life of 6 hours - indicates active or recent muscle traum

  • Persistent increase = ongoing myonecrosis

Excessive haemorrhage Screening Tests

1. CBC including platelet count

2. Biochemistry

3. PT/APTT

Prothrombin Time (PT)

• Measures the activity of extrinsic (factor 7) and common pathways

• low fibrinogen will prolong PT independent of other clotting factor deficiencies

• must have adequate blood collected into citrate otherwise PT may be prolonged

• factors need to be <30% of normal to result in prolonged clotting times

Prolonged PT, normal APTT

• inherited factor 7 deficiency - reported in some dog breeds (does not cause haemorrhage)

• early vitamin K deficiency

• DIC especially in cats

• liver failure

Prolonged PT, Prolonged APTT

• multiple factor deficiencies, affecting both extrinsic and intrinsic pathays or common pathway factor deficiencies

  • vitamin K deficiency - e.g., rodenticide toxicity

  • Disseminated intravascular coagulopathy

  • Liver failure

  • Hypofibrinogenaemia

Activated Partial Thromboplastin Time (APTT)

• Measures the activities of intrinsic (12, 11, 9, 10) and common pathways

• low fibrinogen may prolong APTT independent of other clotting factor deficiencies

• must have adequate blood collected into citrate, otherwise APTT may be prolonged\

• factors need to be <30% of normal to result in prolonged clotting times

• mild deficiencies in multiple factors in the intrinsic or common pathways can prolong APTT, despite individual factors not being < 30%

Prolonged APTT, normal PT

• artefact - difficult venipuncture

• iatrogenic unfractiouned heparin therapy

• inherited deficiency - haemophilia A, and haemophilia B in dogs, factor 12 deficiency in cats (common and as not required for physiologic haemostasis will not cause haemorrhage)

• DIC - especially in dogs

• dogs with systemic lupus erythematosis due to anti-phosphoplipid antibodies

• liver failure

Prolonged APTT, prolonged PT

• Multiple factor deficiencies affected both extrinsic and intrinsic pathways, or common pathway factor deficiencies

  • vitamin K deficiency

  • DIC

  • liver failure

  • hypofribrinogenemia

activated coagulation time (ACT)

• similar to APTT, except that factors need to be <10% of normal to result in prolonged clotting times

• also requires platelets: <10000/uL may result in mildly prolonged diatomaceous earth-based ACT (by 10-20 seconds)