clinical pathology for diagnosis of equine diseases

Yongbaek Kim, DVM, PhD. Dipl. ACVP

Seoul National University, College of Vet. Medicine,

Laboratory of Clinical Pathology

yongbaek@snu.ac.kr

Clinical Pathology for

Diagnosis of Equine Diseases

Clinical Pathology

Hematology (complete blood count, CBC)

Cellular components : RBC, WBC, Platelets

Proteins

Clinical Chemistry

Enzymology

Biomarkers

Fluid Analysis

Urinalysis

Abdominal and thoracic, CSF, Joint

Cytology

Mass

Tracheal and bronchial washing

Blood Sampling: Indications

Diagnosis of clinical disease

Infectious, parasitic

Specific organ dysfunction

Routine management

Routine neonatal foal examinations

Performance horse monitoring

Preanesthetic checks

Prepurchase examinations

Insurance examinations

Immunoglobulin G [IgG] tests for neonatal foals

Import/export examinations

Blood Sampling and Testing

Blood Testing

Complete Blood Count

Clinical Chemistry

Coagulation Tests

EDTA Heparin Serum Citrate

Blood Sampling

Accurately label with the horse's and owner's names

and the date of collection

Analyses should be performed as soon as possible

Some delay is inevitable

Prudent to carry samples in an insulated 'cool bin' (12 to

20°C) to avoid the deleterious effects of excessive

temperature fluctuations that may occur in a vehicle

Blood Sampling

Whole blood samples

Should not be refrigerated: results in hemolysis rendering

hematological tests and many serum enzyme analyses

inaccurate

Ideally, serum should be separated from clotted

samples as soon as practical

Centrifugation or simple standing and pipetting may be

used

Serum separation tubes

Once separated, serum or plasma may be refrigerated or

frozen for storage purposes

Blood Profiles

Profile type Profile content

36 hours Hematology, serum proteins, fibrinogen, PV, SAA, serum IgG

Weanling and

yearling

Hematology, serum proteins and electrophoresis, fibrinogen,

SAP,

serum calcium and phosphate, urine phosphate fractional

clearance ratios

Training horse Hematology, serum proteins and electrophoresis, PV,

fibrinogen, SAA, AST, CK, urine phosphate fractional

clearance ratios

Mature horse Hematology, serum proteins and electrophoresis, fibrinogen,

PV, SAA, AST, CK, GLDH, LD, SAP, IAP, GGT, urea,

creatinine

Basic Screen Hematology, Serum proteins, fibrinogen, AST, CK, GGT, SAP,

urea, creatinine AST Aspartate aminotransferase, CK Creatine kinase, GGT L-gamma glutamyl transferase,

GLDH Glutamate dehydrogenase, IAP Intestinal alkaline phosphatase, LD Lactate dehydrogenase,

PV Plasma viscosity, SAA Serum amyloid A, SAP Serum alkaline phosphatase

Blood Profiles

Profile type Profile content

Inflammatory

profile

Hematology, serum proteins, PV, fibrinogen, SAA

Liver profile Hematology, serum proteins and electrophoresis, fibrinogen,

PV, SAA, AST, GLDH, LD, SAP, IAP

Renal Hematology, Serum proteins, fibrinogen, Urea, Creatinine, Ca,

PO4

Muscle Hematology, AST, CK, Urea, Creatinine

Intestinal profile

Hematology, serum proteins and electrophoresis, fibrinogen,

PV, SAA, SAP, IAP

Electrolytes Na, K, Cl, Ca, PO4, Mg

Endocrine ACTH, insulin, Glucose, Triglycerides

AST Aspartate aminotransferase, CK Creatine kinase, GGT L-gamma glutamyl transferase,

GLDH Glutamate dehydrogenase, IAP Intestinal alkaline phosphatase, LD Lactate dehydrogenase,

PV Plasma viscosity, SAA Serum amyloid A, SAP Serum alkaline phosphatase

Complete Blood Count (CBC)

Hematology analyzer

•Cell counts

•Hemoglobin

•Cell parameters

Microhematocrit tube

•Packed cell volume

•Total plasma protein

•+/- fibrinogen

Blood smear

•WBC differential

•Confirm platelet

count

•Cell morphology

+/- Reticulocyte count

•Some hematology

analyzers can perform

•Only needed in anemic

patients

Erythrogram Hemoglobin** Red cell count** Hct MCV** MCH MCHC RDW Morphology

**Directly measured, all others calculated

Platelets Count** MPV** PDW Morphology

Leukogram TNCC (WBC count)** (Differential cell count) Morphology

Protein Total protein** Albumin** Globulin

Complete Blood Count (CBC)

Complete Blood Count (CBC)

Diseases Blood tests

Anemia Low red blood cell count, packed cell volume

and hemoglobin level

Hemoconcentration

/dehydration

Raised red blood cell count, packed cell

volume and hemoglobin level

Bacterial infections High white blood cell count and neutrophilia

Viral infections Low white blood cell count and neutropenia

during the acute phase

Parasitic or allergic

conditions

Eosinophilia in some cases

ANEMIA

Regenerative anemia

Acute blood loss, hemolytic anemia, and mild/moderate

intestinal parasitism

Reticulocytes: increased (some automated machine)

Macrocytic anemia

Non-regenerative anemia

Generalized bone marrow suppression, chronic inflammation,

neoplasia, chronic renal disease, or iron deficiency

Positive direct antiglobulin (Coombs) test

Diagnostic of immune-mediated anemia: idiopathic immune-

mediated anemia, neonatal isoerythrolysis and equine

infectious anemia.

LEUKOCYTOSIS

Physiological leukocytosis Excitement or stress

Increased blood pressure, heart rate and splenic contraction

Associated with intense exercise, fear or excitement

Transient, return to normal in an hour

Pathological leukocytosis with neutrophilia Infectious and non-infectious inflammation

Circulating immature neutrophils (ie, metamyelocyte and 'band' neutrophils)

Severe acute bacterial infections or septicemic processes

Toxic band cells: neonatal septicemia and adult colitis or enterotoxaemia

LEUKOPENIA

Increased emigration to inflamed tissues

Sequestration to highly vascular organs: pneumonia

Sequestration to body cavities: suppurative peritonitis

or pleuritis

Destruction in the microcirculation

Reduced neutrophil production in bone marrow

suppression: viral infections

Endotoxin shock: acute strangulation, intussusception,

salmonellosis, neonatal bacteremia and septicemia

LYMPHOCYTOSIS

Chronic viral infections

Chronic immune stimulation

Lymphoid neoplasia

May be dramatically elevated (as high as 100 x 109/l) in

cases of generalized lymphoma

None of these conditions consistently produces a

lymphocytosis.

EOSINOPHILIA

Antigen-antibody response in tissues rich in mast

cells

Skin, lung, gastrointestinal tract and female genital tract

Parasitism

Eosinophilic leukemia with eosinophil counts as

high as 2-5 x 109/l (25% on differential leucocyte

count).

THROMBOCYTOPENIA

Increased platelet destruction: immune-mediated

Primary or secondary to other diseases including

infections, neoplasia and drug treatments

Increased platelet usage: hemorrhage or trauma

Transient and reversible thrombocytopenia

DIC: severe and prolonged thrombocytopenia

EDTA-related pseudothrombocytopenia

Clumping of the platelets

Examination of extremities of the blood film

Heparinized or citrated blood should give normal platelet

numbers.

BLOOD BIOCHEMISTRY

Proteins

Plasma viscosity

Plasma fibrinogen

Serum enzymes

Other biochemical tests

Electrolytes

Proteins

Total proteins

Albumin

Globulin

Inflammatory proteins

Proteins: Albumin

Hyperalbuminemia: hemoconcentration and dehydration

Hypoalbuminemia: kidney, intestine, and liver diseases

Protein losing enteropathy: most common

Acute: salmonellosis or cyathostomiasis during the larval

emergence phase

Chronic: heavy cyathostomiasis or mixed large and small

stronglye burden, or a progressive infiltrative lesion of the

intestinal mucosa

Renal failure

Pigment-induced nephropathy, glomerulonephritis and

neoplasia

Liver failure: reduced production of albumin

Confirmed by increased serum liver enzymes and bile acids

Proteins: Globulin

alpha (α), beta (β) and gamma (ϒ) globulin fractions

α2-globulin: acute phase tissue proteins

elevated in acute inflammation and parasitism

(especially cyathostomiasis)

β1-globulin elevated in large and mixed strongyle

larval migration

β2-globulin raised in horses with hepatopathy

fibrinogen levels difficult in heparinized plasma samples

Proteins: Globulin

ϒ-globulin raised in response to antibody production

chronic infection or inflammation

Dramatic β2- or ϒ-globulin monoclonal 'spikes' in

cases with lymphoma or plasma cell myeloma

Hypogammaglobulinemia in neonatal foals with failed

passive immunity transfer

collected between 18 and 36 hours of age

Proteins: INFLAMMATORY PROTEINS

Serum amyloid A

sensitive, rapidly reacting, acute phase protein

help to detect early responses to infection and monitor the response to treatment

rises within hours and peaks at two days

Plasma fibrinogen

acute phase protein in horses with tissue damage

more chronic inflammation (eg, internal abscessation, chronic parasitism or infections)

crudely by subtraction of protein levels between fresh paired serum and plasma samples

direct coagulometry on a citrated blood sample

peak by 10 days and returns to normal slowly

Proteins: INFLAMMATORY PROTEINS

MUSCLE ENZYMES: AST, CK

Aspartate aminotransferase or serum glutamic-oxaloacetic aminotransferase (SGOT)

Rise in acute hepatopathy or myopathy peak at around 24 to 48 hour

return to baseline by 10 to 21 days

Creatine kinase (CK; creatine phosphokinase, CPK)

In acute myopathy, peak at six to 12 hours and return to baseline by three to four days

Exercise-induced myopathy

Serum before, and two to three hours after, submaximal exercise

Viral infections: EHV-1

clinical signs such as fatigue and stiffness.

SERUM ENZYMES: AST, CK

Cytoplasmic (“leakage or hepatocellular enzymes”) Enzymes are released from damaged cells or upon cell

death (necrosis)

AST (Aspartate aminotranferase, SGOT)

SDH (Sorbitol dehydrogenase)

GLDH (Glutamate dehyrogenase) ***ALT in horse: limited activity in liver

Induced (“cholestatic”) Enzymes are produced by cells (increased synthesis of an

enzyme)

GGT (gamma-glutamyl tranferase): preferable

ALP (alkaline phosphatase, “alk phos”, AP)

Hepatic Enzymes

Liver specific High concentration in liver but low in other tissues

Primarily in central lobular region

Mitochondrial rather than cytosolic

Released only with irreversible cell injury

Stable for a month at ~20C

More sensitive than SDH for liver diseases Valuable for the diagnosis of acute liver injury in horses

Limited availability of the assay in US

Cytoplasmic Enzymes: GLDH

SDH: less than 12 hours

SDH < GLDH <AST

AST: several days

Hepatic Enzymes: Half-life

Reference Interval

SDH

AST & GLDH

2 10 6 4 8

Days after following severe Liver injury with recovery

Present in multiple tissues Relatively low in liver but liver ALP is found in serum Relatively high in intestine, but not in serum Bound to bile canalicular surface membranes

Serum ALP mainly from bone and liver Higher in growing horse: 100 fold greater than in adults Half life: about 3 days similar to GGT

Increased ALP by cholestasis Cholangitis, biliary cirrhosis, extrahepatic bile duct

obstruction

Hepatobiliary Enzymes: ALP

Most serum GGT activity originates from liver

Present high in liver, kidney, intestine and pancreas

Half life: about 3 days similar to ALP

Increased with cholestasis and biliary hyperplasia

GGT is more sensitive than ALP for cholestatic diseases

In horse with cholestasis: 9 fold increased GGT, 2-fold

in ALP

No significant difference in GGT activity pre and post

suckling colostrum

Hepatobiliary Enzymes: GGT

Hepatic diseases: Toxins

Pyrrolizidine alkaloids

Megalocytosis, periacinar necrosis, biliary hyperplasia,

periprotal fibrosis

Enzymes elevated before clinical signs

SDH, GLD may be within RI: chronic

Increased GGT and ALP

Hepatic diseases: Toxins

Mycotoxins (aflatoxins, fumonicin) Megalocytosis, centrilobular hepatocellular necrosis,

biliary hyperplasia

Increased SDH, GLD

Increased GGT and ALP: in chronic

Iron toxicosis Foals are more susceptible

Massive periacinar necrosis and deposition of iron in Kupffer cells

Ferrous fumarate or sulfate containing vitamin supplements

Increased SDH, GLD

Hepatic diseases: Infections

Tyzzer’s disease (Clostridium piliforme)

One to six week old foal- acute or peracute hepatitis

Death within hours or days

Multifocal to confluent centeral coagulation necrosis

with inflammatory cells

Filamentous bacteria within hepatocytes: Warthin starry

stain

Marked increase in SDH, moderate to marked

hyperbilirubinemia

Moderate increase in GGT; ALP is not increased

Hepatic diseases: Infections

Cholangiohepatitis

Ascending infection from the intestinal tract: Salmonella,

E.coli, Actinobacillus equuli, etc

Inflammation of biliary tract and adjacent liver

High GGT and ALP relative to SDH and GLD

Hyperglobulinemia in chronic (>3 weeks)

Cholelithiasis is frequent, possible due to decreased

biliary flow

Hepatic diseases: parasites

Parascaris equorum, strongyle larvae

Migrate through liver: hemorrhagic tract with

inflammatory cells (eosinophils), necrosis and fibrosis

Liver flukes (Fasciola hepatica)

Multifocal granuloma with eosinophilic and lymphocytic

inflammation

Elevated GLD and GGT three to five month post

infection

Renal Markers: Urea, Creatinine

Urea: By-product of protein metabolism

Increased by high protein diet or urea supplementation

Increased in fasting or prolonged excerecise:

catabolism

Decreased in protein poor diets or liver failure

Creatinine: By-product of muscle metabolism

Correlated with total muscle mass

Falsely increased in non-creatinine chromogens:

glucose, ketones, cephalosporin antibiotics

Falsely low in hyperbilirubinemia

BUN:creatinine ratio

Not useful to differentiate prerenal, renal, postrenal

Renal Markers: Electrolytes

Hyponatremia, hypochloremia, hyper- or

hypocalcemia, hypophosphatemia, hyper- or

hypokalemia, decreased or increased bicarbonate

Hypercalcemia: commonly in chronic renal failure

Hypocalcemia: commonly in acute renal failure

Renal diseases

ARF CRF Dehydration

Azotemia + + +

Urea/Creat >1:10 <1:10 Variable

USG 1.008 - 1.012 1.008 - 1.012 >1.035

Proteinuria + + _

Others Glycosuria Nonregenerative

anemia

Clinical signs

High FE HyperMg

Hyper Mg Hyperlipidemia

Enzymuria Hypoalbuminemia

Urine casts and

WBCs

Metabolic acidosis

Metabolic acidosis

Colic: Negative Prognostic Indicator

Adopted from Practical Guide to

Equine Colic, by Southwood

Thank you for your attention!!!