Microanatomy in Clinical Practice — Sampling, Interpretation & Diagnosis

Definition of Disease

  • “A departure from health due to abnormality of structure or function.”

  • Core diagnostic principle:

    • Must first understand what is normal to recognise what is abnormal.

Principles of Diagnosis

  • Four classic pillars:

    • Signalment

    • History

    • Physical Examination

    • Ancillary Tests

  • Ancillary (paraclinical) tests extend diagnostic reach beyond what signalment, history and PE can reveal in both live and dead animals.

    • Diagnostic imaging

    • Laboratory investigations (many based on microscopic evaluation of cells, tissues, fluids)

Laboratory Investigation Overview

  • Clinical Pathology

    • Analysis of blood, urine, faeces, milk, wool, aspirated “lumps,” etc.

  • Anatomic Pathology

    • Biopsies & necropsies: evaluate structural change (lesions) in tissues/organs.

  • Microbiology

    • Subsidiary disciplines: Bacteriology, Virology, Mycology, Parasitology—detect infectious aetiologies.

Antemortem Sampling Methods

  • Blood

    • Whole blood, blood-cell fractions, plasma, serum.

  • Bone marrow aspirate/biopsy.

  • Excreta: urine, faeces, milk.

  • Body fluids

    • Pleural, peritoneal, pericardial, synovial, cerebrospinal (CSF), semen, joint, plus cavitary blood.

  • “Washes”

    • Tracheal / bronchial lavage.

  • Tissue contacts

    • Needle aspirates, impression smears, scrapings, cytobrushes, punch/needle/incisional biopsies.

Clinical Pathology — Haematology ("Erythrogram" & "Leucogram")

  • Example reference intervals (adult horse):

    • RBC count (RCC)=5.5–8.5×1012/L\text{RBC count (RCC)} = 5.5\text{–}8.5\times10^{12}/L

    • Hb=120–180 g/L\text{Hb} = 120\text{–}180\ g/L

    • PCV=37–55%\text{PCV} = 37\text{–}55\%

    • MCV=60–77 fL\text{MCV} = 60\text{–}77\ fL

    • MCH=19–24 pg\text{MCH} = 19\text{–}24\ pg

    • MCHC=320–360 g/L\text{MCHC} = 320\text{–}360\ g/L

  • Leucogram:

    • WBC=6–17×109/L\text{WBC} = 6\text{–}17\times10^{9}/L

    • Differential (\% and absolute):

    • Neutrophils 3–11.53\text{–}11.5

    • Bands <0.4

    • Lymphocytes 1–4.81\text{–}4.8

    • Monocytes 0.1–1.30.1\text{–}1.3

    • Eosinophils 0.1–1.30.1\text{–}1.3

  • Platelets: 200–500×109/L200\text{–}500\times10^{9}/L.

  • Morphology assessment on blood smear remains mandatory; automated counters cannot detect inclusions, parasites, atypical cells.

Clinical Pathology — Biochemistry (general adult-equine references)

  • Electrolytes & Acid–Base:

    • Na+=138–153 mmol/L\text{Na}^+=138\text{–}153\ mmol/L

    • K+=3.9–5.7 mmol/L\text{K}^+=3.9\text{–}5.7\ mmol/L

    • Cl−=101–114 mmol/L\text{Cl}^-=101\text{–}114\ mmol/L

    • \text{HCO_3^-}=15\text{–}24\ mmol/L

  • Calcium–phosphate balance: Ca2+=1.9–2.9 mmol/L\text{Ca}^{2+}=1.9\text{–}2.9\ mmol/L ; P=0.87–2.1 mmol/L\text{P}=0.87\text{–}2.1\ mmol/L.

  • Renal: Urea=2.5–9.5 mmol/L\text{Urea}=2.5\text{–}9.5\ mmol/L ; Creatinine=44–150 μmol/L\text{Creatinine}=44\text{–}150\ \mu mol/L.

  • Pancreatic enzymes: Amylase=180–1300 U/L\text{Amylase}=180\text{–}1300\ U/L ; \text{Lipase}<255\ U/L.

  • Hepatobiliary:

    • ALP=10–120 U/L\text{ALP}=10\text{–}120\ U/L

    • ALT=5–80 U/L\text{ALT}=5\text{–}80\ U/L

    • GGT=1–10 U/L\text{GGT}=1\text{–}10\ U/L

    • AST=10–80 U/L\text{AST}=10\text{–}80\ U/L

    • Bilirubin=2–15 μmol/L\text{Bilirubin}=2\text{–}15\ \mu mol/L

  • Muscle leakage: CK=50–400 U/L\text{CK}=50\text{–}400\ U/L.

  • Proteins: Total=54–78 g/L\text{Total}=54\text{–}78\ g/L ; Albumin=24–38 g/L\text{Albumin}=24\text{–}38\ g/L ; Globulin=24–42 g/L\text{Globulin}=24\text{–}42\ g/L.

  • Lipid & endocrine markers: Cholesterol=3.9–7.8 mmol/L\text{Cholesterol}=3.9\text{–}7.8\ mmol/L ; Glucose=3.3–6.7 mmol/L\text{Glucose}=3.3\text{–}6.7\ mmol/L.

Urinalysis

  • Gross parameters: colour, clarity, odour, volume.

  • Specific gravity measured via refractometer. Example scale in transcript spans 1.000–1.0501.000\text{–}1.050.

  • Chemical dip-stick: pH, protein, glucose, ketones, blood, bilirubin.

  • Microscopy: cells, crystals, casts, bacteria, parasites.

  • Quantitative Protein:Creatinine ratio (\text{Pr/Cr}). Trend illustrated from 6.54→06.54\rightarrow 0 across SG axis in slide.

Cytology

  • Definition: Microscopic examination of cells—stained or unstained—harvested from surfaces, lumina, or solid tissues.

  • Sampling tools: fine-needle aspirate (FNA), impression smear, scrape, brush, wash/lavage, fluid centrifugate.

  • Quick, low-cost, minimally invasive; distinguishes inflammation, hyperplasia, neoplasia.

Faecal Analysis

  • Detects parasites (ova, larvae), occult blood, maldigestion products, bacterial overgrowth/toxins.

Histopathology & Tissue Processing

  • Biopsy: tissue sample from a living animal. Biospecimen (>1 cm³) from a cadaver.

  • Fixation: 10 % neutral-buffered formalin in a volume ≈ 10× tissue volume to preserve structure & prevent autolysis.

  • Processing steps:

    1. Ascending alcohol series—dehydrate.

    2. Xylene—clears alcohol.

    3. Paraffin wax—impregnates & supports tissue.

  • Sectioning: microtome cuts 1–7 μm1\text{–}7\ \mu m ribbons. Sections floated on a warm water bath, mounted on glass slides.

Gross vs Microscopic Anatomy

  • Gross organs listed on slide: oesophagus, stomach, small & large intestines, liver, spleen, lungs, trachea, heart, kidney, colon, caecum.

  • Microscopic correlates: cell layers, parenchyma vs stroma, vascular/duct systems.

Staining Techniques

  • Routine contrast: Hematoxylin & Eosin (H\&E).

    • Hematoxylin: basic dye → nuclei blue/purple.

    • Eosin: acidic dye → cytoplasm & extracellular proteins pink/red.

Cytology vs Histopathology

  • Cytology: cells only; rapid; less invasive; good for screening.

  • Histopathology: preserves architecture; essential for tumour grading, margin assessment, complex structural lesions.

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Ethical & Practical Significance

  • Illustrates importance of:

    • Systematic diagnostic workflow (clinical ➜ laboratory ➜ imaging ➜ cytology ➜ histopathology ➜ necropsy).

    • Recognising when ancillary tests surpass the limits of physical examination.

    • Early cytologic detection of neoplasia guiding client decisions (prognosis, economics, welfare).

    • Post-mortem as a final audit of diagnostic accuracy and as a teaching resource.

Key Take-Home Messages

  • Know normals: interpretation of any lab data hinges on reliable reference intervals.

  • Combine data sources: no single test is definitive; integrate CBC, biochem, imaging, cytology, histology.

  • Cytology is rapid and minimally invasive but cannot replace architecture-based histopathology when tumour type/grading is required.

  • Adequate sample handling (fixation, smears, fluid EDTA vs plain) is essential to avoid artefact and diagnostic failure.

  • Post-mortem examination remains the gold standard when ante-mortem diagnostics are inconclusive or when animals die/euthanised.