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Clinical Pathology

Courtesy of Nanette Walker Smith, RVT, CVT and Elizabeth Warren, RVT

Blood collection tubes
Color ContentUses
purple (lavender)  EDTA (ethylenediaminetetraacetic acid)whole blood studies (CBC)
BlueCitrateCoagulation studies
GreenHeparinChemistries (whole blood or plasma)
Green marble top (tiger top)HeparinPlasma Separation
Graydiatomaceous earth   ACT (activated clotting time)
RedNoneSerum chemistries
Red marble topSeparation gelSerum separation

Blood components:
RBCRed Blood Cell (erythrocyte)non-nucleated cells in blood, carry oxygen and CO2 on the hemoglobin. RBCs make up from 40-55% of the blood volume.
WBC:White Blood Cell (leukocyte)nucleated cells of immune system in blood, combat insults to the body and fight infection
>granulocytesEosinophilSomewhat rare, Notable granules in cytoplasm, stain pink/red on Diff Quik (Wright's stain), common if parasite infestation present
BasophilRare, granules in cytoplasm, stain blue/black on Diff Quik (Wright's stain)
NeutrophilMost common granulocyte, granules in cytoplasm do not take stain
>agranulocytesLymphocytesMay range in size from RBC size to Neutrophil size, large nucleus, small amount of blue cytoplasm
MonocytesLargest WBC, often vacuoles and light bluish foamy cytoplasm.
PlateletsThrombocytesNon nucleated, disk shaped elements released into blood system by cells in bone marrow. Smaller than RBCs, large thromobocytes may indicate disease Platelets are important in hemostasis (blood clotting)
PlasmaLiquid of bloodBlood cells are suspended in this. Normally @ 45-60% of blood volume

CBC (complete blood count) Measurements

Hematocrit tubes (tubes may be heparinized to prevent clotting)
* fill with whole blood, seal with clay plug, spin in a high-speed microhematocrit centrifuge and compare to standard graph

PCVPacked Cell Volume% red blood cells in blood
HctHematocrit = PCV
BCBuffy CoatEstimate of # of WBCs and platelets in blood
*under 10X microscope observe for microfilaria at the BC/plasma or BC/serum interface
PPPlasma ProteinSolids in plasma of a heparinized sample
Measure by breaking tube above BC and put plasma drop on refractometer – read g/dl
*Will be slightly higher than a TP/TS due to clotting factors present in plasma
TP or TSTotal Protein, Total SolidsSolids in serum of non-heparinized sample
Measure by breaking tube above BC and put serum drop on refractometer – read g/dl
HgbHemoglobin@ PCV x 1/3 (if normal), best measured by spectrophotometric analysis or by calculation

Volumetric measurements:
MCVMean corpuscular volumeAverage size of RBCs measured in femtoliters
Calculated by the formula (hematocrit X 10/RBC).
A high MCV indicates macrocytosis; low MCV indicates microcytosis
MCHMean corpuscular hemoglobinAverage amount of Hgb in the RBC measured in picograms,
Calculated by the formula (hemoglobin X 10/RBC)
MCHCMean corpuscular hemoglobin concentration% concentration of an RBC, calculated by the formula (hemoglobin/hematocrit X 100)
A high MCHC is usually an artifact; low MCHC indicates hypochromasia
RDWRBC distribution widthIndicator of the variation in sizes of RBCs
Small RDW = uniform population, no information about the size of the cells;
A large RDW = anisocytosis.

Counting blood cells:

Estimated = viewing blood cells via microscope or similar methods.

  1. Viewing entire blood smear slide
  2. Quantitative Buffy Coat (QBC) method - capillary tube that is coated with dye, tube is filled with blood, mixed, centrifuged, blood components separate and stain cell type. QBC machine measures layers of buffy coat. Provides estimation, not an actual enumeration of cells.

Manually = Hemacytometer with whole blood measured and mixed in lysing solution specific for cell type (Unopette), counted on cytometer grid.

Cell Count = (average number of cells counted x dilution factor) divided by (number of squares counted x volume of each square).

Enumerated = automatic counter

  1. Automated counters - Cell counters use electrical impedance to individually measure cells via electric current; must be set for each species; most accurate; minimal time.

The blood smear:

Preparation - Place a drop of blood on one end of a microscope slide, use edge of second slide to smear the drop across the surface of the first slide.

  • Ideal = even distribution of blood over @ 2/3 length of slide, a feathered edge, and a rainbow effect (indicates one cell layer)
  • Stain = modified Wright¹s stain (Diff-quick, Dip-stat).

Evaluation and WBC estimate

  • On 10x, examine peripheral edge of smear for platelet clumps, microfilaria, abnormal WBCs, etc.
    • Choose a one-cell layer area, no overlapping/crowding of cells and count WBCs present in several fields
    • Estimated # of WBCs per ml blood = (average # of WBCs per field x 1000) x 1/4.

Differential WBC count - Count 100 white blood cells (oil immersion-100x), tally each WBC cell type. This gives you the % of each type.

Absolute WBC Count - Total WBC x % on differential for EACH WBC type.

Platelet estimate - Count number of platelets (oil immersion-100x) in several fields.

  • The average # of platelets seen x 15-20,000 = Estimate of total number of platelets per microliter of blood.
  • 8-20 platelets per hpf (high power field) = adequate platelet number. **Do not estimate if platelet clumping is present

RBC morphology - evaluate the red blood cells (oil immersion-100x) for variations from normal (size, color, shape, inclusions).

  • Report as: few = less than 10% of cells affected; 1+ = 25%; 2+ = 50%; 3+ = 75%; and 4+ = all cells affected.
  • Very rarely will you see 3+ or 4+ of any single abnormality.

Blood Cell Parasites:

Hemobartonella felis - small, dark purple cocci on the periphery of feline RBCs
Hemobartonella canis - rare, across surface of RBCs
Cytauxzoon felis - rare, pale rings within all cells
Babesia canis/gibsoni - teardrop shapes within RBC
Ehrlichia spp. - small round clusters/clumps within monocytes and neutrophils

Serum Chemistry

  • Spectrophotography analyzes serum, whole blood, or plasma
  • Factors affecting results include: hemolysis (contents of cells in serum), lipemia (fat), chemicals, sample handling, testing methods, etc.

Liver Function TestsExamples of causes of increased or decreased results
ALBAlbuminProduced in liver; Decreased = hepatic insufficiency or malnutrition, or loss in urine or GI tract.
ALPAlkaline phosphataseElevation = liver disease, cholestasis (bile obstruction), certain drugs;
Elevation normal in young animals due to growing bones
ALTAlanine aminotransferaseLiver and also in kidney or pancreatic damage;
elevated = liver damage, not extent of damage
BILBilirubinIncreased = intravascular hemolysis, liver disease.
Decreased = bone marrow depression (hypoplasia).
Total = conjugated + unconjugated, direct = conjugated, indirect = unconjugated.
GGTGamma glutamyltranspeptidaseNot commonly used to evaluate liver disease in small animals due to low blood levels (normal)
GLBGlobulinGroup of proteins made in liver and immune system
Globulin = TP – albumin
Increased = antibody production; Decreased = dehydration
SGOTSerum glutamic-oxaloacetic transaminase=AST
SGPTSerum glutamic
pyruvic transaminase
=ALT
Kidney Function Tests
BUNBlood urea nitrogenMeasures ability of the kidney to filter nitrogenous waste from the blood; Elevated = high protein, kidney disease (75% kidney destruction), dehydration, lower urinary tract obstruction;
Decrease = severe liver disease
CREACreatinineCreated by energy generation;
Elevation = 75% kidney destruction, other diseases
IphosInorganic PhosphorusElectrolyte, measured as an enzyme; Increased Iphos leads to calcium decrease; Decreased Iphos leads to calcium increase;
Changes = diet, disease (esp. kidney and calcium)
Pancreatic Tests
AMYAmylaseIncrease = pancreatitis; results vary with test method;
use reference ranges for test method
LIPLipaseIncrease = pancreatitis (more reliable than amylase); Affected by lipemia
GLUGlucoseIncrease = diabetes, pancreatitis; stress
Decreased = hyperinsulinism, insulin overdose, starvation, some diseases
Other
CALCalciumElectrolyte, measured as an enzyme; Increase = neoplastic disease, elevated protein, decreased phosphorus;
Decreased = eclampsia, trauma, glycol toxicity, etc.
CHOCholesterolIncreased = diabetes, pancreatitis, liver disease, increased dietary intake, hypothyroidism;
Decreased = malabsorption, low dietary intake
CKCreatinine kinaseProduced by muscle cells;
Increased = muscle damage (any muscle)
Na+SodiumIon – maintenance of plasma volume;
Increased = dehydration, diabetes, heart disease;
Decreased = V/D (fluid loss), acute renal failure, overhydration, congestive heart failure, etc.
K+PotassiumIon – regulates intracellular water balance;
Increased = dehydration, acute renal failure, acidosis, hemolysis, shock;
Decreased = profuse V/D (fluid loss), intestinal obstruction, alkalosis etc.
Cl-ChlorideIon – maintains electrolyte balance;
Increased = dehydration, diabetes, heart disease;
Decreased = V/D (fluid loss), acute renal failure, over hydration, congestive heart failure, etc.
HCO3-BicarbonateIon – blood buffer, helps regulate pH
Calculated Measurements
OsmolalityMeasure of tonicity/water balanceIncrease = cells swell, possible seizures;
Decrease = leads to shock/death; related to sodium levels; 
2(Na + K) + (glucose/18) + (BUN/2.8)
A/G ratioAlbumin/GlobulinThe ratio of albumin to globulin in blood serum, plasma, or urine
C CaCorrected calciumCalcium – Albumin + 3.5
Anion Gap(Na +K) – (Cl + HCO3)Screens for acidosis when blood gas analysis not available.
Increase = acidosis.
Cl/PhosChloride/Phosphorus ratioIncreased = primary hyperparathyroidism;
Decreased = malignancy
CaPCalcium x PhosphorusShould be < 60.
Elevated = tissue calcification
Na/K ratioSodium/Potassium ratioLow < 23 = may be diluted serum;
if no obvious cause consider Addisons

Blood gases

  • Patient's respiratory function and acid-base status.
  • Respiratory compromise or acid-base imbalances impede treatment.
  • Use hand-held potentiometric analyzers for easy in-hospital determination.
  • Evaluations of oxygen = arterial blood sample
  • All other blood gas parameters = venous blood (use correct reference ranges)
  • Read the instructions for your unit (IRMA and I-Stat are most commonly used analyzers), have test cartridges at proper temperature.

Arterial puncture: femoral artery (most common), dorsal metatarsal, brachial, aural, or lingual artery. (can be painful, difficult to perform)

  • Coat syringe with heparin (do not use sodium heparin if ionized calcium or Na+ is being analyzed)
  • Draw plunger back to amount of sample needed.
  • Arteries can't be visualized and are deeper than veins, so technique is based on palpable pulse;
  • Hold syringe in a pencil grasp, puncture at 45-90 degree angle.
  • Arterial blood is under pressure and will fill the syringe without suction.
  • Withdraw needle and hold firm pressure for a few minutes to prevent hematoma.
  • Immediately expel air and plug needle to prevent room air contamination (a rubber tube stopper works well).
  • Test immediately or place syringe in cup of crushed ice and test within 20-30 minutes.

Venipuncture: recommended unless oxygen paramaters are required

  • Test immediately or place syringe in cup of crushed ice and test within 20-30 minutes.

5 step evaluation method:

1. Evaluate PaO2 if applicable to determine if the patient needs oxygen
2. Evaluate the pH to determine if acidotic or alkalotic
3. Evaluate PaCO2, if the same as pH (acidotic or alkalotic), then result is primary respiratory
4. Evaluate HCO3, if same as pH, then result is primary metabolic.
5. Evaluate the parameter NOT responsible for pH change to assess compensation.

Gas Normal Abnormal Definitions and Examples
Blood pH 7.35-7.45 Acidosis <7.35 Metabolic DKA, Addison's, antifreeze poisoning, aspirin, lactic acidosis, diarrhea
Respiratory Upper airway obstruction, anesthesia, pulmonary disease
Alkalosis >7.45 Metabolic GDV, vomiting, diuresis, Cushings
Respiratory Panting, excessive ventilation
PaO2 90 mm Hg Room air (20% O2) Use arterial blood only
Hypoxemia (< 85 mm Hg) Requires oxygen supplementation; give slow/shallow breaths, pulmonary disease, severe anemia.
Hyperoxemia Oxygen poisoning, occurs with manual ventilation of O2. Supplemental O2 best at 40% O2.
PaCO2 35-45 mm Hg Hypoventilation Hypercapnea (> 45 mm Hg) = respiratory acidosis
Hyperventilation Hypocapnea (< 35 mm Hg) = respiratory alkalosis
CO2 = trigger for respiratory rate/rhythm Changes in CNS disorders, pulmonary disease
Respiratory rate changes to compensate for metabolic acidosis or alkalosis
HCO3 Bicarbonate Increased (Metabolic Alkalosis) Regulated by kidneys (movement of hydrogen ions)
Decreased (Metabolic Acidosis) Regulated by kidneys (movement of hydrogen ions)
Base Deficit or Excess The amount of base that must be added to return an animal to normal acid-base status.
Alkalotic (+4) Base excess
Acidotic (-4) Base deficits

Hemostasis and Transfusion
Bleeding timeMeasures the platelets¹ ability to plug a small wound.
Low platelet # or abnormal platelet function (ie Von Willibrand¹s factor deficiency) = prolonged bleeding time
  • Buccal mucosal bleeding time (BMBT) and toenail bleeding time are options. Small cut made in gum tissue or nail quick, allow to bleed freely (do not touch wound directly). Bleeding should stop within about 5 minutes. (test is relatively insensitive).
  • ACT (activated clotting time) - Evaluates the function of and detects severe abnormalities in the intrinsic and/or common pathways of the coagulation cascade. Use special vacutainer tube containing diatomaceous earth. Exact testing procedure must be used. Warm ACT tube to body temperature before test begins.
    • Exactly 2 mls of blood collected via ³clean stick² venipuncture and put into the tube immediately and gently inverted to mix blood and reagent.
    • The tube is maintained at body temperature (in a heat block or held in the armpit) for 30 seconds.
    • Checks for clot formation and immediately returns the tube to the heat source.
    • Check every 10 seconds until a clot forms.  
    • Normal ACT= 60-110 seconds (k9) and 60-75 seconds (feline).
    • Additional testing is required to determine the exact cause of a prolonged ACT.
Blood Transfusionknow animal's blood type and/or to perform compatibility tests with the donor and the recipient¹s blood
(see crossmatching steps below)
Blood typingCanine blood typing kits test for DEA 1.1 antigen
  • reagent containing DEA 1.1 antibody added to blood containing DEA 1.1 antigen will cause agglutination).
  • Dogs who are DEA 1.1 negative will have an immune reaction against DEA 1.1 positive red cells (if sensitized).

Feline blood typing kits test both A and B antigens.

  • Type B = strong naturally-occurring antibodies to type A blood.
  • Type A cats have a weaker antibody to type B blood.
  • Highly recommended to give type-specific blood to feline patients.
  • Type B is fairly uncommon and occurs primarily in purebred cats, including the Asian breeds.
CrossmatchMAJOR crossmatch = recipient serum mixed with donor RBCs to detect antibodies in the recipient against the donor¹s cells
MINOR crossmatch = recipient RBCs mixed with donor serum to detect antibodies in the donor¹s serum against the recipient¹s RBCs (can be important in whole blood transfusions)
FDPsFibrin Degradation Products = increased breakdown of fibrin clots, as occurs in DIC (disseminated intravascular coagulation).
(Commercial kit available)
FibrinogenIn-house - centrifuge 2 mHCT tubes.
Measure protein from one tube, heat second tube to 56 degrees (to cause precipitation of fibrinogen) for 3 minutes, then centrifuge.
Measured protein from second tube, difference between two tubes @ = fibrinogen concentration.
Low = clot formation; Elevated = inflammation
Each 0.lg/dl reading on TP scale is @ 100mg/dl of fibrinogen
Platelet countUse manual, automated counter, or estimation on a blood smear.
Platelet clumping can affect count.
Elevated on automatic count may result if platelets same size as RBCs.
**Verify by visual of blood smear.
Decreased clotting action if low # or intravascular clumping
Primary HemostasisFormation of a platelet plug. Platelet number and function are important in initial stages
PTProthrombin Time - The PT evaluates the function of the extrinsic and common coagulation pathways.
PTTPartial Thromboplastin Time - The PTT evaluates the function of the intrinsic and common coagulation pathways
  • Send out: blood is drawn into a citrate (blue top) tube, need to fill tube precisely as directed (check the tube label). Plasma must be separated from the cells within 30 minutes and kept frozen for lab transport. Tests must be performed within 48 hours of draw if plasma frozen and within 6 hours if refrigerated plasma.
  • In-house: follow directions for system used (may use citrate tube or whole blood)
Secondary hemostasisActivation of 12 different clotting factors plus several other chemicals in the intrinsic, extrinsic, and common pathways (activation of a clot or the "Coagulation Cascade")

Crossmatch - Need one tube of serum and 1 tube of plasma from the donor and from the recipient. Do each of the following steps for both samples.

  1. Centrifuge and separate cells from serum.
  2. Place 0.5cc of red cells in a 12 x 75 mm test tube, fill the tube with saline, mixing to suspend the cells in the saline.
  3. Centrifuge test tubes for 1 minute, then pour off the saline supernatant, resuspend cells with fresh saline.
  4. Repeat this "wash" cycle 3 times, pouring off the supernatant after the each wash.
  5. Resuspend the cells with saline to give a ³weak tomato juice² appearance (2-4% solution).
  6. Make the following mixtures in labeled test tubes:
    1. MAJOR-2 drops patient (recipient) serum plus 2 drops donor RBC suspension,
    2. MINOR-2 drops donor serum plus 2 drops patient RBC suspension.
  7. Incubate tubes at 37 degrees for 30 minutes, then centrifuge at 1000 rpm for 1 minute.
  8. Examine both tubes for gross hemolysis/agglutination.
  9. If no agglutination is seen, place a drop of the mixture on a slide and examine under the microscope.
  10. Agglutination or hemolysis = presence of antibodies, and the incompatibility of the blood types.
  11. The absence of agglutination or hemolysis does not ensure that the animals have compatible blood. Test only shows if antibodies were already present.

Immunology/Serology - in hospital

ELISA (enzyme-linked immunosorbant assay) testing used to identify antigens or antibodies in an animal's blood or body fluids.

  • The patient sample added to reagent containing antibody or antigen.
  • If the corresponding antibody or antigen is present in the patient sample, a complex of Ab/Ag forms.
  • This mixture is added to a system containing an enzyme-linked antibody.
  • If Ab/Ag complex is present, the enzyme-linked antibody attaches to the complex and causes a color change in the test system.

Urine collection and processing

  1. Urine samples should be at least 1cc, and preferably 12cc.
  2. Urine must be examined within 1/2 hour, or the urine constituents start to change.
  3. If the sample cannot be analyzed right away, it should be refrigerated. The sample will only be good for 24 hours when refrigerated.

Catheterizationinsert catheter into the bladder via the urethraRelatively clean sample. This method practical for male dogs,
May be contaminated with cells from the lower urinary tract, lubricant, and/or powder from gloves
Cystocentesiswithdraw urine from bladder with a needle'cleanest' sample, with no contaminants from the lower urinary tract.
Microbiological tests must be performed on cystocentesis samples.
Voided sampleFree catch urine in sterile basinContains contaminants from the lower urinary tract, cannot be used for culture.
If collected from a litter pan, table top or cage floor, contaminated with dirt and environmental bacteria

Urinalysis

 NormalAbnormal
1. Evaluation of physical characteristics
Coloryellow, due to the presence of urochromeDilute = pale to colorless
Concentrated = amber colored
Hemoglobinuria = clear red urine contains hemoglobin
Hematuria = cloudy red urine contains RBCs
Bilirubinuria = brown caused by bilirubin (as in hemolytic conditions)
Odorfresh urine has a characteristic aromaodors include: sweet, ammonia, putrid or foul smelling.
specific gravity1.015-1.050 (refractometer)Dilute (<1.015) can indicate overhydration or kidney damage
Concentrated (>1.050) may reflect severe dehydration fluid loss through other means, etc.
Isosthenuria or USG of 1.008-1.012 = kidney damage
transparencyclearHaziness to cloudiness can result from the presence of mucus, cells, crystals, and/or bacteria
2. Evaluation of chemical characteristics - Reagent Strips (i.e. Chemstrips or MultiStix)
*do not allow urine to flow from pad to pad
**blot strip to remove excess urine
***read results at time indicated on side of bottle
BilirubinNot presentPresence = liver disease, bile duct disease, or hemolysis.
A small amount is normal in male dogs.
Bilirubin breaks down on exposure to light.
BloodVery small amountsIncreased = infection, kidney or urinary tract trauma.
Free hemoglobin will be present with hemolysis
Myoglobin occurs with muscle damage.
GlucoseNot presentGlucosuria occurs when blood glucose exceeds 180mg/dL (diabetes mellitus, recent high carbohydrate meal, fear response, IV fluids with dextrose, vitamin C, aspirin, morphine, penicillin, etc.)
KetonesNot presentOccur when the body metabolizes fats incompletely, as in uncontrolled diabetes and starvation
LeukocytesNot presentPresence = possible infection (verify by microscopic sediment evaluation)
Absence does not rule out infection. False negatives common and this test not accurate for cats (false positives)
NitriteNot presentPresence = infection with certain types of bacteria (those that convert nitrate to nitrite).
Absence does not rule out infection.
PH5.5-7.0 (turns more alkaline on standing)Diet directly influences pH - plant-based foods cause alkaline urine / meats create acid urine
Low pH acidosis, fever, activity, certain drugs
High pH- alkalosis, UTI, drugs, urine retention, bacteria
ProteinTrace amountsIncreased protein is an indicator of kidney damage, UTI, trauma, neoplasia, etc
Blood cells cause a positive protein test
Alkaline urine may cause false positive results.
UrobilinogenTrace amountsIncreased = hepatic or hemolytic disease
3. Microscopic evaluation of sediment
  1. The urine sample centrifuged at 1000 rpm for 5 minutes
  2. Supernatant is poured off, the remaining sediment re-suspended in the last drop of urine
  3. A drop of Sedi-stain or NMB (new methylene blue) can be added at this time (if desired).
  4. One drop of sediment is placed on a slide and cover slipped
  5. Low power lens examination, scan slide for larger elements such as crystals, casts, and cells. (lpf = low power field)
  6. High dry lens - enumerate elements. (hpf = high power field)
  7. Alternatively, a drop of sediment can be smeared onto a slide (like a blood smear), allowed to dry, and stained with Dif-quick. Cellular morphology, presence of bacteria, etc may be more easily determined using this method.
Bacteria0 per hpfMay be seen if free catch sample (few to 1+),
Abnormal in "clean" samples. Bacteria may occur as rod or cocci shape.
CastsRare - 2 per hpfOccur when protein precipitates in renal tubules, indicates degree of urine stasis and possible kidney damage. May need to use low light to see hyaline.
Hyaline casts = pure protein, pale cylinders in the urine.
Granular casts = contain cellular debris, may indicate kidney damage
Cellular casts = contain cells, indicates severe kidney damage
Fatty casts = contains fat droplets, indicates severe kidney damage
Waxy casts = waxy appearance, indicates severe kidney damage
Crystals0-2 per hpfPresence in the urine depends on urine pH. Triple phosphate (struvite) crystals are common in  cat urine, while calcium oxalate crystals are typical in dalmatians. Calcium oxalate crystals also occur in ethylene glycol poisoning. Leucine and tyrosine crystals are seen with liver disease. Cystine crystals may be associated with calculi (stones).
RBC0-2 per hpfLarge numbers of RBCs in the urine is called hematuria and indicates trauma or disease
Renal EpithelialFew per hpfLarge numbers of renal tubular epithelial cells may indicate kidney damage,
SpermatozoaIn males, recently bred females 
Squamous Epithelial4-8 per lpfModerate numbers of squamous and transitional epithelial cells are normal (less if cystocentesis)
WBC0-5 per hpfMay be increased in urinary tract infections

Internal Parasites (endoparasites)
Gastrointestinal system:
CoccidiaIsospora spp.These small eggs can be recovered via float or direct smear
Coccidia - protozoanCryptosporidium spp.Coccidia found in dogs, cats, and many other species including humans.
Very small eggs can be found on float, smear, special stain
EimeriaEimeria spp.Small intestinal parasite of large animals, but may be seen in small animal fecal floats if feces have been ingested.
Esophageal wormSpirocera lupiEggs recovered via fecal float exam or float of vomitus
GiardiaGiardia spp.Flagellates found in dogs, cats, and many other species including humans.
Trophozoites rare on direct smear of very fresh feces;
Cysts via direct smear or floatation with zinc sulfate solution.
Immunologic fecal tests are also available
HookwormsAncylostoma caninum (dog)
Ancylostoma tubaforme (cat)
Ancylostoma braziliense (dog and cat)
Uncinaria stenocephala (dog)
Small intestinal nematodes of dogs/cats,
May be found attached to intestinal wall (hooked) or passed in feces
Eggs recovered via fecal floatation.
Intestinal FlukeNanophyetus salmincolaSalmon poisoning fluke of dogs
Intestinal FlukeAlaria spp.Intestinal flukes of dogs and cats
Liver FlukePlatynosomum fastosumLizard poisoning fluke of cats
RoundwormsToxocaris canis (dog)
Toxocaris cati (cat)
Toxocara leonina (cat)
Ascarids found in small intestine. Large, spaghetti-like adults found in vomitus/feces.
Eggs can be recovered via fecal float
Stomach wormPhysalopteraUsually found attached to stomach wall of dogs/cats.
Adults found in vomitus may be confused with ascarids.
Eggs may be seen in fecal float.
TapewormsDipylidium caninumMost common dog and cat tapeworm, transmitted by flea ingestion.
Diagnosis made by recovery of proglottids (~rice grain) in feces, less often via fecal float.
*other varieties of tapeworm; some may be zoonotic; familiarize with parasites in your area.
ToxoplasmosisToxoplasma gondiiFound in cats and uncooked meat. Diagnosis by blood tests, less often by fecal float/smear
WhipwormsTrichuris vulpis (dog)
Trichuris campanula (cat)
Trichuris serrata (cat)
Small, whip-shaped nematodes in the large intestine of dogs (common) and cats (rare)
Eggs recovered via fecal float.
Circulatory system:
Dipetalonema Dipetalonema reconditumMicrofilaria commonly mistaken as heartworms, found in subcutaneous tissue, diagnosis by modified Knotts, Millipore, or filtration tests used for heartworm
HeartwormsDirofilaria immitisMicrofilaria can be found in blood, adults live in heart and pulmonary artery
Diagnosis of occult infection is made via serologic test
LungwormsAelurostrongylus abstrusus (cat)
Filaroides species (dog)
When eggs hatch, characteristic larvae can be found via fecal flotation or tracheal wash.
LungwormsCapillaria aerophilaFound in the trachea and lungs. Eggs can be recovered in fecal floats.
LungwormsParagonimus kellicottiFound in the lungs. Eggs recovered in feces or sputum
Intracellular
BabesiaBabesia canisIntracellular (RBC) organisms found inside the cells on a blood smear
HemobartonellaHemobartonella felisIn anemic cats, small round or rod-shaped structures that stain dark on Wright's or Romanowsky stain. May need to view several slides before ruling out its presence.
Urogenital system
Giant kidney wormDioctophyma renaleIn dogs--eggs recovered in urine sediment
Bladder wormCapillaria plica (dog) and feliscati (cat)Live in the bladder, eggs recovered in urine sediment

Fecal tests

Fecal float - method of choice for eggs, cysts

  1. Mix sample of fresh feces with a sugar or salt solution (hypertonic) in a vial.
  2. Bring level of fluid to just above the top of the vial, and place a coverslip over the vial.
  3. Allow sample to 'float' for 10-15 minutes (depending on solution used)
  4. Place coverslip on a slide and examine under low power (10X) for parasitic ova.
  5. Giardia cysts precipitate in zinc sulfate flotation solution

Fecal smear - the method of choice for identifying protozoans, coccidia, cryptosporidium, and some types of bacteria (spirochetes, clostridium), etc.

  1. Mix a very small amount of fresh feces with a drop of normal saline on a slide
  2. Add a cover slip
  3. The slide is examined on high power (40X).
  4. Add a drop of Lugol¹s iodine to stain Giardia cysts and trophozoites.
  5. Cells such as RBCs, WBCs, and epithelial cells should be noted.

Fecal stained smear - Aids visualization of bacteria characteristics and cellular morphology

  1. Make a thin smear of feces on a slide
  2. Heat-fix and stain with Dif-quick.
  3. The slide is then evaluated on 100X (oil lens)

External Parasites (ectoparasites)

Fleas, ticks, lice, and mites are common external parasites (they live on the surface of the host).
*Diagnosis is by identification of the adult or recovery of eggs/larvae.

Skin scraping -

  1. Use a dull #10 blade coated with mineral oil and scrape across surface of a suspected lesion (especially at edges)
  2. Light scrape surface scales and crusts for mites such as Cheyletellia,
  3. Deeper scrape (until just before it bleeds) for burrowing mites such as Sarcoptes
  4. Spread material in a drop of mineral oil on a slide and examined with a low power lens under the microscope.

Microbiology

The diagnosis of infection with microbes (bacteria, fungi, viruses) is typically made by sending samples to a reference laboratory

They are identified using special growth media and testing methods

Sensitivity testing to antimicrobial drugs is done.

Preliminary tests done in-house can help determine what tests the reference lab should perform.

Sample collection:

  1. Use aseptic technique
  2. Sample immediately placed into transport medium so that microbes are preserved until testing.
    1. Use specific transport medium for anerobic samples (oxygen-free organisms).
    2. Follow instructions from laboratory for collection and submission of samples.

Primary identification of bacteria:

Color - determined by Gram's stain result

  • Positive = purple
  • Negative = red

Shape -

  • cocci = round
    • Gram Positive - Staphylococcus spp., Streptococcus spp., Pneumococci (encapsulated) spp.
    • Gram Negative - Moraxella spp., Neisseria spp.
  • bacilli = rods
    • Gram Positive - Clostridium (spore-forming) spp., Bacillus anthracis (spore-forming), Listeria spp., Erysipelothrix spp.
    • Gram Negative - Escherichia coli, Salmonella spp., Klebsiella spp., Hemophilus spp., Actinobacillus spp.
    • Aerobic - Pseudomonas spp., Brucella spp., Francisella spp., Pasteurella spp., Bordetella spp.
    • Anaerobic - Bacteroides nodosus
  • spiral
    • Gram Negative - Campylobacter spp., Leptospira spp., Borrelia spp.

Arrangement -

  • single
  • paired - (diplo)
  • clusters - (Staphylococcus)
  • chain - (Streptococcus)

Motility -

  • motile (has progressive forward motion)
  • non-motile (no motion; *Brownian motion should not be mistaken for motility)

Spores - present or not present

Other: Bacteria resembling fungi - Actinomyces (gram positive bacilli), Nocardia asteroides (gram positive bacilli), Mycoplasma (gram negative pleomorphic), Mycobacterium (acid fast bacilli)

Gram stain

1. Prepare a thin smear on a slide and allow to dry
2. Heat fix the slide
3. Flood slide with crystal violet and let stand for 1 minute
4. Rinse with water
5. Flood slide with iodine solution and let stand for 1 minute
6. Rinse with decolorizer for 5-10 seconds
7. Rinse with water
8. Flood slide with safranin counterstain and let stand for 1 minute
9. Rinse with water, blot dry, examine under oil immersion lens.
10. Gram positives appear purple or dark blue. Gram negatives appear red.

Fungal Tests

Wood's lampTurn on 10 minutes prior to exam
Infected hairs (esp. at edges of lesions) may fluoresce (glow) under the UV light
*Negative reaction does not rule out ringworm
Direct microscopyPlace suspect hairs / skin from the lesion on microscope slide with a drop of 20% KOH
Place coverslip on top, warm, and allow to sit for 10-15 minutes
Examine under low (10X) and high dry power (40X) for identification of fungal bodies
Fungal culture
(DTM plate)
Collect and place samples aseptically on an agar plate
Store innoculated media in warm, dark place and evaluate for growth daily for 10 days
pH indicator cause medium to turn red in the presence of dermatophytes

Cytology

Usually stained with Wright's stain and examined microscopically

Swabs = surface samples (ear canal, vagina, rectum, conjunctiva, etc) rolled over the surface of a slide to spread the material into a thin layer.

Scrapings = samples are beneath the surface of the tissue (hard masses, skin, etc). Material is spread onto the surface of the slide with the blade.

Aspirates = samples are fluids, tissues, and internal organs.

FNA (fine needle aspirates)

  • use a needle (22-25 gauge) attached directly to a syringe;
  • apply suction to plunger with the needle placed in the area of interest
  • For delicate tissues
    • Attach 12 cc syringe to IV extension line and a needle.
    • Draw plunger to about 6cc prior to insertion of the needle; this provides gentle suction.
  • Move needle back and forth quickly several times within the area of interest
  • Expel sample onto slide through needle spread via a smear or pull-apart technique.
    • Fluid may be centrifuged prior to evaluation under the microscope and sediment examined.

Biopsies

  • Solid tissue - may be cut in half, blotted dry, and then pressed onto a slide for impression smear cytology (before sample is placed into preservative).
    • Microscopic evaluation:
      Inflammation -WBCs and organisms
      Neoplasia - variable cell sizes (anisocytosis), variable cell forms (pleomorphism), variable cytoplasmic staining intensity, variable nuclear sizes (anisokaryosis), multinucleation, variable nucleolar sizes and shapes (may be multiple per cell), variable nucleus to cytoplasm ratio (may be less than normal), mitotic figures, coarse chromatin pattern.
  • Histopathology samples in a biopsy jar with 10 to 1 formalin to sample ratio

Courtesy of Nanette Walker Smith, RVT, CVT and Elizabeth Warren, RVT

The Nerd Book
The VSPN Nerdbook was created by veterinary technicians and veterinary support staff for their colleagues. The Nerdbook provides information that veterinary technicians and support staff need in practice, but is not meant to contain everything. Procedures and policies vary among practices, so feel free to modify your Nerdbook fit your facility.
Volume One
Contributors
Introduction
You are hereClinical Pathology
Critical Care - Triage
Emergency-Receptionist tips
Medical Calculations
Medical Records
Medical Terminology
Pharmacology
Physical Exam
Preventative Health
Radiology

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