31 August 2016 Urinalysis - a review

Transcription

1 31 August 2016 Urinalysis - a review Bradley Galgut, BVSc (Hons), DACVP Specialist Veterinary Clinical Pathologist

2 Urinalysis A Review Bradley Galgut, BVSc (Hons), DACVP Specialist Veterinary Clinical Pathologist ASAP Laboratory, VIC Functions of the Urinary System Kidneys: Eliminates liquid waste (i.e. metabolic by-products such as urea and creatinine, drugs, toxins etc) in the form of urine Regulates electrolytes (potassium, sodium, chloride, calcium etc) Regulates acid/base balance Controls water balance Kidneys help regulate blood pressure Produce erythropoietin, which controls red blood cell production in the bone marrow Key role in Vitamin D metabolism Urinary Tract Anatomy Urinalysis Vital component of routine diagnostic evaluation along with complete blood count (CBC) and biochemistry Liquid gold Often overlooked/ignored/forgotten ~1 million nephrons in each kidney kidney_function.htm Recommend obtaining a urine sample Detects disease of the urinary system and some metabolic disturbances As urine is a filtrate of blood, we can assess urine for drug by-products, toxins/venoms etc Urine Composition Determined by 3 major factors: Blood/plasma presented to the kidneys Renal functions Filtration Resorption Secretion Added during flow In the kidneys After the kidneys (bladder, urethra, skin) Always consider method of collection Voided, cystocentesis, catheter, off floor Important to note on lab submission form

3 Urine Specimen Storage Urine must be collected into clean (preferably sterile) container (usually yellow-top urine containers) Ideally urine should be analysed immediately after collection ph may change over time Cells may deteriorate Crystals may dissolve / precipitate Casts may break up Bacteria may proliferate with time If analysis will be delayed, refrigerate immediately Urinalysis Procedure 1) Physical evaluation Colour Clarity Urine specific gravity (USG) 2) Chemical evaluation Dipstick (reagent pad) analysis 3) Microscopic examination Sediment evaluation Allow sample to return to room temp before analysis Physical Evaluation - Colour Normal yellow colour due to urochromes Poorly defined group of pigments Generally urine concentration can be predicted by colour Pale yellow = dilute Dark yellow = concentrated Physical Evaluation - Colour Pigmenturia (abnormal urine colour) Red, clear Haemoglobinuria, myoglobinuria Red, cloudy Haematuria Orange-brown Bilirubinuria Coffee-brown Myoglobinuria Colour Red/Brown Urine Haematuria Red, cloudy urine; supernatant is clear or only slightly red RBC s in sediment Positive blood and protein on dipstick Normal plasma colour (clear) RBC s may lyse if urine ph > 7.5 Myoglobinuria Occurs due to muscle injury/necrosis Red-brown supernatant; few or no RBCs in sediment Positive blood and protein on dipstick Plasma normal colour (clear) Increased CK and AST (snake bite, trauma, tying up in horses) Haemoglobinuria Occurs due to intravascular haemolysis Red, clear supernatant Few / no RBC s in sediment Positive blood and protein on dipstick Red plasma (haemoglobinaemia) Haemolytic anaemia (CBC) Clarity Definition: Clearness / transparency Clear Expected in all healthy dogs and cats Hazy or cloudy/turbid Reflects the presence of suspended particles Crystals, cells, mucus, casts, spermatozoa, lipid, other material Equine urine is often cloudy due to mucoproteins

4 Solute Concentration Dissolved ions and molecules in urine Electrolytes and metabolic products (urea, creatinine etc) Concentrations modified by tubular resorption or secretion of solutes and by resorption of H2O (i.e. normal kidney functions) Analytical concepts Osmolality (what we re most interested in!) Solute concentration in a liquid Can t measure this easily (specialised testing) Specific gravity Ratio of solution s weight (density) to that of an equal volume of water solute concentration density SG Rarely performed Refractive index determined by refractometer Estimate of the specific gravity of the solution Light waves slow down and bend (refract) when they enter the solution Degree of refraction s proportionately to the solute concentration Urine Specific Gravity (USGref) Refractometers generally calibrated for human urine Veterinary refractometers available Canine/large animals, feline Calibration scale for dogs is slightly different from calibration scale for people Calibration scale for cats is moderately different from calibration scale for people Urine Specific Gravity (USGref) Refractive index is an estimation of USG Dependent on: Solute concentration Chemical composition of the solute Temperature Refrigerated / cold urine false increase in USG Suspended, non-dissolved particles (cells, casts, crystals etc) do NOT refract light and do NOT alter refractive index Do NOT use dipstick USG pad poor accuracy Urine Specific Gravity (USGref) The higher the number, the more concentrated the sample. The lower the number, the more dilute the sample. Maximal urine dilution ~1.001 (jargon = 10,01) Maximal urine concentration Cats: > Dogs: ~1.060 Usual USG values in health when water intake is adequate Cats: Dogs: Urine Specific Gravity (USGref) Isosthenuria Urine osmolality plasma osmolality Kidneys have not concentrated nor diluted the tubular fluid USG Hypersthenuria Urine osmolality > plasma osmolality Implies that the kidneys have concentrated the tubular fluid USG > Hyposthenuria Urine osmolality < plasma osmolality Implies that the kidneys have actively diluted the tubular fluid i.e. NOT renal failure USG Chemical Evaluation

5 Semiquantitative Dipstick evaluation Dipstick Evaluation Correct technique Urine should be at room temperature (enzymatic reactions) Prevent spill-over from one pad to the next Chemical properties may differ before and after centrifugation E.g. If RBCs are present protein and blood/haem reaction may differ Best to perform dipstick analysis before centrifuging Dipstick Evaluation Correct technique Urine should be at room temperature (enzymatic reactions) Prevent spill-over from one pad to the next Dipstick evaluation Correct technique Read at appropriate times X X Gloves should always be worn!! Dipstick Evaluation USG and Dipstick results Reliable Protein Glucose Ketones ph Bilirubin Blood/Haem Unreliable (Do not use) Urobilinogen Product of bacterial degradation of bilirubin in gut; used in people not dogs/cats Nitrite Screen for bacteriuria in people; not reliable in dogs/cats Leukocyte esterase Screen for inflammatory cells in people; not reliable in dogs/cats False negatives in dogs False positives in cats Specific gravity Protein Neg. Trace 1 30 mg/dl 100 ml 300 mg/dl USG Protein = 300 mg 300 ml 100 mg/dl USG Protein = 300 mg 200 ml water = 600 ml water = mg/dl mg/dl 300 ml 100 mg/dl USG Protein = 300 mg 900 ml 33 mg/dl USG Protein = 300 mg Same amount of protein in each container Degree of proteinuria same as

6 Dipstick Evaluation - ph Effected by Significance of a positive reaction needs to be determined in light of the reaction strength and the USG Diet (carnivores usually have acidic urine ph ) Systemic acid-base balance Delayed analysis / exposure to air Aciduria (ph <7) Metabolic / respiratory acidoses Urinary acidifying agents Protein catabolic states Paradoxical aciduria (severe vomiting) Alkalinuria (ph >7) Proteinuria Chemical Methods Reagent strip method Negatively charged proteins bind dye colour change Detects albumin better than globulins False positive in alkaline urine (ph > 7) Proteinuria 4 main causes 1) Prerenal (overflow) proteinuria Excessive filtration of small proteins exceeds tubular resorptive capacity Haemoglobinuria (haemoglobinaemia), myoglobinuria 2) Glomerular proteinuria Leakage of plasma proteins through permeable/damaged glomeruli Most severe proteinurias /- hypoproteinaemia Glomerulonephritis, amyloidosis Some bacterial infections (e.g. Staph, Proteus) Alkalosis Following a meal (alkaline tide) Exposure of sample to air for extended periods of time Proteinuria Large proteins do not pass through the glomerulus filtration barrier in health Small proteins may pass through the glomerular filtration barrier almost completed resorbed by proximal tubules Interpret in light of USG Healthy dogs may have small amount of protein (trace or 1) in concentrated urine in the absence of evidence of urinary tract disease Proteinuria in cats is always pathologic False positives in alkaline urine (ph > 7) Proteinuria 4 main causes 3) Tubular proteinuria Defective/damaged proximal tubular cells Congenital (e.g. Fanconi syndrome) Tubular injury (toxicosis and hypoxia) Small filtered proteins lost in the urine 4) Haemorrhagic or inflammatory proteinuria Confirm with urine sediment examination Voided sample consider reproductive tract sources

7 Glucosuria Glucose is filtered at the glomerulus and then reabsorbed in the proximal tubule in health Glucosuria Chemical Methods Hyperglycaemic glucosuria Filtered glucose exceeds renal threshold Dog: ~ 10 mmol/l Cat: ~ 16 mmol/l Stress (cats), diabetes mellitus, pheochromocytoma Renal glucosuria Glucosuria but normal blood glucose Proximal tubular disease/transport defect (filtered glucose not resorbed) Congenital Fanconi syndrome (Basenji) Primary renal glucosuria (Basenji, Norwegian Elkhound, Scottish Terriers) Acquired (toxic/hypoxic) Ketonuria Ketones = acetoacetate, acetone, β-hydroxybutyrate Ketones are alternative fuels for the body that are made when glucose is in short supply (i.e cells are starved of glucose for energy). They are made in the liver from the breakdown of fats. Excess ketone production ketonaemia ketonuria Causes Diabetes mellitus Starvation/prolonged fasting Carbohydrate-poor diet Persistent hypoglycaemia Glucose oxidase reaction False positives - hydrogen peroxide (H2O2) - bleach (sodium hypochlorite) False negatives in cold urine Ketonuria Chemical Methods Reagent strip method Acetoacetate and to a lesser extent acetone react with nitroprusside colour change β-hydroxybutyrate does not react Acetone is highly volatile (rapidly lost) Haem in Urine (blood) Reagent strip method: Peroxidase activity of haem enzymatic colour change Detects haemoglobin and myoglobin Intact RBCs speckling Free haem diffuse colour change Differentiate haematuria, haemoglobinuria and myoglobinuria Examine urine sediment for RBCs (haematuria) CBC/biochem for evidence of haemolytic anaemia (haemoglobinuria) Biochem (CK) for evidence of muscle damage (myoglobinuria) Bilirubinuria Physiology Haem degradation processed in the liver excreted via bile If excessive bilirubin is present in the blood bilirubinuria Dogs Low renal threshold for bilirubin Small () or moderate () reactions in concentrated canine urine (USG >1.025) in health Bilirubinuria may occur before hyperbilirubinaemia Causes of pathologic bilirubinuria Haemolytic disease Impaired excretion of bilirubin from the liver (cholestasis, bile duct obstruction etc)

8 Bilirubinuria Chemical Methods Reagent strip method Bilirubin diazonium salt colour change May be difficult to interpret colour change in highly concentrated or discoloured urine Ictotest (tablet) method Based on same principle Lower detection limit Confirmatory test Microscopic Sediment Examination Perform on fresh urine (ideally <2 hours old) Cells/casts degenerate rapidly at room temperature Crystals precipitate/dissolve Bacteria proliferate/die Refrigerate immediately if cannot be evaluated in this time Note urine collection method Ideally use standardised urine volume for sedimentation Semiquantitative, comparison of results Thoroughly mix sample if pouring off for centrifugation Standardise speed & duration of centrifugation ~450G for 5 mins (~1500 rpm) Exposure to UV light bilirubin degradation false negative Microscopic Sediment Examination Use of a sediment stain (Sedi-stain ) is optional Lower microscope condenser to improve contrast Low-power field (10x objective) Casts Crystals Sediment Exam Leukocytes (WBCs) Pyuria (WBCs in urine)= >5 WBCs per HPF Usually infectious (UTI) Can be sterile (neoplasia, necrosis, urolithiasis etc) Unstained WBCs appear as round granular cells times diameter of RBC Usually neutrophils Consider collection method when interpreting High-power field (40x objective) Erythrocytes (RBCs) Leukocytes (WBCs) Epithelial cells Bacteria Sediment Exam Erythrocytes (RBCs) Haematuria = > 5 RBCs per HPF RBC morphology depends on urine concentration and time Consider collection method Lysis of erythrocytes can occur in poorly-concentrated or alkaline urine samples. Crenated RBC Cystocentesis sample localises inflammation to urinary tract RBC WBC Sediment exam Epithelial Cells Squamous cells Contamination from distal urethra, genital tract, skin Transitional cells May slough for inflamed or hyperplastic mucosa Transitional cell carcinoma cytology best tool Renal epithelial cells (rare) RBC May be difficult to differentiate

9 Sediment Exam - Bacteria Voided/catheterised samples may have few bacteria Cystocentesis samples should have none Bacteriuria due to infection usually accompanied by pyuria (but not always!) Can confirm bacterial presence via urine cytology Centrifugation of sample does NOT concentrate bacteria Detection of bacteria in urine Rods > 10,000 / ml Cocci > 100,000 /ml Sediment Exam - Casts Cylindrical molds that form in the tubules Tamm-Horsfall mucoprotein Named and classified according to composition Cells/material that is within or adhered to the mucoprotein matrix Cellular, granular & waxy casts represent different stages of cell degeneration How a cast appears in the urine largely depends upon the length of time it was in the tubules prior to being shed Degenerate in urine with time Large numbers of casts indicate renal tubular disease Usually acute damage Sediment Exam - Casts Hyaline casts Composed of protein only Low numbers may be normal Increased numbers suggest glomerular proteinuria Epithelial casts, coarse and finely granular cellular casts, waxy casts Ischaemic or toxic renal tubular injury White cell casts Suggest pyelonephritis Red cell casts Suggest glomerular or tubular haemorrhage Sediment Exam - Crystals Precipitation depends on: ph Ion concentration Temperature Prolonged storage/refrigeration enhances formation of some crystals and dissolution of others Crystalluria is a common finding Insignificant? Pathologic state causing change in urine ph or ion concentration? Risk factor for development of urolithiasis Most animals with crystalluria do not have uroliths Some with uroliths do not have crystalluria Crystalluria Struvite Calcium oxalate (dihydrate) Crystal Significance Urine ph Ammonium biurate Amorphous urates Common in Dalmation and English Bulldogs, liver dysfunction and portosystemic shunts. ph<7 ph>7 Bilirubin Bilirubinuria Calcium oxalate dihydrate Common in healthy animals, ethylene glycol toxicity, hypercalcaemia Ammonium biurate Calcium oxalate monohydrate Cystine Magnesium ammonium phosphate (struvite) Ethylene glycol toxicity, hypercalcaemia Congenital/hereditary cystinuria, liver disease Common in healthy animals with alkalinuria. May be associated with urease-producing bacteria (UTI). Uric acid As with ammonium biurate Bilirubin Cystine Calcium oxalate (monohydrate)

10 The End!