Clinical Biochemistry 3

Clinical Biochemistry 3

1. Protein

   • Dipsticks may give false positives in alkaline urine or in the presence of detergents – this may occur if the dipstick is placed on the work surface in a lab
   • Dipstick and sulphosalicylic acid techniques predominantly measure albumin
   • Protein in urine will indicate kidney failure
   • Protein : creatinine ratio
     • Ratio removes the influence of concentration
   • Influence of cells on protein levels
     • If there is enough blood to make the urine go pink, it will probably be affecting the urine
     • If it’s pink, don’t trust the UPr:Cr – get more measures
       • UPr:Cr will not exceed 0.4 at the point where the sample is visibly pink

2. Dipstick

   • Manual reading or automated systems to read the sticks
   • Pipette urine on to dipsticks so as not to contaminate the urine sample
   • Leucocyte esterase panel
     • Sensitivity of 77% and a specificity of 34% (POOR)
   • Nitrites
     • Generally more specific than sensitive – many non- nitrite producing urinary tract infections exist
     • Show that things have been broken down
   • pH
     • Rapidly affected by storage of the sample
     • Poor accuracy with dipstick tests
   • Dipsticks should be checked between batches or in doubt
   • No ability to distinguish intact red cells, haemoglobin and myoglobin: microscopy required to see if intact red cells, other clinical finding for myoglobinuria

3. Specific gravity

   • Dipsticks for specific gravity are not much use – much better to use a refractometer
     • Measure by refractometer – most accurate way of testing
   • Quality control: measure specific gravity of distilled water (should be 0.0), 5% NaCl should be 1.022)
   • Should be measured at room temperature
   • Measure solutes, glucose if diabetic
   • Is animal capable of concentrating its urine? Is kidney function normal?
   • Isothenuric – sp. gravity 1.008 – 1.013 (filtered, same as plasma)
   • Hyposthenuric – sp. gravity < 1.008 – urine has been diluted by kidneys (you have renal function)
   • The range of values for SG can vary according to water intake and hydration status
   • Urine specific gravity can be used to assess if an azotaemia is pre-renal or or renal in origin
     • In order to do this the urine has to be collected prior to starting fluid therapy
   • In a dehydrated/azotaemic animal an USG > 1.035 (cat); >1.030/1.025 (dog, also same threshold for horses) suggests good renal function
     • Concentrating urine in response to dehydration
   • USG 1.007-1.025 in an azotaemic animal suggest primary intrinsic renal failure (acute or chronic)

4. Samples

   • Collection methods
     • Results vary enormously, particularly for sediment and culture
     • Off floor
     • Clean container (plastic dustbin)
     • Free catch
     • Bladder squeeze
     • Catheterisation
     • Cystocentesis
       • Direct bladder puncture – fine needle aspirate through body wall
     • Need to balance invasiveness to the animal vs. the usefulness of the result. Point of consideration, especially with respect to catheterisation/cystocentesis
     • Only cystocentesis or sterile catheterised samples appropriate for culture
       • Point needle caudally when collecting sample
       • Even in these may get gel or glove powder artefact
         • Starch from glove powder can be in sample
   • Storage
     • Analyse within 1 hour of collection, as composition will change with time
     • Refrigeration preserves physical and chemical properties of urine, slows bacterial overgrowth and maintains cellular elements, (preserves costs)
       • But refrigeration increases the numbers of crystals present (significantly for CaOx)
         • These are the crystals most commonly looked for to indicate disease
   • Sterile universal vs boric acid
     • Sterile universal needed for sediment examination
       • Big tube, white top
     • Boric acid can be used to ‘suspend’ the number of bacteria in free catch samples, but not recommended for cystocentesis samples
       • Small tube, red top
       • Stop bacteria multiplying or dying off
       • Can miss low numbers of bacteria
     • Makes test less sensitive
     • Must fill to the line
       • Nasty environment for bacteria if undefiled
   • Gross examination
     • Record appearance including turbidity (how cloudy), colour and any odour (not necessary)

5. Sample examination

   • Crystals
     • Magnesium ammonium phosphate
       • Struvite
       • May also form fern –frond structures
       • May be seen in normal animals, those with uroliths (any type), or other urinary tract disease including UTI
       • Dissolve if acidified
       • Mixed with mucous = blockage
       • Can be pathological, but can have them with no clinical signs
     • Calcium phosphate
       • Many forms: amorphous; clear prisms, clear elongated type may form rosettes
       • Normal dogs, those with alkalinised urine, with or without uroliths
       • Not seen as frequently
     • Calcium oxalate dihydrate
       • Squares with intersecting lines from corner
       • May occur singly or as conglomerate structures
       • With or without calcium oxalate uroliths
       • In any pH urine
     • Calcium oxalate monohydrate
       • In hypercalcaemia and excessive oxaluria
       • Ethylene glycol toxicity – antifreeze
       • Causes renal failure, acid/base disturbances, neurological signs quite early on
       • Rhubarb leaf toxicity
       • Seen alone or with calcium oxalate dihydrate
       • Polarising light helps to identify – present also in renal histological slides
     • Ammonium biurate
       • ‘Thorn apple’ morphology
       • With liver function compromise, particularly portosystemic shunts
       • Increased incidence in dalmations
     • Amorphous urates
       • Similar circumstances to ammonium biurate
       • Other amorphous crystals include amorphous phosphates – differentiate with alkali: phosphates dissolve, urates remain
     • Uric acid
       • May occur singly or in sheaves/rosettes
       • Diamond or rhomboid
       • Significance as for ammonium biurate
       • Can be grown from amorphous urates by adding acetic acid – helps confirm identity
     • Bilirubin
       • May be normal in concentrated urine in dogs – renal bilirubin conjugation
       • Abnormal in cats
       • May see increased amounts in pre-hepatic, hepatic or post hepatic origin icterus
     • Cystine
       • Clear hexagons, often stacked
       • Cystinuria (defect in renal transport of cystine in the tubule)
       • Many breeds, reported autosomal recessive in some
       • Newfoundlands
       • Can lead to uroliths
       • In acidic urine
     • Leucine
       • Brownish spherical with ‘target’ lamination
       • Possibly associated with aminoaciduria or severe liver disease
   • Cells
     • Erythrocytes
       • Usually < 5 per 400x field
       • Haemolyse in low s.g. urine (<1.008), but also affected by other factors (time, temperature)
       • Biconcave discs, may be crenated or swollen
       • If ruptured, membrane only noted (ghost cell)
       • Few expected in cysto sample
       • Have a ‘wrinkled’ edge after they have been in urine for a while
     • Leucocytes
       • Usually <5 per 400x field
       • 1.5 – 2 x size of erythrocytes
       • Granular cytoplasm, lobed or segmented nucleii
       • Difficult to distinguish from epithelial cells – consider air drying a sample and Giemsa stain
       • Denote inflammation but may be difficult to tell visually if septic or sterile (e.g. in response to uroliths)
       • Look grainy
     • Transitional epithelial cells
       • Large cells (up to 40um) Leucocytes 12 - 14
       • Smallest basal layer more columnar than round
       • Transitional cell carcinoma difficult to diagnose without nuclear staining – to differentiate neoplasia from dysplasia need high cell yield and air dried cytological staining (e.g. Giemsa, rapid stains)
       • Changes in morphology may also be caused by exposure to urine
       • Pink and grainy, often form sheets (characteristic)
     • Renal epithelial cells
       • Proximal tubular cells rectangular, some may show microvillous border
       • Vary widely in size
       • Difficult to differentiate from other epithelial cells or leucocytes
       • Normal turnover releases some, higher numbers expected in disease
     • Squamous epithelial cells
       • Originate externally or the distal urethra
       • Large, flattened cell, usually anucleated
       • May be folded or rolled to imitate casts
       • If external in origin, may have attached bacteria
       • Flat looking cells
       • Contact with outside of animal – bear in mind for culture results – sample not sterile
   • Casts
     • Urinary casts
       • Tamm – Horsfall mucoprotein secreted from loop of Henle, distal tubules, collecting duct: this alone may become a hyaline cast
       • Cells may become snared in the protein: possibly degenerate as continue down tubule/duct
       • Low numbers unremarkable, high numbers consistent with renal insult: hypoxia, ischaemia although absence is not a rule out
     • Leucocyte cast
       • May need to confirm identity of cells with air dried
       • Must have renal involvement to mould the cells into cylinders – tubular moulding
       • May have concurrent lower UTI
     • Erythrocyte cast
       • Denotes renal bleeding
       • Individual erythrocytes visible within the hyaline material
       • Idiopathic renal haematuria, trauma
   • Lipid
     • Normal in cats
     • Lipid droplets float on a higher plane than much of sediment
     • Result of normal tubular degeneration
     • No direct relationship between lipidaemia/lipiduria
   • Yeast/fungi
     • May be pathological especially in immunocompromised animals on chronic antibiotics
     • Candida or systemic fungal (e.g. Cryptococcus)
     • If no inflammation, suspect contamination and overgrowth
   • Parasites
     • Capillaria plica or Capillaria felis cati – bipolar plugs and lemon shape
     • Differentiate from faecal contamination with Trichuris vulpis
     • Also Dioctophyme renale and Dirofiliria microfilaria
   • Contaminants
   • Examine under reduced light : drop the condenser or reduce iris size
     • Want to see edges
   • 100 x scanning overview and to allow initial quantification of elements
     • 400 x to allow identification of morphology and bacteria
   • Numerical scores either per high power field (40 x objective) or subjective low, medium, high for bacteria, crystals, sperm, parasites

6. Sediment examination

   • Mix sample
   • Transfer standard volume (5ml) - to allow sample to sample comparison - to a conical tipped capped centrifuge tube
     • Centrifuge at 1500 rpm for 5 minutes
   • Decant supernatant with a pipette leaving a small amount (0.5ml) for resuspension
   • A lot of subjectivity in terms of interpretation
   • Add stain if desired, usually at equal volume with sediment – remember will alter quantification. Consider doing both stained and unstained.
     • Resuspend by flicking the pot until well mixed
       • The sample can then be examined in either a commercial graticule or a single drop under a standard square coverslip to allow quantification