Urinalysis

Common urine specimen types for urinalysis

Random, midstream, catheter collection

What is the recommended specimen for urinalysis?

First morning - momst concentrated urine

What is clean catch urine?

MSU urine but genitals are cleaned to reduce bacterial contaminaiton

How fast should urines be processed for urinalysis

Ideally, 2 hours

Why is quick testing of urinalysis beneficial?

biluribin, urobilinogen and pH of urine can be instable.

Bacteria can multiply, cells can degrade

Pros and cons of refrigerating urine for urinalysis

Pros - prevent bacterial growth

Cons - promote crystal formation

How old does a urinalysis specimen have to be to be rejected

>24 hours

The 3 components of routine urinalysis

Physical examination

Chemical examination

Microscopic examination

What is judged during physical examination of urine

Clarity and color

What can cause cloudiness in urine

Amorphous urates in acidic urine

Amorphous phosphates in alkaline urine

Significant WBC or RBCs

What affects urine color

Urine concentration, presence of excreted metabolites, medications, other chemicals

Normal urine color

Pale yellow to dark amber

How is chemical examination performed for urine

Urine reagent test strips composed of multiple pads with enzymes/chemicals to detect analytes

How is urine reagent test strips red?

Detected manually by color change or reflectance spectrophotometry

What chemicals are tested in urinalysis

Leukocyte esterase

Nitrite

Urobilinogen

Protein

pH

Blood

Specific gravity

Ketones

Bilirubin

Glucose

Clinical significance of glucose in urine

Not normally found in urine - seen when blood glucose levels exceed renal threshold (11 mmol/L)

Seen in uncontrolled diabetes mellitus, advanced renal disease, pregancy

False positives of glucose in urine

Oxidizing agents, peroxide contamination

False negatives of glucose in urine

- Sensitivity decreased by high urine SG and low temp

- High ketone levels

- High concentrations of ascorbic acid

- Bacterial glycolysis

Clinical significance of bilirubin in urine

Not a normal constituent of urine

- Early indicator of liver disease: hepatitis, cirrhosis

- Bile duct obstruction: gallstones, tumors

what form of bilirubin is found in urine

Conjugated (water-soluble) bilirubin

False positive of bilirubin in urine

Colored urines

False negative of bilirubin in urine

- Photo-labile and temp sensitive

- High concentrations of ascorbic acid

Urobilinogen and Bilirubin urine results in hemolytic disroders

Urobilinogen: high

Bilirubin: negative

Urobilinogen and Bilirubin urine results in liver disease

Urobilinogen: high, but not as high as hemolytic disorders

Bilirubin: positive/negative

Urobilinogen and Bilirubin urine results in hepatobiliary obstruction

Urobilinogen: normal

Bilirubin: high

Clinical significance of ketones in the body

Indicates metabolism of fatty acids, which comes if no carbs available:

- Indicates uncontrolled diabetes mellitus

- Starvation, fasting

- pregnancy

- Strenuous exercise

- Vomiting, dehydration

False positive of ketones in urine

Colored urines

Large amounts of compounds containing sulfhydryl groups (MESNA, Captopril)

False negative of ketones in urine

Improper storage conditions, acetoacetic acid can be broken down by bacteria

What ketone body is detected by urine reagent strip

Acetoacetic acid

What is specific gravity

Density of solution compared to the density of an equal volume of deionized water

What effects specific gravity

Solute mass, solute number

Normal range of urine SG

1.005, 1.030

Clinical significance of urine specific gravity

Can monitor patient hydration and kidney ability to concentrate urine

- SG of 1.00 - specimen adulteration

- Increased SG correlates with high ketones

Does glucose and urea cause increased SG for dipstick method?

No - they are non-ionic (test only reads ions)

How can specific gravity be tested for urine?

Dipstick, or refractometry

How does a refractometer work

Measures specific gravity indirectly by comparing the refractive index of light in the air (1.000) to urine

How to calibrate refractometer

Using water and NaCl solutions

Clinical significance of hematuria (blood cells in urine)

Renal calculi (kidney stones)

Glomerulonephritis

Pyelonephritis

Transient hematuria from strenuous exercise

Clinical significance of hemoglobinuria

- Occurs when free Hgb exceeds binding capacity of haptoglobin

- Seen in intravascular hemolysis, transfusion reactions, severe burns, infection

Clinical significance myoglobinuria

- Seen in rhabdomyolysis, trauma and crush injuries

- Myoglobin is toxic to nephron tubules and may cause acute renal failure

False positives of blood in urine

- Menstrual contamination

- Sensitive to myoglobin and hemoglobin

False negatives of blood in urine

- Increased SG

- Erythrocytes settled in tube (improper mixing)

Normal pH of urine

4.5-8.0

Clinical significance of urine pH

If kidney function is normal:

- Urine is acidic in resp and metabolic acidosis

- Urine is alkaline in res and matabolic alkalosis

False alkalinity of urine pH

Urine is not fresh or refrigerated if delayed testing

- pH > 8.5 = improper storage

False acidity of urine pH

Excess urine on the reagent strip can was protein reagent pad buffer on the pad

Is protein seen in urine?

Yes in very small amounts (<0.15g) - reagent strips do not detect this

Clinical significance of protein in urine

associated with renal diseases such as glomerulonephritis and nephrotic syndrome

False positives of protein in urine

- Highly buffered alkaline urines

- High SG

- Prolonged dipping of reagent strip (removes biffer)

- colored urines

False negatives of protei in urine

Pad is not as sensitive to globulin proteins, and mucoproteins. Neg result may not have albumin, but has these

Cause of pre-renal proteinuria

Low MW plasma proteins excreted in urea - possibly acute phase reactants, hemoglobin, myoglobin, or monoclonal free light chains

Selective glomerular leakage

Slits of glomerular podocytes larger than normal, allowing albumin to pass through. Still size selectivity

Non-selective glomerular leakage

proteins of any size can pass through damaged glomerulus

Tubular proteinuria

Glomeruli heavy, renal tubules cannot reabsorb low MW proteins such as B2-microglobulin and immunoglobulin.

What can cause tubular proteinuria

Heavy metal poisoning and nephrotoxic drugs (cadmium, gentamicin)

Post renal proteinuria

Proteins found in urinary tract due to inflammation, malignancy, or injury

Can also be due to uromodulin

Clinical significance of urobilinogen in urine

Can be normally found in urine (<16 umol/L) as a water soluble bilirubin degradation product

Increased amounts seen in hepatic and hemolytic disorders

False positives of urobilinogen in urine

Colored urine, porphobilinogen

False negatives of urobilinogen in urine

Degradation by acidic urine, light, and RT storage

What four tests require reflex to microscopy if positive on the urine test strip?

Protein, Blood, Nitrites, Leukocytes

Clinical significance of nitrites in urine

Indicates presence of bacteria in urine

False positives of nitrites in urine

Colored urines, improper storage can cause bacterial growth

False negatives of nitrites in urine

- Urine not held in bladder for min. 4 hours

- Bacteria not producing nitrate reductase

- No dietary nitrates

- High ascorbic acid

- High SG

Clinical significance of leukocytes in urine

Seen in infections and inflammatory diseases such as UTIs and Pyelonephritis

False pos of leukocytes in urine

Colored urines

False negatives of leukocytes in urine

- Protein >5 g/L

- Glucose >30 g/L

- Ascorbic acid

- High specific gravity

- Antibiotics: cephalexin, cephalothin, gentamicin, tetracycline

- Lymphocytes do not produce leukocyte esterase and are not detected

- Leukocytes will settle to the bottom of a sample, proper mixing before testing is essential

How is pH determined on dipstick

Based on double indicator system: pad has bromothymol blue and methyl red

- Protons in urine react with anionic indicator dye to reduce the dye and cause color change

How is leukocytes determined on dipstick

Leukocyte esterase activity: esterase reacts with ester on reagent pad to produce aromatic compound

- at acidic pH, aromatic compound reacts with diazonium salt to produce azo dye

How is Nitrite determined on dipstick

At acidic pH, nitrites in urine react with aromatic amine to produce diazonium salt

- Diazonium salt reacts with aromatic compound to produce azo dye

How is proteins determined on dipstick

At acidic pH, proteins in urine are anionic. Indicator dyes on the reagent pad release protons in response to proteins

- Indicator changes color due to proton loss

How is glucose determined on dipstick

Glucose in the presence of oxygen reacts with glucose oxidase on reagent pad to produce gluconic acid and H2O2

- H2O2 + reagent peroxidase react with chromagen to produce oxidized colored chromagen

How are ketones determined on dipstick

At alkaline pH, acetoacetate reacts with sodium nitroprusside to produce color

- In the presence of glycine, acetone can also react with sodium nitroprusside

How is blood in urine determined on dipstick

Blood has pseudoperoxidase that reacts with H2O2 in strip to oxidize a chromogen on reagent pad to colored form

How is urobilinogen determined on dipstick

At acidic pH, urobilinogen reacts with Erhlich's reagent (p-dimethylaminobenzaldehyde) to produce red color

At what time of day does urobilinogen peak in urine

Afternoon

How is bilirubin determined on dipstick

In acidic medium, aromatic conjugated bilirubin reacts with diazonium salt to form colored azobilirubin

isothenuria

urine with a SG of 1.010

Hypothenuria

Urine with SG<1.010

Hypersthenuria

Urine with SG >1.010

How is specific gravity determined on dipstick

Dipstick measures ionic SG

- at alkaline pH, ions in urine react with polyelectrolyte and pH indicator. Pads ionize, and protons are released, lowering pH of pad.

urinalysis biochemical results of UTIs

Leukocyte - pos

Nitrite - pos

Urobilinogen - normal

Protein - neg

pH - alkaline

Blood - pos

SG - normal

Ketones - neg

Bilirubin - neg

Glucose neg

Urinalysis biochemical results of pyelonephritis, glomerulonephritis

Leukocytes - neg

Nitrites - neg

Urobilinogen - normal

Protein - pos

pH - normal

Blood - pos

SG - normal to low and fixed

Ketones - neg

Bilirubin - neg

Glucose - neg

Microscopic results of UTI

Increased WBCs

Increased Bacteria

increased RBC

Increased transitional epithelial cells

Microsopci results of Pyelonephritis/Glomerulonephritis

Increased WBCs

Increased RBC

Increased renal tubular epithelial cells

Increased RBCs casts

Occasional WBCs, renal cell casts

Urinalysis biochemical results of Nephrotic syndrome

Leukocytes - neg

Nitrite - neg

Urobilinogen - normal

Protein - POS (severe)

pH - normal

Blood - pos (small)

SG - normal

Ketones - neg

Bilirubin - neg

Glucose - neg

Microscopic results of nephrotic syndrome

Lipiduria, oval fat bodies

Increased RBC

increased Casts (esp fatty)

Increased renal epithelial cells

What stains can be used to help visualize fatty casts or oval fat bodies

Oil Red O, Sudan III

How is urinalysis microscopic examination standardized

Using constant specimen, centrifugal force, and sediment volume. A standardized system (Urisystem kit) is used as well

What is urine sediment stained with

Supravital stain (crystal violet and safranin O)

Usual procedure of urine microscopci analysis

Observe 10 fields at 10X and 10 fields at 40X for cells, crystals, casts, microorganisms

Use of polarizing light microscopy for urinalysis

Detect birefringent urine elements such as uric acid and choleserol

Theory of polarizing light microscopy

microscope light passes through a polarizing filter which restricts the light to a single plane; the polarized light passes the urine sample to another polarizing filter (analyzer) which is positioned at 90° to the first polarizing filter.

How does birefringence work for polarizing light microscopy

polarized source light is refracted and passes through the second polarizing filter.

How are casts formed

Uromodulin (renal protein) congeals in distal tubules and collecting ducts of kidney, typically during renal stasis