Chemical Urinalysis

Common errors in urine dipstick testing

• sample is not mixed well enough

• the strip is submerged for too long

• reactions are read at the wrong time or in the wrong lighting

isothenuria

patient is producing urine with a specific gravity equal to plasma (1.010)

• due to chronic kidney disease or kidney damage

• are the kidneys doing anything?

hyposthenuria

patient is producing urine with a specific gravity less than plasma (<1.010)

• urine is less dense than plasma

• due to

  • increased fluid consumption

  • diuretics

  • kidney infection

  • renal disease

  • diabetes insipidus (type 1)

hypersthenuria

patient is producing urine with a specific gravity greater than plasma (>1.010)

• urine is more dense than plasma

• due to

  • dehydration

  • vomiting or diarrhea

  • diabetes mellitus (type 2)

  • nephrosis

What test method measures urine specific gravity?

pKa electrolyte dipstick test

pKa electrolyte dipstick test

• pKa polyelectrolyte on the stick combines with bromothymol blue dye in an alkaline solution to produce a pH change

• reactions range from blue (alkaline) to green-yellow (acidic)

• the higher the urine concentration, the more H+ are released, therefore lowering the pH

What are some causes of falsely elevated and decreased results in the pKa electrolyte test?

• falsely elevated results

  • urine is high in protein (protein steals H+)

• falsely decreased results

  • urine has a high pH (>6.5)

normal urine pH range

4.5 to 8.0

Determining the pH of urine can aid in ___ ___

crystal identification

What test method is used for determining urine pH?

double indicator reaction

• utilizes methyl red and bromothymol blue dyes

• methyl red changes from red to yellow in acidic conditions

• bromothymol blue changes from yellow to blue in alkaline conditions

Causes of acidic urine

• metabolic or respiratory acidosis

• UTIs with acid-producing bacteria

• chronic kidney failure

• diet, food

• medications

Causes of alkaline urine

• metabolic or respiratory alkalosis

• UTIs with urease producing bacteria

• chronic kidney failure

• diet, food

• medications

What is the first indicator of kidney disease?

proteinuria

• the kidneys are supposed to reabsorb proteins, not excrete them

Normal urine protein levels

no protein or less than 10 mg/dL

• normal urine proteins

  • albumin, Tamm-Horsfall protein (natural defense to infection), or Bence Jones protein

What test method is used for determining urine protein?

protein error of indicators reaction

Protein Error of Indication test

• dipstick indicators change color in the presence of protein

  • protein + H⁺ = color change

• albumin reacts strongly

• reported as:

  • negative, trace (<10), 1+, 2+, 3+, or 4+

Causes of falsely elevated and falsely decreased results in the protein error of indicators test

• falsely elevated results

  • urine is alkaline, has a high specific gravity, or dipstick is left in for too long

• falsely decreased results

  • proteins in the specimen are NOT albumin

What is the confirmatory test used for positive “protein error of indicator” test results?

sulfosalicylic acid (SSA) precipitation test

• SSA reacts with ALL proteins in the specimen

• proteins precipitate out of the urine and the urine is graded by turbidity

  • negative, trace, 1+ through 4+

What is pre-renal proteinuria?

• proteinuria caused by conditions affecting the plasma before reaching the kidneys

• not indicative of actual renal disease

Causes of pre-renal proteinuria

• multiple myeloma (Bence Jones proteins in urine)

• hemoglobinuria and myoglobinuria

  • excess Hgb in the blood due to increased RBC destruction

• sepsis

renal proteinuria

• proteinuria due to true renal disease

  • glomerular damage or tubular dysfunction

Causes of renal proteinuria

• glomerular or tubular disorders

• dehydration

• strenuous exercise

• hypertension

• preeclampsia

• Fanconi Syndrome

  • inherited tubular malreabsorption disorder

What is post-renal proteinuria and what causes it?

• proteins present in the urine after reaching the kidneys (excreted into the urine in the ureters, bladder, or urethra)

• causes

  • lower UTIs

  • prostatic or seminal fluid

  • blood from menstruation or injury

What is the purpose of microalbumin tests?

• to aid in diagnosing diabetic nephropathy

  • reduced glomerular filtration leads to eventual renal failure

• the presence of microalbumin in urine can predict the onset of renal complications

pre-renal glucosuria

• serum glucose levels exceed the renal threshold (160-180 mg/dL)

• seen in diabetes mellitus (type 2), pregnancy, pancreatitis, or simply eating high-sugar meals

renal glucosuria

• poor tubular reabsorption leads to glucose in urine

• seen in end-stage renal disease

What is the test method used for determining urine glucose and how does it work?

• glucose oxidation enzyme reaction

  • glucose oxidase converts glucose to gluconic acid and hydrogen peroxide, producing a color change for measurement

  • color change ranges from blue to green to brown

Causes of falsely elevated and falsely decreased glucose oxidation enzyme reaction tests

• falsely elevated results

  • contamination with bleach or hydrogen peroxide, improper dipstick storage

• falsely decreased results

  • patient takes vitamin C

What is the confirmatory test performed after a positive glucose oxidation test?

• Clinitest

• Benedict’s copper reduction reaction

  • detects glucose, fructose, pentose, lactose, and galactose (galactosuria is an inborn error of metabolism)

What are ketones?

• products of fat metabolism

• normal urine has no ketones

  • ketones are present in urine when fat is metabolized for energy instead of carbs

Why is monitoring ketones in urine valuable? What instances can produce ketonuria?

• valuable for monitoring diabetes mellitus patients

• seen in:

  • vomiting, starvation, malabsorption, and keto diets

What test method is used to determine urine ketone levels?

• sodium nitroprusside reaction

  • reaction causes a color change from tan to pink to purple

  • reported as negative, trace, small, moderate, or large

Limitations to the sodium nitroprusside test method for determining urine ketones

• falsely elevated results

  • highly pigmented urine

• falsely decreased results

  • delayed testing (ketones rapidly deteriorate)

Confirmatory test for urine ketones

• Acetest

  • utilizes the same sodium nitroprusside reaction PLUS lactose for better color differentiation

  • replaced with BHOB tests

If a patient has positive glucose AND positive ketones in their urine, what might be suspected?

• diabetic ketoacidosis

  • since type 1 diabetics have little to no insulin production, hyperglycemia occurs

    • plasma glucose reaches the renal threshold and is excreted in urine

  • ketones are produced because fat is being metabolized for energy

    • insulin helps the body use glucose, but no insulin = no glucose usage

Causes of blood in urine

• kidney stones

• glomerular disease

• pyelonephritis

• strenuous exercise

• menstruation

Causes of hemoglobinuria

• RBC lysis in the urinary tract

• intravascular hemolysis

  • hemolytic anemias, transfusion reactions, burns, infections

Causes of myoglobinuria

• rhabdomyolysis (muscle cell damage) due to:

  • trauma, crush injuries, seizures (extreme muscle tensing), extreme physical exertion

What is the test method used for determining blood in urine?

pseudoperoxidase activity of hemoglobin

How does the pseudoperoxidase activity of hemoglobin test method work?

• hemoglobin and peroxidase oxidize chromogen resulting in a color change (yellow to dark green)

  • diffuse (solid) color reaction: Hgb or myoglobin is free in the urine

  • speckled color reaction: Hgb is inside intact RBCs

• reported as negative, trace, small, moderate, or large

What are some causes of falsely decreased hematuria test reactions?

• high specific gravity

• vitamin C

• specimen is not mixed well

How is bilirubin formed? How does it become urobilinogen?

• old RBCs are broken down in the liver and spleen

• free hemoglobin is broken down into iron, protein, and protoporphyrin

• protoporphyrin is converted to UNCONJUGATED bilirubin and released into circulation where it binds to albumin and is transported to the liver

• in the liver, the unconjugated bilirubin is metabolized into CONJUGATED bilirubin

• conjugated bilirubin is transported to the intestines

• intestinal bacteria metabolize the conjugated bilirubin into urobilinogen

unconjugated bilirubin is/is not excreted by the kidneys

IS NOT

conjugated bilirubin can/cannot be excreted by the kidneys

CAN

What does bilirubinuria indicate?

• liver damage or bile duct damage

  • normal bilirubin metabolism in the GI tract is defective

  • therefore, conjugated bilirubin is excreted through the renal system

What is the test method used for determining urine bilirubin?

• Diazo reaction

  • conjugated bilirubin + diazonium salt = azo dye

  • color change ranges from tan to pink to violet

  • reported as negative, small, moderate, or large

Causes of falsely elevated and falsely decreased results of the diazo bilirubin test

• falsely elevated results

  • pigmented urine (AZO or blood)

• falsely decreased

  • light exposure, vitamin C, or increased nitrates

What is the confirmatory test for positive diazo bilirubin reactions?

Ictotest

• more sensitive, less interference with highly pigmented urine

normal urine urobilinogen levels

less than 1 mg/dL

abnormal urine contains over 1 mg/dL

Why would urobilinogen be present in urine?

• intravascular hemolysis

• liver disease

• gallbladder obstruction

• constipation

T/F: An overworked or diseased liver does not reabsorb urobilinogen efficiently, therefore excess urobilinogen is excreted through the kidneys.

true

What test method is used for determining urine urobilinogen?

Ehrlich reaction

• Ehrlich’s reagent + acid = color change (red and pink)

• reported as normal (0.1-1 mg/dL) or abnormal (2, 4, or 8 mg/dL)

Causes of falsely elevated and falsely decreased results of the Ehrlich reaction for urobilinogen

• falsely elevated results

  • pigmented urine

• falsely decreased results

  • specimen is old, elevated nitrates, presence of formalin

What clinical condition might explain a positive urine bilirubin but a negative/normal urine urobilinogen?

bile duct obstruction

• conjugated bilirubin is not reaching the intestines (duct is blocked)

• bilirubin is not converted to urobilinogen

• the liver can still conjugate the bilirubin, but it is excreted in the urine

What clinical condition might explain a positive urine bilirubin and a positive urine urobilinogen?

liver damage

• liver cells are damaged and cannot effectively conjugate bilirubin

• the liver relies on the kidneys for excretion of both conjugated bilirubin and urobilinogen

What clinical condition might explain a negative urine bilirubin but a positive urine urobilinogen?

hemolytic anemia/processes

• increased RBC destruction — increased Hgb degradation — increased unconjugated bilirubin in the blood

• the liver can still conjugate the bilirubin

• intestinal metabolism is increased and urine urobilinogen is positive

Why might urine be positive for nitrite?

bacterial infection, indicative of a UTI

What test method is used for determining urine nitrites?

Greiss reaction

• nitrite + aromatic amine = diazo salt

• diazo salt + tetrahydrobenzoquinolin = color change (white to pink)

• reported as negative, trace, or positive

Causes of falsely elevated and falsely decreased urine nitrite

• falsely elevated

  • highly pigmented urine

• falsely decreased

  • non-nitrite producing bacteria (gram pos), vitamin C, or antibiotics

Leukocyturia indicates

inflammation and infection of the upper or lower urinary tract

Where is leukocyte esterase produced?

• WBC granules (NOT lymphocytes)

  • therefore, a negative leukocyte esterase test does not always mean the absence of WBCs

What test method is used for determining urine leukocyte esterase?

leukocyte esterase and diazo reaction

• color change ranges from white to pale to dark purple