Chemical Analysis of Urine

Disadvantage of using reagent test strips

Results are qualitative. Cannot obtain fully quantitative results

Causes of myoglobinuria

Rhabdomylosis (muscle destruction) caused by trauma, comas, convulsions, muscular diseases, alcohol and drug abuse, excessive exertion, or cholesterol medications

Confirmation test for multiple myeloma

Serum protein electrophoresis

How does hematuria appear on a blood reagent strip?

Speckled pattern

How to store reagent strips

Store with a desiccant in an opaque, tightly sealed container in a cool, dry area below 30 degrees C.

Confirmation Test for Protein

Sulfosalicylic Acid (SSA) precipitation. Must be performed on centrifuged specimens to remove any extraneous contamination.

Benign glomerular proteinuria

Temporary glomerular proteinuria caused by strenuous exercise, high fever, dehydration, or exposure to cold

What is the clinical significance of ketones in urine?

The body is unable to metabolize carbohydrates for ATP, metabolizes fat instead. By-product is ketones, which are secreted in the urine

With regard to pH, what is different about a first morning specimen versus other specimens?

First morning specimen is slightly acidic, between 5.0 and 6.0

Bilirubin

Formed as a normal product from the degradation of hemoglobin in the liver and spleen. Is water insoluble when unconjugated, water soluble when conjugated.

Hemoglobinuria

Free hemoglobin in urine (result from lysed red blood cells), causes urine to appear red and clear

Unconjugated bilirubin

Free, unbound bilirubin. Water insoluble.

What is the most frequent chemical analysis performed on urine?

Glucose

Glycosuria

Glucose in the urine

How does the nitrite test work?

Greiss reaction

What hormone do pregnancy tests detect?

HCG hormone produce by the placenta.

How is bilirubin formed?

Hemoglobin is broken down into the globin and heme, and iron is removed from heme (resulting in protoporphyrin). This protoporphyrin is further degraded into bilirubin.

What can cause false negatives in urine blood test?

High concentrations of ascorbic acid, high specific gravity, formalin, captopril, high concentrations of nitrite, unmixed specimens

What can cause false positive for SG chemical test?

High concentrations of protein

What can cause false negatives with LE test?

High concentrations of protein, glucose, oxalic acid, or ascorbic acid, crenation from high SG, inaccurate timing of reading, antibiotics

What can interfere with the protein reaction on the reagent strip?

Highly buffered alkaline urine overriding the acid buffer system, resulting in a color change unrelated to protein concentration; highly pigmented urine, high specific gravity, quaternary ammonium compounds, detergents, antiseptics, chlorohexidine

How is the HCG hormone detected for pregnancy tests?

Immunoassay

What can cause false negatives for ketone test?

Improperly preserved specimens, as bacteria metabolize ketones for energy

Hemosiderin

yellow-brown granules in sediment. It is an iron-storing molecule found in cells (as opposed to circulating blood)

What two organs regulate the blood's pH?

Lungs and kidneys

What can cause false positives in blood test for urine?

Menstrual contamination, strong oxidizing agents, and bacterial peroxidases

Clinical significance of specific gravity test?

Monitors patient hydration, detects loss of renal concentrating ability, detects diabetes insipidus, detects unsatisfactory specimens

Two major types of urinalysis reagent strips

Multistix and Chemstrip

Why does myoglobinuria result in a positive test for blood?

Myoglobinuria has a similar pseudooxidase property to hemoglobin, thus can catalyze the same reaction and produce the same results.

Glomerular proteinuria

Occurs when damage to glomerular membrane occurs, impairing selective filtration. This results in protein entering the ultrafiltrate and being secreted in urine. Also results in abnormal substances being deposited on the damaged membrane (typically in immune disorders such as lupus and glomerulonephritis)

What can cause false negatives in bilirubin test?

Old specimen (bilirubin gets oxidized into biliverdin, which does not react), ascorbic acid, nitrite (combines with diazonium salt and blocks reaction)

What can cause false positives for nitrite test?

Old specimens, pigmented urine, color interference (in automated readers)

How often should positive and negative controls of the reagent strip be run?

Once per 24 hours

What can cause false positives with LE test?

Oxidizing agents, formalin, pigmented urine

What can cause false positives in the glucose test reaction?

Peroxide or other oxidizing detergents

Myoglobinuria

Presence of myoglobin in urine, results in clear, red-brown urine

Nitrite

Produced by nitrate-reducing bacteria who reduce nitrate into nitrite.

How does the blood test on urine test strip work?

Pseudoperoxidase activity of hemoglobin catalyzing the reaction between peroxide and the chromogenic reagent to produce color change

When should additional controls be run on the reagent strips?

When a new bottle of strips is opened, when the results are questionable, or when there are concerns over the strip integrity.

What measurement of urine protein is considered proteinuria (and thus a signal of pathological condition)

When it measures at 30mg/dL or 300mg/24h

Postrenal Proteinuria

When protein enters the urine after filtration in the lower urinary and genitourinary tract. Can be caused by microbial infections, menstrual contamination, semen/prostatic fluid, vaginal secretions, or traumatic injury

Microalbuminuria

When small levels of albumin is present in the urine (between 30 and 300mg/dL). Associated with type 1 and 2 diabetes mellitus, which causes reduced glomerular filtration (resulting in albuminuria) and eventual renal failure. Also associated with increased risk of cardiovascular disease

Tubular proteinuria

When tubular damage (by acute tubular necrosis) affects the reabsorptive ability of nephron tubules. Caused by toxic substances, heavy metals, viral infections, or Fanconi syndrome, all of which cause damage to the tubules.

Myoglobin

heme-containing muscle protein

What should be done if a urine with a pH above 8.5 is encountered?

pH above 8.5 is most likely due to a specimen that is old and/or improperly preserved, so one must obtain a fresh specimen

List of tests on urinalysis reagent strip

pH, protein, glucose, ketones, blood, bilirubin, urobilinogen, nitrite, leukocyte esterase, and specific gravity

What amount of blood in urine is considered clinically significant?

Any amount of blood greater than five cells per microliter of urine

Renal threshold of glucose

160 to 180mg/dL

Normal range of urine pH

4.5 to 8.0

Normal amount of urobilinogen in urine

<1mg/dL (there is also a small amount of urobilinogen filter and excreted by the kidneys)

What factors can influence blood (and thus urine) pH?

Acid-base content of blood, patient's renal function, patient's diet, age of specimen, and the presence of a urinary tract infection

What is the primary protein we measure in urine?

Albumin

What protein is typically detected for in the reagent strip?

Albumin

How does ascorbic acid interfere with the nitrite, bilirubin, urobilinogen, and leukocyte esterase tests?

All these tests involve reactions with diazonium salt. Ascorbic acid combines with the diazonium salt and inhibits the reactions.

What must be done to refrigerated samples prior to using a reagent test strip?

Allow the urine sample to return to room temperature, because the cold will inhibit the enzymes involved in the reactions

Acetest

Another form of the sodium nitroprusside test for detecting ketones; not usually used as confirmation test in laboratories

What can cause false negatives for nitrite test?

Bacteria without reductase enzyme (can still cause infection without reducing nitrate), insufficient contact time between bacteria and nitrate, lack of urinary nitrate, bacteria further converting nitrite to nitrogen, antibiotics, ascorbic acid, high specific gravity

What should always follow a suspicious negative nitrite test?

Bacterial culture of urine

How does one collect urine to test for orthostatic proteinuria?

Before bed, empty bladder. Collect specimen immediately on rising and right after first morning specimen. Negative reading will be seen on the first morning specimen, but a positive result will be seen in the second specimen (if present)

What can cause conjugated bilirubin to appear in urine?

Bile duct obstruction, liver disease or liver damage. Bilirubin backs up into circulation (since it cannot be processed by liver in this manner) where it eventually gets excreted in urine. No urobilinogen forms when this happens.

What happens to bilirubin after formation in the liver/spleen?

Bilirubin is bound to albumin, which cannot be excreted by the kidneys under normal conditions. The conjugated bilirubin then travels to the liver to be processed for excretion.

Conjugated bilirubin

Bilirubin that is conjugated with glucuronic acid or albumin.

Nondiabetic glycosuria

Can be caused by pancreatitis, pancreatic cancer, hyperthyroidism, cerebral trauma, myocardial infarction. These conditions result in increase of hormones that convert glycogen back into glucose (working against insulin)

Renal proteinuria

Caused by glomerular or tubular damage

Orthostatic (Postural) proteinuria

Caused by increased pressure on the renal vein when in the vertical position. Usually disappears when body moves to horizontal position.

Nondiabetic ketonuria

Caused by poor carbohydrate intake in diet, vomiting, weight loss, eating disorders, frequent strenuous exercise

Bence Jones Protein (BJP)

Composed of immunoglobulin light chains, precipitates at temperatures between 40 and 60 degrees Celsius, dissolves at 100 degrees C. Found in multiple myeloma

Prerenal proteinura

Conditions in which proteinuria is caused by an increase in low-molecular-weight proteins in the blood plasma that exceeds reabsorptive capacity. Rarely seen on a reagent strip (not albumin)

Ictotest

Confirmation test for bilirubin, uses diazonium salt reagent to react with bilirubin. Blue/purple color is positive.

Function of insulin

Convert glucose into glycogen

Clinitest

Copper reduction reaction used to screen for metabolic disorders in newborns by detecting galactose or other abnormal reducing sugars in the urine. Can also be used to detect glucose, but is ineffective due to interference from other reducing sugars such as galactose, lactose, and fructose

How does the glucose reaction work in the test strip?

Enzyme glucose oxidase, which oxidizes glucose into gluconic acid and peroxide, and peroxidase, which combines the peroxide and the chromogenic reagent, producing a color change

What can cause hematuria?

Damage to renal system caused by renal calculi, glomerular disease, tumors, trauma, pyelonephritis, toxic chemicals, and anticoagulants

Leukocyte esterase test

Detects leukocytes by enzymatic activity of leukocyte esterase using diazonium salt. Can even detect lysed cells (as enzymes are still present)

Causes for ketonuria?

Diabetes mellitus, vomiting, starvation, malabsorption, certain diets

How does the urobilinogen test work in the Chemstrip test strip?

Diazodium salt reaction

How does bilirubin test work?

Diazonium salt reaction with bilirubin produces color change

What is useful about the microscopic examination of blood after the chemical detection of blood?

Differentiating between hemoglobinuria and hematuria

How does the specific gravity chemical test work?

Dissociation constant (pKa) of polyelectrolyte in alkaline medium

What is the clinical significance of >1mg/dL of urobilinogen in urine?

Early sign of liver disease, liver damage, and hemolytic disorders

What does proteinuria usually indicate?

Early stages of renal disease

How does the urobilinogen test work in the Multistix test strip?

Ehrlich's aldehyde reaction

What can cause false negatives in the glucose test reaction?

Enzymatic reaction interference, ascorbic acid and other strong reducing agents, high levels of ketones, high specific gravity, age of specimen

How do hemolytic disorders increase urobilinogen levels in urine?

Increase bilirubin production, leading to increased urobilinogen production. Excess urobilinogen reenters circulation where it eventually reaches and is filtered by glomerulus.

Diabetic ketoacidosis

Increased accumulation of ketones in the blood. Leads to ketonuria. Caused by electrolyte imbalance, dehydration, and diabetic coma (all caused by diabetes mellitus)

Causes for glomerular proteinuria

Increased pressure on the filtration mechanism caused by hypertension, strenuous exercise, dehydration, or pregnancy

Hematuria

Intact red blood cells in urine, causes urine to appear red and cloudy

What happens when unconjugated or albumin-bound bilirubin (conjugated) travels to the liver?

It becomes conjugated with glucuronic acid, and then travels to the intestines where it is excreted. Intestinal bacteria reduce the conjugated bilirubin to urobilinogen, stercobilinogen, and urobilin, and the products are excreted in feces.

What is the significance of urine pH medically?

It can detect respiratory or metabolic ketoacidosis, respiratory or metabolic alkalosis, defects in renal tubular secretion and reabsorption of acids and bases, renal calculi formation, identification of precipitates and crystals, or unsatisfactory specimens.

What is the clinical significance for urine glucose?

It is a major screening tool to detect diabetes mellitus.

How does the ketone reaction work in the reagent strip?

Ketones react with sodium nitroprusside to give a red color.

Why can age of the specimen result in false negatives for glucose?

Letting a specimen sit for too long allows any bacteria in the sample to consume the glucose for ATP

What can cause false positives to occur in ketone test?

Levodopa (in large doses), medications containing sulfhydryl groups, improperly timed readings

Why should you never use distilled water as a negative control for the reagent strips?

The reactions are specifically designed for urine ionic concentration.

What can cause hemoglobinuria?

Transfusion reactions, hemolytic anemias, severe burns, infections (such as malaria), strenuous exercise, brown recluse spider bites, lysis of red blood cells in alkaline urine

Renal glycosuria

Tubular reabsorption disorder where glucose is not reabsorbed by the tubules. Associated with end-stage renal disease, cystinosis, and Fanconi syndrome

What does a positive leukocyte esterase test indicate?

Urinary tract infection

What does a positive nitrite test indicate?

Urinary tract infection

What can cause false positives in bilirubin test?

Urine pigments, pyridium, drugs, and dyes such as indican or iodine

What gives feces its brown color?

Urobilin (oxidized from urobilinogen) and stercobilin (oxidized from stercobilinogen)