Clinical Urinalysis Review Austin Community College Medical Laboratory Technology Clinical II Spring...

Clinical Urinalysis Review

Austin Community College

Medical Laboratory Technology

Clinical II Spring 07

Urine Blood Testing

http://library.med.utah.edu/WebPath/TUTORIAL/URINE/URINE.html

Chemical Exam of Urine


Reagent strip manufactures Bayer Corporation- Diagnostics Division

(formerly Ames) produces Multistix

Boehringer-Mannheim Corporation which produces Chemstrip

Behring Diagnostics which produces Rapignost


Reagent strip precautions and source of errors

Normal dipstick procedure: Dip strip briefly, but completely into well mixed,

room temperature urine sample. Withdraw strip, blot briefly on its side Keeping the strip flat, read results at the

appropriate times by comparing the color to the appropriate color on the chart provided.


Sources of error (& preventions) Testing cold specimens

would result in a slowing down of reactions; test specimens when fresh or bring them to RT before testing

Inadequate mixing of specimen could result in false reduced or negative reactions to

blood and leukocyte tests; mix specimens well before dipping

Over-dipping of reagent strip will result in leaching of reagents out of pads; briefly,

but completely dip the reagent strip into the urine


Inadequate blotting & Failure to keep strip horizontal will result in over-run or mixing of reagents

between the different reaction pads; blot excess urine off the strip and keep strip horizontal. If dipping from the tube, can run the side of the strip along the rim to remove excess urine.

Improper timing of tests over development of reagent pad colors leading

to falsely increased results; follow manufacturer’s recommendations


Inadequate light misinterpretation of results; use good lighting

Mis-using color chart misinterpretation of results; hold strip just over

color chart and match colors as close as possible, consider use of back-up tests, if needed, especially if urine’s color masks reaction colors.


Handling and Storage Keep strips in original container, stored at RT Protect from moisture and volatile fumes Use before expiration date Do not touch reagent pad areas


Quality Control - use appropriate, commercially prepared positive and negative controls. Use commercially prepared pos and neg

controls, at least once per 24 hours, and anytime a new bottle is opened, or question of validity of results. Readings should agree with published results ± one color block.

Urine Glucose Testing

Normal : no glucose detected

Clinical significance of abnormal results (Glucosuria) Plasma glucose level exceeds renal threshold

(160-189 mg/dL) Diabetes mellitus

Renal tubular dysfunction Filtered glucose not being reabsorbed in tubules


Dipstick Testing Method Glucose initiates reaction

Coupled reaction Glucose oxidase – oxidizes glucose to gluconic acid and

concurrently reduces oxygen to hydrogen peroxide. Hydrogen peroxide in presence of the enzyme peroxidase

will oxidize an indicator, giving a colored reaction. Chromogens

Potassium iodide or Tetramethylbenzindine


Sensitivity @ 50-100 mg (compared to Clinitest’s 250) SO- Can

have a positive dipstick but a neg Clinitest

Specificity - is specific for glucose only. not affected by other sugars or reducing substances.


Interfering substances

High specific gravity and high pH may depress color. Ascorbic acid-false neg Bleach or peroxide may give false positive

Urine Bilirubin Testing

Normal : no bilirubin detected

Clinical significance of abnormal results (Bilirubinuria) Jaundice - Condition when serum bilirubin

becomes greater than the liver can handle, and there is an abnormal collection of bilirubin in the tissues giving them a yellow color


Prehepatic / Hemolytic jaundice Excessive hemolysis of RBC; beyond what the

liver can process Type of bilirubin? Is bilirubin found in the urine? YES/NO? Explain.


Prehepatic / Hemolytic jaundice Type of bilirubin? – indirect, insoluble,

unconjugated Is bilirubin found in the urine? – No, the bilirubin

is not water soluble


Hepatic jaundice Liver’s cells malfunctioning

Ie. viral hepatitis, cirrhosis etc.

Both (direct) bilirubin and urobilinogen found in urine.


Post hepatic (regurgative or obstructive) hepatitis Obstruction to outflow of bile – some type of

blockage Gall stones Tumor Edema

Conjugated bilirubin backed up into blood (Bilirubinuria) and passes into urine


Testing method Urine dipsticks for bilirubin – a diazo reaction

Impregnated with stabilized diazotized 2,4 dichloraniline

Color goes from buff to brown also shades of pink – violet

If urine is strongly colored, look for change in pad color after dipping. Use Ictotest for backup.


Interfering substances

Medication metabolites, pigments and indican may obscure readings

False negatives due to aged specimens, especially those exposed to light and oxidation.

Urine Ketone Testing

Ketone Bodies Origin - not normally present

Products of fat catabolism - breakdown of fat into CO2 and H2O

What are the 3 ketone bodies?


Acetone 2%. -Acetone is volatile, & excreted primarily through the

lungs

Diacetic Acid (Acetoacetic) the first formed, 20 % of the total the form detected by most ketone test procedures

Beta hydroxybutyric Acid majority formed, but not detected by routine tests. Only Hart’s test, an old ‘wet chemical’ test will detect this one.


Definitions Ketonuria - ketones in the urine Ketonemia - ketones in the blood Ketosis - disease state, when patient has

increased amount of ketones. Acidosis - state when blood pH is decreased,

an accumulation of acids; commonly occurs as a result of ketosis

Urine Ketone Testing Clinical significance

Health – formed in liver and completely metabolized Disease – excessive formation and accumulation

Disturbance of carbohydrate metabolism when there is a decrease of carbohydrate metabolism, then the

body stores of fat must be metabolized to supply energy. As a result of this increased fat metabolism ketones will be found

in the urine. Ex. low carbohydrate diets, diabetes

Starvation Vomiting and diarrhea in children Van Gierke's Disease – glycogen storage disease

High fat diet


Clinical effects Metabolic acidosis

Lowering of blood & urine pH Brain toxicity


Testing most use nitroprusside

detects diacetic acid and a small amount of acetone, but does not detect β-hydroxybutyric acid.

Produces purple color Can be used on urine or blood

Urine Specific Gravity Testing The specific gravity is a measure of the

weight of urine compared to an equal amount of water.

Specific gravity it proportional to urine osmolality which is a measure of concentration.

Urine Specific Gravity Testing The specific gravity will always be greater

than 1.000 and will increase as more materials are dissolved in the urine.

The value changes throughout the day depending on fluid intake.

Urine Specific Gravity Testing Specific gravity between 1.002 & 1.035 on a

random sample is normal if kidney function is normal. Specific gravity in Bowman’s capsule fluid is @

1.007 Any reading below this indicates hydration Any reading above this indicates some degree of

dehydration

Urine Specific Gravity Testing Again dilute urine will have values less than

1.010. Fixed specific gravity = 1.010; isothenuria Diabetes insipidus End-stage renal disease

And concentrated urine will have values usually over 1.020. Usually due to dehydration and can be seen in

well population as well as sick.

Urine Specific Gravity Testing Increased urine specific gravity may indicate / be seen in: * Dehydration * Diarrhea * Excessive sweating * Glucosuria * Heart failure (related to decreased blood flow to the kidneys) * Renal arterial stenosis * Syndrome of inappropriate antidiuretic hormone secretion

(SIADH) * Vomiting * Water restriction

Urine Specific Gravity Testing Decreased urine specific gravity may indicate

/ be seen in: * Excessive fluid intake * Diabetes insipidus – central or nephrogenic * Renal failure (that is, loss of ability to

reabsorb water) * Pyelonephritis

Urine Specific Gravity Testing Specific gravity > 1.035 (refractometer)

Could have very high glucose levels Could contain radiographic dye

Urine Specific Gravity Testing Testing

Polyelectrolytes , pH indicator (bromthymol blue measures the pH change), and alkaline buffer

Urine Specific Gravity Testing Interfering substances

False elevation of results may be seen in samples with increased protein concentration.

Some reports of reduced specific gravity results on alkaline specimens.

Lipids may also effect results

Urine Blood Testing

Normally not found in urine Hemoglobinuria – free hemoglobin in urine

Circulating free hemoglobin normally picked up by haptoglobin preventing loss in urine

When serum levels of hemoglobin > 100 mg/dL threshold is exceeded

Hematuria – RBCs in the urine Trauma / irritation of renal organs

Urine Blood Testing

Urine Blood Testing

Testing dipstick reaction

HGB H2O2peroxidase OxygenHGB H2O2peroxidase Oxygen

Oxygen + Gum guaiac, benzidine or orthotolidine green or blue

oxidation products

Urine Blood Testing

‘Blood’ test detects Free Hemoglobin RBCs – get lysed on the pad & their hemoglobin

reacts Myoglobin – muscle hemoglobin

Principle based on the peroxidase-like activity of the heme portion of the molecule

Urine Blood Testing

Sensitivity – can detect at levels of 5-10 cells/uL

Interfering substances Ascorbic acid Nitrates Oxidizing agents (ie bleach) Contaminate blood (menstrual)

Urine pH Testing

Normal: kidneys capable of 4.5 – 8.0 Factors effecting pH

Diet – general & specific foods Time of day Metabolic disorders Drugs / medications

Dipstick capable: 4.5 – 9.0

Urine pH Testing

Test method Dipstick indicators – methyl red and bromthymol blue Range 4.5-9.0

Caution – other chemicals on dipstick can effect pH reading

Urine Protein Testing

Normally not found in measurable amounts on dipstick (<150 mg/dL /day) Permeability of glomerulus

Damage to glom capularies Changes in glom blood flow

Albumin excretions may be increased temporarily due to exercise, uti, and acute illness with fever.

Dipstick results of >@ 1+ (30mg/dL) would equal to @ 500 mg/dL (clinical proteinuria)


Only albumin detectable by dipstick Sensitivity (@15-30 ml/dL)


New testing for microalbumin & creatinine Results:

Protein 20-200 mg/dL (30-300 mg/dL /24 hr) Creatinine 10-300 mg/dL

Albumin/creatinine ratio Normally albumin in the urine is less than 30 mg/ gram

creatinine


Principle - Protein error of indicators at fixed pH, certain indicators show one color in the

presence of protein and another in absence of protein - the “error” of the indicator.

Indicator – tetrabromphenol blue - can be hard to read at the trace end

Citrate Buffer – maintains pH 3 -quite acid


Sources of error Sensitive only to albumin Urine with strong / unusual color makes reading

difficult Highly alkaline or buffered urine will neutralize

acid buffer and lead to increased erroneous results.

Urine container contamination would interfere


Urine back up test 3% sulfosalicylic acid

Added to the supernatant to detect any kind of protein. Urine will turn cloudy if protein is present.

Urine Urobilinogen Testing

Normally found in small amounts, especially in early afternoon

Increased amounts may indicate liver disease or be seen as result of hemolytic disorders

Decreased amounts: If intestinal bacteria destroyed Liver doesn’t conjugate bilirubin Biliary obstruction – failure of bilirubin to reach

small intestine

Urine Urobilinogen Testing

Test principle based on Ehrilich’s reaction

Para-dimethylaminobenzaldehyde = Ehrlich's reagent.

Must protect specimen from light and test immediately

Urine Nitrate Testing

Nitrate Detects presence of certain types of bacteria screening for presence of UTI. Certain species of bacteria convert nitrate (normal

constituent of urine) to nitrite Escherichia - most common cause of UTI Klebsiella Proteus Pseudomonas Enterobacter Citrobacter


Aromatic amine in reagent strip reacts with nitrite; producing a diazonium salt

The diazonium salt reacts with sulfanilic acid and acetic acid to produce a pink azo dye


Limitations reported as positive or negative Not all UTI causing bacteria convert nitrate to nitrite

Haemophilus Staphylococcus Streptococcus


Fresh first morning specimen is preferred - besides being the most concentrated specimen, the urine has been in the bladder longer, allowing bacteria time and opportunity to convert the nitrates to nitrites.

Urine Leukocyte Testing

Leukocyte esterase testing is another test used as a means of screening for urinary tract infection.

Does not measure concentration of leukocytes

Will detect presence of lysed leukocytes as well as intact WBCs


test principle: Leukocyte esterase, an enzyme present in

granulocytes, hydrolyzes indoxylcarbonic acid esterase to produce indoxyl, which reacts with a diazonium salt to create a purple color usually in 2 min.


Reaction interference False positives - oxidizing detergents False negatives - greatly increased glucose, protein,

or specific gravity- increased sp gr could cause WBC to crenate preventing their releasing their esterase, So it is possible for the dipstick to be negative when there are WBCs present.

Clinical Urinalysis Review Austin Community College Medical Laboratory Technology Clinical II Spring...

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