clinical chem test 1

routine void

• no patient prep needed

• MOST CONTAMINATED

midstream “clean catch” (CC)

• used for culture

• procedure: clean area w antiseptic, discard initial portion of urinary stream, and collect midstream portion into a clean container

• avoids vaginal discharges/contamination

catheterized collection technique

• usually used for pts having difficulty voiding

• insert sterile catheter thru urethra into bladder, urine flows into bag

• collect by aspiration or directly from port NEVER ACCEPT URINE FROM BAG

  • things accumulate

  • urine in bag can be old - only good for 2 hrs

suprapubic aspiration collection

• use of syringe and needle inserted thru the abdominal wall and directly into bladder

• not usual, LEAST contaminated

collection containers

• use clean, dry container w lid

• sterile container for culture

• pedi-bags for infants

• LABELED PROPERLY on container, not just lid

necessary for proper labeling of urine specimen

• NAME

• DATE

• PT ID NUMBER

• TIME OF COLLECTION

random collection

• collected at any time

• adv: convenient for patiend

• disadv: may be diluted; false picture of health

1st morning collection

• patient collects urine as soon as they wake up

• adv: most concentrated; PERFERRED METHOD

• disadv: none

post-prandial collection

• collected 2-3 hrs after eating

• adv: can be used for detection of carbohydrate metabolism disorders

• disadv: patient responsible for collecting on time

24-hour collection

• 1st void is discarded; collect all urine for 24 hrs

• adv: allows us to quantitate substances (protein, glucose, hormones)

• disadv: pt responsible for collecting on time, may forget

timed collection

• collected at a particular time of day

  • ex: urine collection between 2-4 PM

• adv: allows us to test substances w/ diurnal variation (different concentrations at different times of day, like cortisol being higher in the morning)

• disadv: pt responsible for collecting on time

handling of urine specimen

• delivered to laboratory immediately (ASAP)

• a delay of MORE THAN 2 HRS requires a PRESERVATIVE

• if no preservative and OVER 2 hrs - DISCARD AND RECOLLECT

changes in unpreserved urine

• chemical

• physical

• microscopic

#1 way to preserve urine

• refrigeration

  • gives up to 8 HRS to work w/ urine (follow lab policy)

  • prevents bacterial proliferation

  • slows/inhibits enzymatic processes that break down cell components

what changes occur in urine stored at room temp?

• rapid bacteria multiplication

  • urea splitting bacteria INCREASE PH IN URINE (ammonia)

  • increase in urine pH → decomposition of casts

• glucose is used up by bacteria (false lower/negative reading)

• blood cells and casts deteriorate over time

preservatives - boric acid

• most commonly used

• keeps bacteria stable

• preserves formed elements, INTERFERES W PH READING

• used to preserve urine for culture and sensitivity testing

preservatives - chloroform

• inhibits bacterial growth

• changes CELLULAR SEDIMENT

• may be used for aldosterone testing

preservatives - chlorohexidine

• inhibits bacterial growth

• useful glucose preservative

• affects BILIRUBIN and UROBILINOGEN testing

preservatives - formalin

• preserves urinary sediment

• may PRECIPITATE PROTEIN and give FALSE POSITIVE result for REDUCING SUBSTANCES

preservatives - preservative tablets

• release formaldehyde (not used anymore - toxic to humans)

• interferes w REDUCING SUBSTANCES at high concentrations

• INCREASES S.G.

preservatives - toluene

• preserves ketones, proteins, reducing substances

• not effective against BACTERIA PRESENT

• floats on surface of urine and is flammable

preservatives - thymol

• rarely used

• interferes w ACID PRECEPITATION test for protein, but NOT STRIP TEST

TESTS THAT REQUIRE SPECIMEN TO BE UNPRESERVED

• pregnancy test

• molecular methods

  • GCE and chlamydia tests

  • PCR- related tests

physical changes in unpreserved urine

• color: changes due to oxidation or reduction of substances (darker)

• clarity: falsely decreased due to bacterial growth + solute precipitation (cloudier)

• odor: increased due to bacterial proliferation + decomp. of urea to ammonia (stinkier)

unpreserved urine - glucose

• DECREASES

• it is metabolized by bacteria or yeast

unpreserved urine - ketones

• DECREASES

• ketones volatize

unpreserved urine - bilirubin

• DECREASES

• photo-oxidation and hydrolysis

unpreserved urine - urobilinogen

• DECREASES

• oxidation of urobilinogen

unpreserved urine - pH

• INCREASES

• bacterial decomposition and conversion of urea to NH3

unpreserved urine - nitrites

• INCREASES

• bacteria proliferation (up to a certain point)

unpreserved urine - RBCs, WBCs, casts

• DECREASES

• degeneration by enzymes

urinalysis components

• physical characteristics - quickest/easiest part

• chemical characteristics - dipstick

• microscopic examination - centrifuge → supernatant/sediment → microscope

  • usually omitted if physical and chemical exam meet normal findings

physical characteristics noted

• color

• clarity

• odor

• concentration

• volume received

color

• urochrome and UROBILIN (lipid soluble pigments)

  • formed in large intestines

  • gives urine its characteristic color

  • other possible pigments

• influence of hydration

  • pale to dark yellow

  • intensity correlates w/ concentration

    • darker = more concentrated

  • oxidation can cause color change

colorless - pale yellow urine

• diluted urine

• normal

pale/straw to dark yellow urine

• normal

dark amber urine

• bilirubin

  • also causes yellow foam

orange urine

• bilirubin

• medication

light red/pink urine

• blood, hemoglobin, myoglobin

• menstruation

• beets, rhubarb, blueberries artificial dyes

red-brown uria

• hematuria

• menstruation

• rhabdomyolysis (myoglobin)

• prophyrinuria

• meds

black/brown/tea urine

• homogentisic acid

• melanin

• antibiotics

• senna laxatives

green/blue urine

• biliverdin

• pseudomonas bacterial infection

• amitriptyline (antidepressant); cimetidine (ulcer/acid reflux med.)

• food dyes

clarity

• overall visual appearance

  • degree of transparency

• normal urine = clear

  • indicates hydration and healthy UT

• cloudiness/turbidity

  • due to contaminants

  • pathogenic and nonpathogenic sources

  • clarity and color usually go hand in hand

levels of clarity

• clear - no visible particulate matter

• slightly cloudy - some visible particles, background not obscured

• cloudy - visible particles, background slightly blurred

• turbid - cannot view background through urine

substances causing turbidity - PATHOLOGIC

• RBCs

• WBCs

• bacteria

• yeast

• trichomonas

• renal epithelial cells (should stay in kidneys)

• abnormal crystals

• pus

substances causing turbidity - NON PATHOGENIC

• mucus

• squamous epithelial cells

• normal crystals

• radiographic media

• sperm

• fecal contamination

• powder

• lotion

foam caused by SHAKEN urine

• normal urine foam will dissipate

• stable foam = ABNORMAL

  • stable WHITE = moderate/large amount of albumin (protein)

  • stable YELLOW = bilirubin is present

• note: foam usually not reported, used as support presence/absence of indicated substances

differentiation of acidic urine

• amorphous urates

• radiographic contrast media

differentiation of alkaline urine

• amorphous phosphates

• carbonates

odor

• not reported

• can give clues on abnormal substances

• normal = faintly aromatic

• ammonia (pungent) = bacteria = old urine or uti

• sweet/fruity = ketones

• maple syrup = congenital metabolic disorder

• mousy/musty (in infants) = phenylketonuria

concentration and specific gravity

• amount of solutes present in sample

  • SG of urine consists primarily of urea and electrolytes (Na, K)

  • varies w diet, health, hydration status

  • useful for assessing renal function - kidney’s ability to conserve water as needed

• color is crude indicator

low specific gravity

• hyposthenuria

• range: less than 1.010

• excess fluid status - can indicate renal failure

• possible causes:

  • diabetes insipidus

  • kidney failure

  • pyelonephritis

  • malignant hypertension

  • excess glucose and protein

high specific gravity

• greater than 1.025

• suggests dehydration

• possible causes:

  • volume loss

  • diabetes mellitus

  • diarrhea

  • CHF

  • shock

  • syndrome of inappropriate ADH

  • trauma, stress, drugs

• note: IV radiography dyes/drinks give false elevaiton

specific gravity : diabetes mellitus vs insipidus

• BOTH: HIGH URINARY VOLUME

• Mellitus

  • deficiency of insulin (excess glucose excreted in urine)

  • INCREASED specific gravity (glucose = dense molecule)

• Insipidus

  • deficiency ADH

  • DECREASED specific gravity (kidneys not concentrating urine; more water

osmolality

• another measure of concentrating/diluting ability of kidneys

• better than specific gravity; unaffected by high molecular weight solutes

• changes = DIRECTLY PROPORTIONAL TO AMOUNT OF SOLUTE

• normal reference range = 500-850 mOsm/kg water

direct methods for specific gravity measurements

• urinometer

• falling drop method

• NOT DONE ANYMORE

indirect methods for specific gravity measurements

• reagent strip method

• refractometer

urinometer

• replaced by more accurate methods

• direct method - measures SG at room temp

• no longer recommended

• requires CORRECTION for temp, glucose, protein

• a min. of 15 mL of urine needed

• weighed glass float w long, narrow calibrated stem

falling drop method

• time rate of fall of drop of urine through temperature controlled column of silicone based oil

• rate proportional to mass of solutes present

• more precise and accurate than refractometer and urinometer

• ALREADY TEMP CORRECTED

• CORRECT ONLY FOR PROTEIN AND GLUCOSE

refractometry

• measure refractive index of a solution

  • refractive index = ratio of velocity of light in air to velocity of light in sltn

• based on refractive index of light

• quality control

  • check with water daily - 1.000

  • 5% NaCl - 1.022

reagent strips

• varioud configurations

  • blood

  • pH

  • ketones

  • glucose

  • protein

  • specific gravity

  • bilirubin

  • urobilinogen

  • nitrite

  • leukocyte esterase

pH principle

• normal urine pH: 4.5 - 8.0

• double indicator system

  • enables pad to produce readings over natural range of urine pH - SENSITIVE TO H+ IONS

  • methyl red = acid indicator

  • bromthymol blue = alkaline indicator

    • we read a combined color change

run over effect

• due to excess urine after dipping - “leaching”

• can falsely get an acidic reading

• to fix: dab dipstick on paper towel

why do we avoid testing old urine?

• causes false pH increase do to ammonia formation by bacteria

acidic urine can be caused by

• diet (high protein, cranberry ingestion)

• sleep

• acidifying drugs

• diarrhea (water loss = bicarb loss)

• metabolic acidosis

• respiratory acidosis

• UTI w acid producing bacteria (E.coli)

alkaline urine can be caused by

• diet (vegetarian; low carb)

• alkaline drugs

• acute and chronic renal failure

• metabolic alkalosis

• respiratory alkalosis

• UTI w alkaline producing organism - pseudomonas

protein in urine

• normally, only small amnt of low weight protein is filtered in glomerulus

  • ALBUMIN and HMWP should NOT be in urine

• Tamm-Horsfall protein = normal

  • excreted by tubules, not contained in plasma

  • forms matrix of urinary casts

• SSA test is used for confirmation - detects albumin, globulins, and bence-jones proteins

protein - reagent strip

• detects ALBUMIN only

• pad principle:

  • based on “protein error of indicators”

  • point of color change of some pH indicators is different in presence of protein from that observed in absence of protein

• TETRABROMPHENOL BLUE - most common indicator

  • no protein = buffered at pH 3 = yellow

  • protein = H+ acceptor

  • presence of protein = error occurs in behavior of indicator = color changes to blue/green

protein - false positive

• highly buffered alkaline urine

• prolonged exposure of pad to urine = “leaching”

• container cleaning compounds (bleach contamination)

• urine w blood

protein - false negative

• dilute urines

• proteins other than albumin present (dont get picked up by dipstick)

protein in urine - PATHOLOGIC CAUSES

• glomerular nephritis (inflammation of glomeruli - decreased filtration)

• malignant hypertension (kidney damage)

• kidney disease

• pyelonephritis (UTI) - bacterial infection

• renal vascular disease

protein in urine - PHYSIOLOGIC CAUSES

• usually happens in short amount of time

• physical stress

• exercise

• heat/cold exposure

• fever

albuminuria

• presence of albumin in the urine (aka microalbuminuria)

• DETECTS EARLY SIGNS OF KIDNEY PROBLEMS IN DIABETICS

  • may be positive due to UTI

• tested w sensitive techniques

  • immunoassay and tablet protein error of indicator test

• tests to measure

  • albumin to creatinine ratio in random urine

  • 24 hr collection of urine

orthostatic/postural proteinuria

• occurs following time in VERTICAL POSITION only

  • benign cause

  • more frequent in young adults

• confirmed w timed study:

  • empty bladder before bed

  • collect specimen immediately after rising

  • collect another specimen after in vertical position for several hours

  • NEGATIVE in the MORNING and POSITIVE on specimen collected LATER IN THE DAY

bence-jones protein

• abnormal immunoglobin light chains in urine

• present in MULTIPLE MYELOMA

• heat precipitation test - screening procedure

  • bence jones protein is present when precipitation appears between 40-60^oC (test = 40-60 precipitate → 100 disappears → back to 40-60 precipitate again)

• confirm w immunoelectrophoresis

SSA protein precepitation

• confirmatory test for proteins in urine

• 3% sulfosalicylic acid = detects albumin, globulins, bence-jones proteins

• added to small and equal volume of urine

• acidification causes precipitation of proteins in urine

• semi-quantitated visually or more precisely using photometry

glucose

• normally contained in ultrafiltrate and reabsorbed in the proximal tubule

• NEVER NORMAL IN URINE

• presence in urine: glucosuria

  • amount in urine depends on level in blood, rate of glomerular filtration, degree of tubular reabsorption

  • renal threshold exceeds 160-180 mg/dL

glucose pad principle

• double enzyme rxn

  • enzyme Glucose Oxidase coupled w peroxidase

  • 1st rxn: glucose in urine reacts w enzyme. glucose is oxidized forming gluconic acid and H2O2

  • 2nd rxn: H2O2 reacts w chromogen in presence of peroxidase enzyme. causes visible color change proportional to amnt of glucose in urine

• specific for glucose

glucose false positive

• none in sample

• contamination

  • strong oxidizing agents: bleach, peroxides, cleaning agents

glucose false negatives

• low temp

• high SG

• vitamin C (inhibits enzyme)

• aspirin in rly high amounts

• high ketones

• high bacteria

clinitest

• test for reducing substances

  • sugars occasionally found in urine (galactose, lactose, fructose, maltose)

• copper reduction test

• used to screen for other reducing substances

• TETS DONE ON ALL INFANTS

  • early detection of galactosemia

clinitest principle

• reducing sugars will reduce copper sulfate (CuSO4) to cuprous oxide (Cu+) in the presence of heat and strong alkaline solution

  • blue → green → yellow → orange

clinitest false positive

• nalidixic acid

• cephalosporins

• ascorbic acid (vitamin C)

• urinary preservatives

  • formalin

  • formaldehyde

clinitest false negatives

• technique errors

• “pass through” effect can cause erroneous results

  • 5 drops of urine normally tested nut high glucose levels can pass through quickly and return to blue

  • USE 2 DROP TECHNIQUE and diff chart

ketones

• chemicals when the body breaks down fat for energy (fatty acid metabolism)

• fat used when glucose (carbs) not available

  • ketone bodies produced in urine:

    • 2% acetone

    • 78% beta-hydroxybutyric acid

    • 20% acetoacetic acid (aka diacetic acid)

      • only type of ketone detected by strip test

ketone pad principle

• utilizes sodium nitroprusside and an alkaline buffer

• ketone bodies in urine (acetoacetic acid/diacetic acid) react w sodium nitroprusside to produce magenta/purple color

ketones false positives

• urine highly pigmented (dark colored red urine)

• both high SG and low pH

• phenylketones (red-orange color)

• confirm questionable results w tablet test

ketones false negatives

• controls containing acetone

• breakdown by bacteria - old urine

• volatilization of ketones (time) - old urine

acetest

• used for ketones

• contains sodium nitroprusside, glycine, alkaline buffer, and lactose

  • lactose allows for better color differentiation

  • can be used on serum, urine, plasma, and whole blood

• 10x more sensitive to diacetic acid than acetone

blood

• strip detects hematuria, hemoglobinuria, myoglobinuria

• correlates w urine appearance, microscopic exam, appearance of patient’s plasma, and plasma chemical test

• CAN COEXIST - must confirm and differentiate

blood pad principle

• based on the peroxidase activity of hemoglobin and myoglobin

• catalyze a rxn between peroxide and a chromogen

  • free hemoglobin/myoglobin = uniform color change

  • intact/whole RBCs = speckled pattern

  • color intensity = proportional to concentration

• hemoglobin rxn

  • H2O2 + chromogen → oxidized chromogen + H2O2

    • hemoglobin acts as peroxidase

blood false positives

• presence of oxidizing contaminants (bleach)

• high bacterial content - bacterial peroxidases

• menstrual blood

• povidone-iodine (betadine)

blood false negatives

• high levels of ascorbic acid (vit. C)

• high SG

• proteins

• nitrites

• formalin (preservatives)

blood - hematuria

• RBCs in the urine

• may result from bleeding at any point in urinary system

  • GLOMERULONEPHRITIS, TUMORS, TRAUMA, RENAL CALCULI

  • PYELONEPHRITIS, TOXIC CHEMICALS, DRUGS, UTIs

• early indicator of renal disease

• RED CELLS DISINTEGRATE IN DILUTE URINE (less than 1.010 SG)

• if low RBC quantity present, only detect chemically and microscopically

• can cause color change → pink/red, red/brown, smoky

blood hemoglobinuria

• intravascular hemolysis (red cell components lyse into plasma)

• hemolysis w kidney, lower urinary tract, or in sample

  • transfusion reactions

  • hemolytic anemia

• severe burns

• haptoglobin = protein that binds free hemoglobin

  • when haptoglobin in plasma saturated → see hemoglobin in urine

    • some hemoglobin absorbed into tubular cells of the kidneys as hemosiderin

blood - myoglobinuria

• released after acute destruction of muscle fibers (rhabdomyolysis)

  • injury

  • strenuous exercise in untrained individual

  • convulsions

  • electric shock

• can damage kidneys

• rare

• SERUM NORMAL

• urine is red/brown to black

blood hemoglobin vs myoglobin

• both will produce positive reagent strip blood results

• 1st macroscopic differentiation

  • red plasma + red urine = hemoglobinuria

  • clear plasma + red urine = myoglobinuria

myoglobin screen principle

• hemoglobin is precipitated by ammonium sulfate

  • mix 2.8 g ammonium sulfate in 5ml of urine

  • let stand for 5 mins then filter

  • test filtrate w strip (positive = pink/myoglobin ; negative = clear/hemoglobin)

bilirubin

• formed by breakdown of hemoglobin in the RES (reticuloendothelial system)

• normally, NO DETECATABLE AMOUNT SHOULD BE DETECTED

• if present = SIGN OF LIVER OR BILIARY SYSTEM DISEASE

• only water soluble (unconjugated bilirubin) can be excreted by urine

bilirubin pad principle

• coupled rxn of a diazonium salt w bilirubin in an acid medium

• produces tannish-pink/purple color

• often hard to read

• bilirubin rxn:

  • bilirubin + diazide → azobilirubin