Tayag, Merylea Kelly A.

AUBF BSMT-3B

INTRODUCTION TO SPECIMEN COLLECTION

URINALYSIS Containers:
Clean, dry, leak-proof, wide mouth and flat
bottom, disposable, made with clear material,
O U T L I N E
capable of containing 50 ml.
Urinalysis 9. Suprapubic Specimen Handling:
Urine Volume Aspiration Labels, Recommended time for testing,
Variations: 10. Glass technique contamination, volume and transportation.
a. Oliguria 11. Pediatric Specimen
b. Anuria 12. Drug Specimen S P E C I M E N C O L L E C T I O N :
c. Nocturia Analysis of Urine T Y P E S O F U R I N E S P E C I M E N
d. Polyuria a. Macroscopic Random specimen – most commonly received;
Specimen Collection b. Chemical can be collected any time of the day
Types of Urine Examination of Disadvantage: may lead to false-positive
Specimen: Urine result.
1. Random Specimen 1. Protein First morning specimen – collected in the
2. First-morning 2. Glucose morning when you wake up; recommended for
Specimen 3. Ketones pregnancy testing
3. Fasting specimen 4. Blood 8-hour Urine specimen
4. 2hr PPBS 5. Bilirubin Fasting specimen/second morning specimen –
5. Glucose Tolerance 6. Urobilinogen second voided specimen after a period of
Specimen 7. Nitrite
fasting
6. Timed Specimen 8. Leukocyte
2-HR PPBS (Postprandial Urine Test) – collect
7. Mid-stream Catch
2 hours after eating
8. Catheterized
Glucose Tolerance specimen – patient will be
instructed to take a powdered sugar
Timed specimen 24-hour urine specimen, 12-
INTRODUCTION TO URINALYSIS hour, 4-hour – for in-patients example:
Urinalysis - examination of urine physically, Day 1 – 7 AM: Patient voids and discards
chemically, and microscopically specimen. Patient collects all urine for the
Importance: used to detect a wide range of next 24 hours.
diseases that arises from human body Day 2 – 7 AM: Patient voids and adds this
urine to the previously collected urine.
U R I N E V O L U M E Mid-stream catch – specimen provides a
safer, less traumatic method for obtaining
Factors affecting urine volume:
urine for bacterial culture
1. Fluid intake - hydration
Catheterized – specimen is collected under
2. Fluid loss from non-renal source -
sterile conditions by passing a hollow tube
dehydration
through the urethra into the bladder
3. Variations in secretion of ADH
Suprapubic aspiration (anaerobic culture) –
NUO: 600-2000ml
urine may be collected by external
Average: 1200-1500ml
introduction of a needle through the
abdomen to the bladder
Variation:
Glass technique/3 glass technique/Prostatic
Oliguria - decreased in urine output
specimen – first container and second
Infants <1ml/kg/hr
container will be voided, third container will
Children <0.5ml/kg/hr
be collected
Adults <400ml/day
Pediatric specimen – random specimen may
Anuria - complete cessation of urine flow
be collected by attaching a soft, clear plastic
Nocturia - increase urine output in nocturnal
bag (wee bag) with adhesive to the general
phase
area of both boys and girls
Polyuria - usual on diabetic patients
Drug specimen collection – must collect 30ml
Children <2.5-3ml/kg/day
with the temperature of 30.5-37.7 (within 4
Adults <2.5L/day
mins)
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A N A L Y S I S O F U R I N E Clinical Correlations:
( M A C R O S C O P I C T E S T ) 1. Isosthenuric
2. Hyposthenuric
Volume – for examination 10-15ml (average:
3. Hypersthenuric
12ml)
Color – straw to amber; indicates the degree
Transparency/Clarity – refers to the turbidity
of hydration and should correlate with urine
of urine specimen
specific gravity
Terms used: clear, hazy, cloudy, turbid, milky
a. increased fluid intake – pale urine,
1. Non-pathological causes of turbidity –
decreased specific gravity (except DM)
squamous epithelial cells (female specimen),
b. decreased fluid – dark urine, increased
mucus threads (male specimen), Amorphous
specific gravity
urates, Apo4, carbonates, urates,
Three Pigments:
spermatozoa, fecal contamination, talcum
1. Urochrome - major responsible for the yellow
powder, vaginal creams
pigment of urine
2. Pathological causes of turbidity – RBC, WBC,
2. Urobilin – dark yellow/orange impart an
bacteria, yeast, non-squamous epithelial cells,
orange brown color to urine which is not fresh
abnormal crystals, lymph fluids, lipids
3. Uroerythrin – pink or reddish pigment found
Odor – not part of urinalysis
in many pathological urine but mostly found
Normal: faint aromatic odor or odorless
in normal urine in a very low quantity (pink
Other conditions:
deposits in amorphous urate in urine
1. Ammoniacal - infection (proteus)
Abnormal Urine Color:
2. Fruity, sweet – presence of ketones
Dark yellow/amber/orange
3. Rotting fish – trimethylaminuria
Indications: conc. urine, presence of bilirubin
4. Rancid butter - tyrosinemia
Drugs: phenazopyridine or azo-gantrisin
5. Sweaty feet - isovaleric acid
compounds
6. Mousy odor - PKU (lacks phennylalanine
Red/pink/brown
hydroxylase, sever mental retardation;
Indications: presence of blood or porphyrins
bacterial inhibition test - Guthrie's)
Drugs: rifampicin, phenolphthalein,
7. Cabbage odor - methionine malabsorption
phenindione, phenothriazines
8. Maple syrup odor - manifests Maple Syrup
Brown/black
Urine Disorder (MSUD - increase amino acid
Indication: presence of melanin or
in blood and urine = increase in leucine,
homogentisic acid
isoleucine, and valine); caramelized sugar,
Drugs: levodopa, methyldopa, phenol
curry
derivatives and metronidazole
9. Bleach - contamination
Blue/green
10. Sulfur - cystine disorder
Indications: Pseudomonas infection (UTI) or
increased urinary indicant in intestinal tract
CHEMICAL EXAMINATION OF URINE
infection
Drugs: clorets, methocarbamol (robaxin), It is used for complex complications or diagnosis.
methylene blue, amitriptyline, IV phenol Importance: used to detect Protein, glucose,
derivatives ketones, blood, bilirubin, urobilinogen, nitrite and
Specific Gravity – density of a solution compared leukocyte.
with the density distilled water with similar Protein
temperature; evaluates ability of kidneys to Normal: <10mg/dL or 100mg/24hrs
concentrate urine Albumin - major serum protein found in urine
Methods of Determination: Serum and tubular microglobulins
1. Urinometry Tamm-horsfall protein or uromodulin (origin
2. Refractometry in ALH)
3. Harmonic oscillation densitometry Proteins for prostatic, seminal, and vaginal
4. Reagent strip disease
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A. PRE-RENAL PROTEINURIA D. POST-RENAL PROTEINURIA

Conditions that affect the plasma prior to 1. Lower UTI or inflammations

reaching the kidneys. 2. Injury or trauma
1. Intravascular hemolysis 3. Menstrual contamination
2. Muscle injury or crush injury (myoglobulin) 4. Prostatic Fluid/spermatozoa
3. Severe infection and inflammation (increased 5. Vaginal secretions
in APRs)
4. Multiple myeloma – proliferation of IG plasma Protein Reagent Strip (60sec) – sensitive to
cells, BIP (Ig light chains); demonstrable by albumin
serum immunoelectrophoresis Principle: protein indicators (sorensen’s)
Urine: ppt at 40-60°C – cloudy Reagents:
Dissolves at 100°C – clear Multistix – Tetrabromophenol blue (indicator)
Chemstrip – Tetrachlorophenol
Bence-Jones Protein seen in the following: tetrabromophenolphthalein (indicator)
Multiple myeloma Sensitivity: Multistix – 15-30mg
Macroglobulinemia Sources of error/ interference:
Malignant lymphomas False-positive:
- Highly buffered alkaline urine
B. RENAL PROTEINURIAL: - Pigmented specimens, phenazopyridine
GLOMERULAR DISORDERS
- Quaternary ammonium compounds
Immune complex disorders (detergents)
Amyloidosis - Antiseptics, chlorhexidine
Toxic agents - Loss of buffer from prolonged exposure of
Diabetic Nephropathy the reagent strip to the specimen
decreased glomerular filtrate = renal failure - High Specific gravity
Microalbuminuria – proteinuria not detected in False-negative:
routine RGT strip - Proteins other than albumin
Micral Test: Correlations with other tests: Blood, Nitrite,
Principle: enzyme immunoassay (antibody Leukocytes, Microscopic
enzyme conjugate)
Sensitivity: 0-10mg/dL Sulfosalisylic Acid Precipitation Test:
Reagents: Gold-labelled Antibody, B- - Cold precipitation test that reacts equally with
galactosidase, chlorophenol red galactoside all forms of protein
Interference: false negative: dilute urine Note: (-) reagent strip but (+) SSA = presence of
Strenuous exercise other proteins
Dehydration
Hypertension SULFOSALISYLIC ACID PRECIPITATION TEST
Pre-edampsia Protein
Orthostatic/Postural proteinuria (postdural Grade Range Turbidity
cader protein) – proteinuria when standing (mg/dL)
due to pressure to renal veins Negative <6 no increase in turbidity
First morning urine – (-) protein
Urine after secondary standing – (+) protein Trace 6-30 noticeable turbidity

distinct turbidity with no

1+ 30-100
C. RENAL PROTEINURIAL: granulation
TUBULAR DISORDERS
turbid with granulation, no
1. Fanconi’s Syndrome 2+ 100-200
floculation
2. Toxic agents or heavy metals
turbid with granulation and
3. Severe viral infections 3+ 200-400
floculation

4+ >400 clumps of protein

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Glucose Copper Reduction Test (Clinitest)

Most frequent analyte tested in urine Test relies on the ability of glucose and other
(CSF=protein). substances to reduce copper sulfate to
Renal Threshold: 160 to 180mg/dL; plasma cuprous oxide in the presence of alkali and
concentration of a substance at which tubular heat.
reabsorption stop and increase amount of A color change progressing from a negative
substance in excreted urine blue (CuSO₄) through green, yellow and
Other sugars in urine: Fructose, Galactose, orange/ red (CuO₂) occurs when the reaction
Lactose, Pentose takes place.
Tablet contains copper sulfate, sodium
Clinical Significance of Glucose in Urine:
carbonate, sodium citrate and sodium
Hyperglycemia associated - increase of blood
hydroxide.
glucose, increase of urine glucose
Pass-through phenomenon may occur if >2
a. Diabetes Mellitus - deficiency of insulin
g/dL sugar present in urine
b. Acromegaly - too much growth hormone
GLUCOSE OXIDASE VS. CLINTEST REACTIONS
c. Cushing's Syndrome - increased cortisol
Glucose
d. Hyperthyroidism - high Thyroxine (T4) and Clintest Interpretation
Oxidase
triiodothyronine (T3)
e. Pheochromocytoma - high catecholamines Non-glucose reducing substance
(adrenalin, epinephrine, norepinephrine) Negative Positive present; possible interfering
substances for RGT strip
f. Pancreatic Cancer
g. Central Nervous System Damage 1+
Negative Small amount of glucose present
h. Pancreatitis positive
i. Stress 4+ Possible oxidizing agent
Negative
j. Gestational Diabetes positive interference on reagent strip
Renal associated - normal blood glucose,
impaired tubular reabsorption of glucose Other sugars in urine:
a. Advance renal disease 1. Fructose or Levulose: high in fruits, honey
b. Fanconi's Syndrome - characterized by syrup; (+) Seliwanoff
defective tubular reabsorption 2. Galactose: galactosemia (Enzyme deficient:
c. Pregnancy Galactose I-PO₄ uridyltransferase deficiency,
Galactokinase deficient)
REAGENT STRIP FOR GLUCOSE
3. Lactose: glucose and galactose; during
Double sequential enzyme reaction lactation; towards end of pregnancy; patient
Principle
(highly specific for glucose)
on strict milk diet, (+) Rubner's Test (lead
Multistix: glucose oxidase, peroxidase, acetate)
potassium iodide 4. Pentose (5-carbons): xylose, arabinose = high
Reagents
Chemstrip: glucose oxidase, peroxidase, in fruits (Benign Pentosuria: Xylulose)
tetramethylbenzidine
5. Sucrose (glucose + fructose): increase intake,
Multistix: 75-125 mg/dL intestinal disease, non-reducing sugar; (-)
Sensitivity
Chemstrip: 40mg/dL copper reduction
False-positive: contamination by Ketones
oxidizing agents and detergents Results from increased fat metabolism due to
False-negative: high levels of ascorbic inability to metabolize carbohydrate, as occurs in
Interference
acid; high levels of ketones; high specific Diabetes Mellitus, increased loss of carbohydrate
gravity; low temperature; improperly
from vomiting, and inadequate intake of
preserved specimens
carbohydrate associated with starvation and
Correlation malabsorption.
with other Ketones
Fruity-odor
tests
78% BHA (beta-hydroxybutyric acid) – major
ketone
20% acetoacetic acid/diacetic acid – parent
ketone
2% acetone
AUBF Page 5

Glucose Hemoglobinuria vs. Myoglobinuria:

Most frequent analyte tested in urine Plasma Examination:
(CSF=protein). Hemoglobin - red or pink (low haptoglobin)
Renal Threshold: 160 to 180mg/dL; plasma Myoglonin - pale yellow (high Creatine
concentration of a substance at which tubular Kinase, high Aldolase)
reabsorption stop and increase amount of Blondheim's Test (ammonium sulfate):
substance in excreted urine Proc: Urine + 2.8 ammonium sulfate (80% sat.)
Other sugars in urine: Fructose, Galactose, →centri/ filter → test supernatant for blood
Lactose, Pentose with rgt strip
Hemoglobin - clear supernatant (with red
Clinical Significance: ppt) = (-) reagent strip blood
Diabetes acidosis Myoglonin - red supernatant = (+) reagent
Insulin dosage monitoring strip blood
Starvation BLOOD REAGENT STRIP
Malabsorption/ pancreatic disorders
Based on Pseudoperoxidase activity of
Strenuous exercise hemoglobin
Vomiting Tetramethylbenzidine (chromogen)
Inborn error of amino acid metabolism Principle H₂O₂ + chromogen→(heme)→ oxidized
chromogen + H₂O
KETONE REAGENT STRIP
= (-) yellow
Principle Sodium Nitroprusside reaction (+) green-blue ↑
Sodium Nitroprusside Multistix:
Reagents
Glycine (chemstrip) Disopropylbenzenedehydroperoxide
Tetramethylbenzidine
Multistix: 5-10 mg/dL acetoacetic acid Reagents
Chemstrip: 2,5-dimethyl-2,5-
Sensitivity Chemstrip: 9mg/dL acetoacetic acid,
dihydroperoxide
70mg/dl acetone
Tetramethylbenzidine
False-positive: Phthalein dye; Highly
pigmented red urine; Levodopa; Multistix: 5-20 RBCs/uL, 0.15 – 0.062
Medication containing free sulfhydryl mg/dL hemoglobin
Interference Sensitivity
groups Chemstrip: 5 RBCs/uL hemoglobin
False-negative: improperly preserved corresponding to 10 RBCs/uL
specimens False-positive: strong oxidizing agents;
Correlation bacterial peroxidases; menstrual
with other Glucose contamination
tests Interference False-negative: high specific gravity;
Formalin; Captopril; High concentration
ACETEST: Sodium Nitroprusside, glycine, disodium of nitrite; Ascorbic acid >25 mg/dL;
phosphate and lactose unmixed specimens

Blood Correlation
Hematuria - intact red cells (cloudy red) with other Protein, Microscopic
tests
Renal calculi, glomerulonephritis, pyelonephritis,
tumors, trauma exposure to toxic chemicals, Hemolysis:
anticoagulants, strenuous exercise. Intravascular Hemolysis:
Hemoglobinuria – clear red, intravascular - 10% aged RBCs
hemolysis - within blood vessels
Transfusion reactions, hemolytic anemias, severe - when C¹ is completely activated
burns, infections/malaria, strenuous exercise/ red Extravascular Hemolysis:
blood cell trauma. - 90% aged RBCs (major)
Myoglobinuria – clear red, rhabdomyolysis (↑ - within RES
muscle destruction - when C¹ is not activated or incompletely
Muscular trauma/ crush syndromes, prolonged activated
coma, convulsions, muscle-wasting diseases,
alcoholism/overdose, drug abuse, extension
exertion.
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Bilirubin UROBILINOGEN REAGENT STRIP

- conjugated bilirubin Erlich’s reaction = urobilinogen + Erlich’s
- early indication of liver disease Principle
rgt (P-DAB) = (+)red

Multistix: PDAB
Clinical Significance:
(paradimethylaminobenzaldehyde)
Hepatitis Reagents Chemstrip: 4-methoxybenzene-
Cirrhosis diazonium tetrafluoroborate(diazo
Biliary obstruction (gallstones, carcinoma) reaction)

BILIRUBIN REAGENT STRIP Multistix: 0.2 mg/dL urobilinogen

Sensitivity
Chemstrip: 0.4 mg/dL urobilinogen
Principle Diazo Reaction
Multistix
Multistix: 2,4-dichloroaniline diazonium
False-positive: Porphobilinogen; Indican;
salt
p-aminosalysilic acid; sulfonamides;
Reagents Chemstrip: 2,6-dichlorobenzene-
methyldopa; procaine; chlorpromazine;
diazonium-tetrafluoroborate
highly pigmented urine
(result: (+) tan or pink to purple)
False-negative: old specimens;
Interference
Multistix: 0.4-0.8 mg/dL bilirubin preservation in formalin
Sensitivity
Chemstrip: 0.5 mg/dL bilirubin Chemstrip
False-positive: Highly pigmented urine
False-positive: Highly pigmented urine,
False-negative: old specimens;
phenazopyridine; Indicant (intestinal
preservation in formalin; highly
disorders); Metabolites of iodine
Interference concentrations of nitrate
False-negative: Specimen exposure to
light; Ascorbic acid >25 mg/dL; High Correlation
concentrations of nitrite with other Bilirubin
tests
Correlation
with other Urobilinogen
tests WATSON SCHWARTZ TEST
For differentiating urobilinogen and
ACETEST: ICTOTEST TABLETS porphobilinogen.
Positive: Blue to purple color W A T S O N S C H W A R T Z T E S T
Reagents: p-nitrobenzene-diazonium-p- Other
Urobilinogen Porphobilinogen Erlich-
toluenesulfonate
reactive
SSA
Sodium bicarbonate Chloroform
colorless red red
Boric Acid reaction

Urine (top
Urobilinogen cherry red colorless colorless
layer)
- Afternoon specimen (2pm-4pm)
Butanol
- Bile pigment that result from hemoglobin red colorless red
extraction
degradation
- Small amount in normal urine (<1 mg/dL or Erlich Butanol
red colorless red
unit) (top)

Urine
colorless red colorless
(bottom)

insoluble to
soluble to insoluble to
chloroform
Solubility chloroform & chloroform &
but soluble
methanol methanol
to butanol
HEMATOLOGY Page 7

HOESCH TEST LEUKOCYTE REAGENT STRIP

Rapid screening test for urine porphobilinogen (≥ Leukocyte Esterase
2mg/dL) 120 sec or 2 minutes: (+) neutron, eosino,
Reagent: Erlich reagent dissolved in 6M HCl Principle baso, mono
(inverse Erlich’s) (-) lympho ; maybe (+) histiocytes,
2 gtts of urine + 2 ml rgt = (+) red trichomonas

Multistix: Derivatizedpyerole AA ester,

BILIRUBIN & UROBILINOGEN IN JAUNDICE diazonium salt
Reagents
Urine Bilirubin Urobilinogen Chemstrip: Indoxylcarbonic acid ester,
diazonium salt (purple)
Hemolytic disease negative +++
Multistix: 5-15 WBC/hpf
Liver Damage + or - ++ Sensitivity
Chemstrip: 10-25 WBC/hpf
Bile Duct False-positive: Strong oxidizing agent;
+++ normal (-/↓)
Obstruction Highly pigmented urine; nitrofurantion
False-negative: High concentrations of
Nitrite Interference
proteins, glucose, oxalic acid, ascorbic
- Detection of bacteriuria, UTI, mostly gram (-) acid, gentamicin, cephalosporins,
bacteria, reduces nitrate to nitrite. tetracyclines

(+) nitrite = corresponds to 100,000 org/ml Correlation

UTI = ≥ 100,000 CFU/ml or 1 x 10⁵ CFU/ml with other Protein, Nitrite, Microscopic
tests
NITRITE REAGENT STRIP
Greiss Reaction (positive nitrite
Principle Note: Strip can detect even lyzed WBCs.
corresponds to 100,000 org/mL)

Multistix: p-arsanilic acid;

Tetrahydronezo(h)quinolin3-ol
Reading Times of Substance on the Strip:
Reagents 30 sec = glucose and bilirubin
Chemstrip: sulfanilamide 3-hydroxy-
1,2,3,4-tetrahydro-7; Benzoquinoline 40 sec = ketones
45 sec = Specific Gravity
Multistix: 0.6 mg/dL nitrite ion
Sensitivity 60 sec = PPBUN (pH, protein, blood,
Chemstrip: 0.5 mg/dL nitrite ion
urobilinogen, nitrite)
False-positive: Improperly preserved
120 sec or 2 minutes = leukocytes
specimens; Highly pigmented urine
False-negative: Nonreductase-
containing bacteria; Insufficient contact Vitamin C or Ascorbic acid is a reducing property
Interference time between bacteria and urinary and results to false (-) rgt strip.
Nitrate: Lack of urinary nitrate; Large BBLNG = blood, bilirubin, leukocyte, nitrite, glucose
quantities of bacteria converting nitrite
to nitrogen; High concentration of
ascorbic acid; High specific gravity

Correlation
with other Protein, Leukocyte, Microscopic
tests

Leukocyte
- Screening of urine culture specimens.

Significance: UTI or inflammation