URINALYSIS
endocrine, body position
Histroy and Importance:
Edwin Smith Surgical Papyrus
 study of urine can be found in the drawings of
cavemen and in Egyptian hieroglyphics
Early physicians
 basic observations as color, turbidity, odor, volume,
viscosity, and even sweetness (by noting that
certain specimens attracted ants).
Hippocrates
 wrote a book on “uroscopy.” (5th century BC)
1140 AD
 color charts had been developed that describedthe
significance of 20 different colors
17th century
 invention of microscope
 examination of urinary sediment
Thomas Addis
 developed methods for quantitating the microscopic
sediment.
Richard Bright
 introduced the concept of urinalysis as part of
adoctor’s routine patient examination in 1827.
1930s
 the number and complexity of the tests performed
in a urinalysis had reached a point of impracticality,
and urinalysis began to disappear from routine
examinations.
TWO UNIQUE CHARACTERISTICS OF URINE
SPECIMEN
1. Urine is a readily available and easily collected
specimen.
2. Urine contains information, which can be obtained by
inexpensive laboratory tests, about many of the body’s
major metabolic functions.
“The testing of urine with procedures commonly
performed in an expeditious, reliable, accurate, safe,
and cost-effective manner.” - the Clinical and
Laboratory Standards Institute (CLSI)
URINE FORMATION
 The kidney is the only organ with such a
noninvasive means by which todirectly evaluate its
status.
 Urine is an ultrafiltrate of plasma
Notes:
1200 ml average urine daily output
170,00 ml of filtered plasma
Normal
a. 95% water
b. 5% solutes
 Solute variations: diet, activity,metabolism,
 Major organic solute is urea (protein,amino acid
breakdown); also creatinine and uric acid
 Urea and creatinine identify a fluid as urine
URINE VOLUME
 Determined by body’s state of hydration
 Influenced by fluid intake, nonrenal fluid loss,
antidiuretic hormone (ADH)variations, excretion of
large amounts of dissolved solids (e.g., glucose)
 Usual daily volume: 1200-1500 mL
 Normal range: 600-2000 mL
Anuria
 cessation of urine flow
 Severe kidney damage, decreased renal blood flow
Oliguria
 decrease in urine output
 causes: vomiting, diarrhea, perspiration, severe
burns
a. < 1ml/kg/hr → Infants
b. < 0.5 ml/kg/hr → Children
c. < 400 ml/day → Adults
Nocturia
 increased urine excretion at night
 normally 2-3 times more excretion in the day
Polyuria
 Increase in daily urine volume
 Associated with Diabetes mellitus and Diabetes
insipidus, artificially induced by diuretics,
caffeineand alcohol
a. >2.5 L/ day → Adults
b. 2.5- 3 mL/kg/day → Children
SPECIMEN COLLECTION AND HANDLING
Specimen container:
Recommended - Disposable Wide mouthed Flat bottom
Screw capsat least 50 ml capacity
Note: for 24 hours specimen “Large plastic container
SPECIMEN LABELLING
Information on label:
Specimen should bear the following information:
✓ Patient’s name
✓ Age
✓ Patient’s ID number
✓ Date and Time of collection
✓ Patient’s location/ward
✓ Attending physician

Requisition form:
Must accompany the specimen
✓ Information must match label
✓ Time of receipt is stamped on requisition
✓ Other information: type of specimen, interfering
medications

SPECIMEN REJECTION:

Ground for specimen rejection:

 Unlabeled containers
 Non-matching labels andrequisitions
 Contaminated specimens:feces, paper
 Contaminated containers
 Insufficient quantit
 Delayed or improper transport:ice, refrigeration

SPECIMEN INTEGRITY:
 Test within 2 hours of collection
 Refrigerate if testing is delayed
 Most problems are caused by bacterial
multiplication

TYPES OF SPECIMEN:

 The composition of urine depends on the patient’s

metabolic state and the timing and procedure used
for collection
 Patients must be instructed when special collection
SPECIMEN PRESERVATION: techniques are required

Ideal preservative is: Random Specimen

 Bactericidal
 inhibits urease
 preserves formed elements
 Does not interfere with chemical testing
Routine is refrigeration; this is a must for culture
specimens
 Causes precipitation of amorphous crystals
 Must return to room temperature for chemical
testing
Commercial transport tubes are available but
they must be compatible with tests
 Most common type received
 Routine screening for obvious abnormalities
 May be collected at any time
 Dietary intake and activity may alter results
 Patients may have to collect a follow-up specimen
First Morning Specimen
 Ideal screening specimen
 More concentrated than a random specimen
 Patient is in a basal state
 Use for orthostatic protein confirmation and urine
pregnancy tests
 Patient collects immediately on arising, delivers to
lab within 2 hours
Fasting Specimen
 Also known as the 8 hour specimen
 Actually is second specimen voided – collected
after the first morningspecimen
 Does not contain metabolites from evening meal
 Recommended for glucose monitoring
2 hour Postprandial Specimen
 Monitors insulin therapy
 Results can be compared with fasting urine
specimen and blood testresults
PROCEDURE:
1. Patient voids before eating routine meal
2. Eats meal
3. Collects next specimen 2 hours after finishing meal
Glucose Tolerance Specimen
 Institutional option for collection with blood glucose
tolerance test – not frequently done
 Specimens are collected at the same intervals as
the blood samples
 Used to correlate renal threshold with patient’s
ability to metabolize glucose
Timed Specimen
 Required for quantitative results
 24-hour specimens are needed for measuring
substances with diurna lvariation (results differ in
a.m. and p.m.) and substances that vary with meals,
activity, and body metabolism
 Shorter timed specimens can be used for
substances with consistent levels
 Accurate timing is critical for accurate results
 Calculation for units per 24 hours includes the
volume in milliliters of urine collected
PRINCIPLE: collection must begin and end with an
empty bladder.
Example (Timed Specimen)
1. 7 AM – patient voids and discards urine
2. Patient begins collecting urine
3. 7 AM second day – patient voids and adds this urine
to the specimen
container
Handling of Timed Specimen
 Thoroughly mix specimen and measure
 Save a large enough aliquot to test, and repeat test
if necessary
 Keep specimen on ice or refrigerated during
collection
 Use appropriate and nontoxic preservatives
 Review instructions with patient
Catheterized Specimens
 Sterile specimen collected from bladder witha
catheter
 Most common test is culture and sensitivity
IMPORTANT
CULTURE FIRST BEFORE PERFORMING ROUTINE
URINALYSIS
Midstream Clean-catch Specimen
 Alternative to catheterized specimen
 Less contaminated than routine collection
 Provide patient with mild cleansing material and
container and instructions:
1. Wash hands
2. Clean genitalia with supplied cleanser
3. Void into toilet, then into container,and finish into toilet
4. Do not touch or contaminate inside of Container
Suprapubic Aspiration
 Completely free of contamination forculture and
cytology
 External needle aspiration from thebladder
 Possible pediatric specimen
Prostatitis Specimen
 Collection similar to midstream clean-catch
 3-glass collection:
1. Container 1 – first urine passed
2. Container 2 – midstream urine
3. Massage prostate to obtain
prostatic fluid
4. Container 3 – remaining urine and
fluid
 Quantitative cultures on all 3 specimens
 Examine 1 and 3 microscopically for WBCs
Stamey-Mears: 4 glass collection
1. Initial voided (VB1)
2. Midstream (VB2)
3. Massaged prostate excretions (EPS)
4. Post-massage urine (VB3)
 Cultures on all specimens
a. VB1 and VB2 positive: urinary infection
b. Negative cultures on VB1 and VB2 and
positive on EPS and VB3 show prostatitis
 EPS examined for WBCs >10-20/ hpf: abnormal
Pre and Post Massage Test
1. Specimen 1 – midstream clean-catch specimen
2. Specimen 2 – post-massage specimen
 Prostatitis is indicated by a quantitative culture
result in the second glass that is 10 times higher
than specimen 1
Pediatric Specimen
 Wee bag: Soft, clear plastic bags, with
hypoallergenic tape applied to genital are
 Monitor bag frequently
 Clean-catch method with sterile bag canbe used
 Bags with tubes to a larger container areavailable
for timed specimens
Drug Specimen Collection
 Proper collection, labeling,handling must be
documented
 Chain of custody –documentation from the time of
specimen collection until the time of receipt of
laboratory results; standardized form always
accompanies specimen
 Specimen must withstand legals crutiny
POINTS TO CONSIDER
a. Photo ID of urine donor or ID by employer
b. No unauthorized access to specimen
c. No adulteration, substitution, or dilution of specimen
ADULTERATION TEST
1. Temperature taken within 4 min must be 32.5-37.7°C
2. Report temperatures outside of range immediately.
3. Collect another specimen ASAP
4. Inspect urine color for anything unusual
 Follow laboratory instructions for labeling,
packaging, and transport
FECALYSIS
PHYSIOLOGY
 Feces are the end product of digestion
 Final digestion occurs in the small intestine aided
by pancreatic enzymes and bile salts
 Majority of fluids involved in digestion are
reabsorbed, with only about 150mL excreted in fece
 Excess water reaching large intestine: diarrhea
DIARRHEA
 Definition: >200 g stool weight per day with
increased liquid and > 3 movements per day
 Mechanisms of diarrhea: secretory, osmotic, altered
motility
 Laboratory tests: fecal sodium,
potassium,osmolarity, and pH
✓ pH <5.6 indicates sugar malabsorption
✓ Sodium, potassium, osmolarity used to calculate fecal
osmotic gap
MECHANISM OF DIARRHEA
Secretory Diarrhea
 Microbial infections increase secretion of water
and electrolytes
✓ E. coli, Clostridium, Vibrio cholerae, Salmonella,
Shigella,
Staphylococcus, Campylobacter, Cryptosporidium
Others include: Drugs, laxatives, inflammatory bowel
disease/colitis, endocrine disorders, malignancy,
collagen vascular disease
Osmotic Diarrhea
 Incomplete digestion or reabsorption of food
increases water retention in large intestine
a. Malnutrition: impaired reabsorption
b. Maldigestion: impaired digestion of foods
Others include: Lactose intolerance, celiac sprue
(malabsorption), amebiasis, antibiotics, laxatives,
antacids
Altered Motility
 Irritable bowel syndrome: hypermotility and
constipation; food, chemicals, stress, and exercise
are causes
 Rapid gastric emptying (dumping syndrome)
divided into early and late dumping syndrome
based on timing of symptoms (30 minutes to 2
hours)
Others include: Gastrectomy, gastric bypass, post-
vagotomy, duodenal ulcer, diabetes mellitus
SPECIMEN COLLECTION:
 Patients need detailed instructions
 Pea sized specimen for routine stool examination
 Use clean container and transfer to laboratory
container
 No toilet water contamination
 Ova and parasite containers are used only for
that purpos
 Quantitative collections are 72 hours
✓ 72 hours: time required to pass
through intestine
PHASES OF STOOL EXAMINATION
Macroscopic Examination
 Color
 Consistency
 Other notations upon seeing stool specimen:
nematodes, cestodes, trematodes, etc.
Microscopic Examination
 Fecal Leukocytes
 >3 Neutrophils/hpf is significant
 Fecal RBC
 Muscle fibers
 Fecal fats
 Parasites, Bacteria
PHASES OF STOOL EXAMINATION
Brown 3. A heel warmer can be used for specimen that can
 Normal withstand a temperature slightly higher than 37˚C.
Examples:
Red Cold agglutinin, cyrofibrinogen, and cyroglobulins
 Bleeding in lower gastrointestinal tract or
consuming ng matataas sa pulang food coloring Chilled specimens
 Chilling slows the metabolic process which could
Green affect the results for some specimen. It should be
 Undigested bile completely submerged in crushed ice and water
 Crohn’s disease slurry during transport and immediately tested or
 Antibiotics refrigerated if needed.
 Pagkain ng madadahon na gulay Examples:
Adrenocorticotropic hormone (ACTH), acetone,
Yellow Angiotensin converting enzyme (ACE), ammonia,
 Problem in gall bladder catecholamines, free fatty acids, gastrin, glucagon,
homocysteine, lactic acid, parathyroid hormone (PTH),
White ph/blood gas (if indicated), pyruvate, renin
 Antacids (Aluminum hydroide)
 Sakit sa atay Light-sensitive specimens
 Problem sa liver  There are cases when exposure to light could affect
the result of a specimen, like bilirubin. The
Black phlebotomist should wrap the tube with aluminum
 Bleeding in upper gastrointestinal tract foil or use light-blocking amber-colored container.
 Pagkain ng karne Examples:
 Iron supplements Bilirubin, Carotene, Red cell folate, serum folate, Vitamin
B2,

CLINICAL CHEMISTRY
Vitamin B6, Vitamin B12, Vitamin C, urine porphyrins,
and urine porphobilinogen

STEPS INVOLVED IN PROCESSING AND HANDLING CRITERIA FOR SPECIMEN REJECTION

DIFFERENT TYPES OF SPECIMENS  The collected specimen is transported to the central
processing or triage for screening and prioritizing.
✓ The result of a test is compromised when the proper The specimen are:
collection procedures, storage, processing, and (1) identified,
transporting protocol were not followed in the pre (2) logged or accessioned,
analytical phase. (3) sorted by department and type of processing, and
✓ Studies show that approximately 48% to 68% of (4) evaluated for specimen suitability which is necessary
laboratory result failures are due to prior to analysis for accurate reasons.
phase mishandling or error
The specimen is rejected if it did not meet a specific
Routine Handling criterion
Phlebotomist should adhere to time limits set for delivery such as:
of  Specimen is not identified properly.
specimen to the laboratory except for cases such as  It has inadequate volume to complete the test. o
emergency specimen or other conditions. Hemolysis
 The use of wrong tube for collection. o Outdated
 Mixing tubes by inversion - usually between 3 to 10 tube
inversions.  Improper handling (improper mixing)
 Transporting specimens - should be transported  Contaminated specimen
with the stopper to (1) avoid contact between  Insufficient specimen or "Quality Not Sufficient"
contents and the stopper, (2) minimize agitation of (QNS)
the specimen, and (3) aid clot formation for serum  Incorrect collection time
tubes.  Exposure to light
 Did not follow testing time limits
Special Handling  Delay or error in processing

Body temperature TIME CONSTRAINTS AND EXCEPTIONS FOR

1. Specimen that precipitate or agglutinate if allowed to
cool below body temperature should be transported at
near body temperature which is 37˚C.
2. The tubes should also be pre-warmed at 37˚C and
portable heat blocks are used during transport which
could hold the temperature for 15 minutes from removal
from the incubator.
DELIVERY AND
PROCESSING OF SPECIMENS
Delivery time limits
✓ The specimen should be transported to the laboratory
immediately after collection. Routine blood specimen is
expected to reach the laboratory within 45 minutes.
✓ For specimen that needs centrifugation, it should be
done in 1 hour.
✓ Hematology specimen with EDTA which are placed in
tubes with lavender or purple stopper should not be
centrifuged
Time Limit Exceptions
The delivery time limit has some exception such as
specimen that are marked as "STAT" or "emergency", it
takes priority over all other specimen in terms of
transportation, processing and testing. Other exceptions
to the time limit rule are as follows:
1. Blood smear from EDTA specimen
2. EDTA specimen for CBC
3. EDTA specimen for erythrocyte sedimentation rate
(ESR)
determination
4. EDTA specimen for reticulocyte counts
5. Glucose test drawn in sodium fluoride tubes
6. Prothrombin time (PT)
OSHA-REQUIRED PROTECTIVE EQUIPMENT WORN
WHEN
PROCESSING SPECIMEN
 When processing specimen in the laboratory, the
health worker is exposed to blood and other
potentially infectious materials. For this reason,
health care institutions should comply with the
appropriate protective equipment required by the
Occupational Safety and Health Administration
(OSHA) which includes wearing gloves to prevent
contact with blood, laboratory gown, laboratory
coats, and masks.
CENTRIFUGATION
 A centrifuge is a apparatus that is used to separate
cells, plasma or serum of blood specimen which is
achieved by spinning the blood tubes inside the
vessel at a high speed such that the centrifugal
force will cause the separation of specimens.
 It is important to leave the stoppers on the tube
before and during centrifugation to avoid
contamination, evaporation, aerosol formation, and
pH changes which will affect the accuracy of the
results.
 Take note that the tubes should be balanced in a
centrifuge, meaning tubes of the same size and
volume of specimen should be placed opposite one
another. A centrifuge should not be repeated.
 The plasma specimen collected in tubes with
anticoagulants should be centrifuged immediately
and without any delay. On the other hand, serum
specimen needs to be completely clotted prior to
centrifugation. Normally, complete clotting takes
around 30 to 60 minutes at room temperature.
 Most of the test needs the stopper to be removed to
obtain the serum or plasma.
 A gauze or tissue is used to cover the stopper to
catch drops of blood that may leak or to catch
aerosol that maybe released during the process.
 The tube stoppers should be removed by pulling it
straight up and off the tube.
ALIQUOT PREPARATION
 An aliquot of specimen refers to a portion of a
sample specimen taken for chemical analysis or
testing.
 This is prepared when multiple tests are ordered on
a single specimen.
 The preparation is done by transferring a portion of
the specimen into one or more tubes that are
labeled with the same information as the original
specimen tube using a disposable transfer pipettes.
 Do not put specimen with different anticoagulants in
the same aliquot tube.
 ake sure to cover the tube as soon as it is filled.
Specimen Type
 whole blood, serum, or plasma
 Whole blood: both the liquid portion of the blood
called plasma and the cellular components (red
blood cells, white blood cells, and platelets).
 Anticoagulated blood
 As whole blood sits, the cells fall toward the bottom,
leaving a clear yellow supernate on top called
plasma.
 Serum: If a tube does not contain an anticoagulant,
the blood’s clotting factors are active to form a clot
incorporating the cells. The clot is encapsulated by
the large protein fibrinogen. The remaining liquid is
called serum
 The major difference between plasma and serum is
that serum does not contain fibrinogen (i.e., there
is less protein in serum than plasma) and some
potassium is released from platelets (serum
potassium is slightly higher in serum than in
plasma).
 It is important that serum samples be allowed to
completely clot (≈20 minutes) before being
centrifuged.
 Plasma samples also require centrifugation but do
not need to allow for clotting time
 Specimens should be centrifuged for approximately
10 minutes
 serum or plasma characteristics: hemolysis and
icterus (increased bilirubin pigment) or the
presence of turbidity often associated with
lipemia(increased lipids)
 Samples should be analyzed within 4 hours; to
minimize the effects of evaporation, samples should
be properly capped and kept away from areas of
rapid airflow, light, and heat.
 refrigeration at 4°C for 8 hours: Many analytes
are stable at this temperature, with the exception of
alkaline phosphatase (increases) and lactate
dehydrogenase (decreases as a result of
temperature labile fractions four and five).
 samples may be frozen at -20°C and stored for
longer periods
 Physiologic variation refers to changes that occur
within the body, such as cyclic changes (diurnal or
circadian variation) or those resulting from exercise,
diet, stress, gender, age, underlying medical
conditions, drugs, or posture
 Most samples are drawn on patients who are
fasting (usually overnight for at least 8 hours)
 smoking: causes an increase in glucose as a result
of the action of nicotine; growth hormone; cortisol;
cholesterol; triglycerides; and urea
 High amounts or chronic consumption of alcohol
causes hypoglycemia, increased triglycerides, and
an increase in the enzyme gamma-
glutamyltransferase and other liver function tests.
 Intramuscular injections increase the enzyme
creatine kinase and the skeletal muscle fraction of
lactate dehydrogenase.
 Opiates, such as morphine or meperidine, cause
increases in liver and pancreatic enzymes
 oral contraceptives may affect many analytic results
 Many drugs affect liver function tests
Lipid Measurement
Lipids and lipoproteins are important indicators of CHD
risk
Cholesterol Measurement
 have fasted for at least 12 hours are preferred for
total cholesterol testing and required for triglyceride
testing
Triglyceride Measurement
 useful in detecting certain genetic and other types
of metabolic disorders, as well as in characterizing
the risk of CVDs
Lipoprotein particles
 chylomicrons [chylos], VLDL, LDL, and HDL
Chylomicrons
 Because of their large size, they scatter light, which
accounts for the turbidity or milky appearance of
postprandial plasma. Because they are so light,
they also readily float to the top of plasma when
stored for hours or overnight at 4°C and form a
creamy layer.
VLDL
 Like chylomicrons, they also reflect light and
account for most of the turbidity observed in fasting
hyperlipidemic plasma specimens, although they do
not form a creamy top layer like chylomicrons,
because they are smaller and less buoyant
Intermediate-Density Lipoproteins
 also referred to as VLDL remnants, normally only
exist transiently during the conversion of VLDL to
LDL.
 risk for peripheral vascular disease (PVD) and
coronary artery disease (CAD)
Low-Density Lipoproteins
 proatherogenic and may be a better marker for
CHD risk
High-Density Lipoproteins
 the ability of HDL to remove cholesterol from cells,
called reverse cholesterol transport, is one of the
main mechanisms proposed to explain the
antiatherogenic property of HDL.
Renal Function
Creatinine clearance
 is derived by mathematically relating the serum
creatinine concentration to the urine creatinine
concentration excreted during a period of time,
usually 24 hours.
 Glomerular filtration assessment
Estimated GFR
 earlier detection of chronic kidney disease (CKD)
Cystatin C
 Levels of cystatin C rise more quickly than
creatinine levels in acute renal failure.
β2-Microglobulin (β2-M)
 Elevated levels in serum indicate increased cellular
turnover as seen in myeloproliferative and
lymphoproliferative disorders, inflammation, and
renal failure.
Myoglobin
 associated with acute skeletal and cardiac muscle
injury
 Blood levels of myoglobin can rise very quickly with
severe muscle injury. In rhabdomyolysis
Urine microalbumin
 measurement is important in the management of
patients with diabetes mellitus, who are at serious
risk for developing nephropathy over their lifetime.
 If detected in this early phase, rigid glucose control,
along with treatment to prevent hypertension, can
be instituted and progression to kidney failure
prevented.
Neutrophil gelatinase–associated lipocalin (NGAL)
 It can be measured in plasma and urine and is
elevated within 2 to 6 hours of acute kidney injury
(AKI).
The Electrolytes
 The electrolytes in the body mainly aid in moving
nutrients in the body and removes wastes in the
cells of the body.
 The POCT uses electrolyte panels to determine the
blood level of sodium (Na+), potassium (K+,
 chloride (Cl-), bicarbonate ion (HCO3), and ionized
calcium (iCa2+).
 Sodium helps keep the normal balance of fluids in
the body and also plays a role in transmitting nerve
impulses. An elevated level of sodium is called
Hypernatremia while reduced level is known as
hyponatremia.
Determination of Sodium
 Specimen: Serum, plasma, and urine are all
acceptable for Na+ measurements.
 When plasma is used, lithium heparin, ammonium
heparin, and lithium oxalate are suitable
anticoagulants.
 The specimen of choice in urine Na+ analyses is a
24-hour collection.
 Sweat is also suitable for analysis.
Potassium
 Collection of Samples
 if a tourniquet is left on the arm too long during
blood collection or if patients excessively clench
their fists or otherwise exercise their forearms
before
 venipuncture, cells may release K+ into the plasma.
 hemolysis: K+ may be falsely elevated
 Specimen: Serum, plasma, and urine may be
acceptable for analysis.
Chloride
 Specimen: Serum or plasma
 Whole blood samples may be used with some
analyzers.
 The specimen of choice in urine Cl− analyses is 24-
hour collection because of the large diurnal
variation.
 Sweat is also suitable for analysis.
Determination of CO2
 Specimen: venous serum or plasma determinations
 Serum or lithium heparin plasma is suitable for
analysis.
Determination of Magnesium
 Specimen: Nonhemolyzed serum or lithium heparin
plasma may be analyzed.
 hemolysis should be avoided
 Oxalate, citrate, and ethylenediaminetetraacetic
acid (EDTA) anticoagulants are unacceptable
because they will bind with Mg2+.
 24-hour urine sample
Determination of Calcium
 Specimen: either serum or lithium heparin plasma
collected without venous stasis.
 For analysis of Ca2+ in urine, an accurately timed
urine collection is preferred.
 no liquid heparin
 use dry heparin
Determination of Inorganic Phosphorus
 Specimen: Serum or lithium heparin plasma is
acceptable for analysis.
 Circulating phosphate levels are subject to
circadian rhythm, with highest levels in late morning
and lowest in the evening.
Determination of Lactate
 Specimen Handling
 Ideally, a tourniquet should not be used because
venous stasis will increase lactate levels.
 Heparinized blood may be used but must be
delivered on ice and the plasma must be quickly
separated.
Arterial Blood Gases
Arterial blood gas (ABG) test:measures the level of
oxygen, carbon dioxide and acid-base (pH) in the
patient's blood which gives the physician an idea about
the status of
the function of your lungs, heart and kidneys.
 ABGs measured by POCT methods include
potential hydrogen (pH), partial pressure of carbon
dioxide (PCO2), partial pressure of oxygen (PO2),
and oxygen saturation (SO2).
pH: The arterial pH test checks the balance of the
acid base level which shows the metabolic and
respiratory
status of the patient.
✓ Normal range is from 7.35 to 7.45 only. ✓ The PCO2
is an indicator on how well air is exchanged between the
blood and lungs. The test shows the measure of
pressure exerted by dissolved CO2in the blood plasma
in proportion to the PO2in the alveoli.
✓ Hypoventilation is when the PCO2 level increased to
an abnormal level while hyperventilation is when it
decreases.
✓ PO2 is representative of the pressure exerted by
the dissolved O2 and the ability of the lungs to diffuse
oxygen through the alveoli which is usually used to
measure the effectiveness of an oxygen therapy. A
normal person exhibits 98% oxygen saturation
Non Protein Nitrogenous Compound
• Urea
• Amino acids
• Uric acid
• Creatinine
• Creatine
• Ammonia
Blood urea nitrogen (BUN): urea determination
 Measurement of urea is used to evaluate renal
function, to assess hydration status, to determine
nitrogen balance, to aid in the diagnosis of renal
disease, and to verify adequacy of dialysis
 Urea concentration may be measured in plasma,
serum, or urine.
Uric acid:
 to confirm diagnosis and monitor treatment of gout,
to prevent uric acid nephropathy during
chemotherapeutic treatment, to assess inherited
disorders of purine metabolism, to detect kidney
dysfunction, and to assist in the diagnosis of renal
calculi
 Specimen Requirements: heparinized plasma,
serum, or urine.
Creatinine
 Measurement of creatinine concentration is used to
determine the sufficiency of kidney function, to
determine the severity of kidney damage, and to
monitor the progression of kidney disease
 Specimen Requirements
 Creatinine may be measured in plasma, serum, or
urine. Hemolyzed and icteric samples should be
avoided, particularly if a Jaffe method is used.
Lipemic samples may produce erroneous results in
some methods
Ammonia
 provides useful information are hepatic failure,
Reye’s syndrome, and inherited deficiencies of urea
cycle enzymes
 Severe liver disease is the most common cause of
disturbed ammonia metabolism.
 collect via venous blood only
 Heparin and EDTA are suitable anticoagulants.
Liver Function Test
 Assessment of Liver Function/Liver Function Tests
 Bilirubin
 Urobilinogen in Urine and Feces
 Serum Bile Acids
 Enzymes
 Tests Measuring Hepatic Synthetic Ability
 Tests Measuring Nitrogen Metabolism
 Hepatitis
 Specimen Collection and Storage
 Total bilirubin methods using a diazotized sulfanilic
acid solution may be performed on either serum or
plasma.
 A fasting sample is preferred as the presence of
lipemia will increase measured bilirubin
concentrations.
 Hemolyzed samples should be avoided
 Bilirubin is very sensitive to and is destroyed by
light; therefore, specimens should be protected
from light.
Determination of Urine Urobilinogen
(Semiquantitative)
• Specimen
• A fresh 2-hour urine specimen is collected. This
specimen should be kept cool and protected
from light.
Fecal Urobilinogen
 It is carried out in an aqueous extract of fresh feces,
and any urobilin present is reduced to urobilinogen
by treatment with alkaline ferrous hydroxide before
Ehrlich’s reagent is added.
Aminotransferases
 The two most common aminotransferases
measured in the clinical laboratory are AST
(formerly referred to as serum glutamic oxaloacetic
transaminase [SGOT]) and ALT (formerly referred
to as serum glutamic pyruvic transaminase [SGPT]).
Phosphatases
 Alkaline Phosphatase
 The clinical utility of ALP lies in its ability to
differentiate hepatobiliary disease from osteogenic
bone disease.
γ-Glutamyltransferase
 GGT is a membrane-localized enzyme found in
high concentrations in the kidney, liver, pancreas,
intestine, and prostate but not in bone
Lactate Dehydrogenase
 LD is an enzyme with a very wide distribution
throughout the body. It is released into circulation
when cells of the body are damaged or destroyed,
serving as a general nonspecific marker of cellular
injury
Diabetes Screening
2-hour Postprandrial Glucose
 This blood test is done to check if the patient is
suffering from diabetes and other metabolic
problems. The 2-hour PP test is also used to
monitor insulin therapy. The principles of 2-hour PP
specimen collection are:
 A high-carbohydrate diet was introduced 2 to 3
earlier.
 The patient should fast at least 10 hours prior to the
test.
 Fasting glucose specimen maybe be collected
before the start of the procedure.
 A special breakfast containing an equivalent of
100g glucose or a glucose beverage will be given
on the day of the test.
 Blood glucose specimen will then be collected 2
hours after the meal.
Glucose Tolerance Test
 A patient who could be suffering from carbohydrate
metabolism problems is subjected to the glucose
tolerance test (GTT) which is also called oral
glucose tolerance test (OGTT) to evaluate the
ability of the body to metabolize glucose by
measuring the tolerance level to high glucose level.
 Insulin response to a measured dose of glucose is
recorded by specimen collection at given intervals.
 The GTT length is 1 hour for gestational diabetes
while it is 3 hours for other evaluations.
 The method of collection should also be consistent
be it venipuncture or skin puncture.
GTT Procedure
 Before the procedure the patient must eat balanced
meal containing approximately 150 grams of
carbohydrates for 3 days and must fast for 12 to 16
hours before the scheduled test. Drinking water is
allowed to avoid dehydration and because urine
specimen is also collected as part of the test. The
steps in the GTT procedure are as follows:
 Begin with the normal identification protocol.
Explain the procedure and advise the patient that
only water is allowed during the whole test period.
 The fasting specimen is drawn and the glucose
level is checked and should be over 200 mg/dL for
the test to proceed.
 The patient should collect a fasting urine specimen
if ordered.
 The patient is given the glucose beverage dose.
Adult dose is 75g while children are given 1g per
kilogram of weight. For gestational diabetes the
dose should be between 50 to 75g.
 The beverage should be ingested within 5 minutes.
 Record the time when the drink was finished then
start timing the test which is collected within 30
minutes, 1 hour, 2 hours and so forth.
 A copy of the collection time is provided to the
patient.
 If applicable the collection time for other specimen
such as urine should coincide with the computed
collection time.
 The exact time collected and the time interval
should be written in the label along with patient
identification information.
 Transport the specimen immediately or within 2
hours for accurate results.
Trace Elements, Toxicology & Drug Testing
 Trace elements tests for presence of aluminum,
arsenic, copper, lead, iron, and zinc.
 They are collected in small amounts and must use
special element free tubes colored royal blue, since
traces of these elements in the glass, plastic or
stopper could trickle into the specimen will affect
the accuracy of the result.
 The type of additive, if any, is indicated in the label.
(red - no additive, lavender - EDTA, and green-
heparin). To avoid contamination, change the
transfer device before filing the tube.
Drug Screening
 Companies, healthcare organizations and sports
associations subject their potential employee to
drug screening as part of their pre employment
requirement.
 The company or organization could also run a
random screening without prior notice.
 The specimen used is urine instead of blood. The
chain of custody protocol is strictly implemented
since legal implications are involved.Patient
preparation requirements
 The purpose and procedure should be explained to
the patient.
 The patient should be advised about his legal rights.
 There should be a witness present when the form is
signed.
 Specimen collection requirements
 A special area should be designated for the
purpose of urine collection.
 During the collection, a proctor is present to ensure
that the specimen came from the correct person.
 Split sample may be used for parallel testing.
 Proper labeling should be followed to establish a
chain of custody.
 Protect the specimen from tampering. After
collection it should be sealed in a lock container
and sent to the laboratory immediately.
Documentation should be handled carefully
Blood Alcohol (Ethanol) Specimens
 Blood alcohol (ethanol [ETOH]) tests are usually
ordered for purposes related to treatment but could
also be for industrial or job-related purposes such
as insurance claims or programs and employee
drug screening.
 The law enforcement department orders blood
alcohol concentration (BAC) for individuals involved
in traffic related accidents.
 The ETOH test for treatment purposes do not
require the chain of custody to be accomplished but
the results of such tests can become evidence in
court. However, BAC for industrial and legal
samples should follow the chain-of-custody protocol.
 The ETOH specimen collection uses aqueous
povidone-iodine and aqueous benzalkonium
chloride (BZK).
 Avoid using isopropyl alcohol and tincture of iodine
as antiseptic because these might affect the results.
 Use gray-top sodium fluoride glass tubes for
specimen collection. These tubes could be with
anticoagulant but it depends on the need of the
required specimen for a specific test.
 The tubes are filled until the vacuum is exhausted.
The tube stopper should remain in place at all times
because alcohol could evaporate.
Therapeutic Drug Monitoring
 The Therapeutic drug monitoring (TDM) measures
drug levels at designated intervals so that the
appropriate dosage can be established and
maintained for the patient thus avoiding toxicity.
 TDM begins with prescription of the initial dosage
appropriate for the patient's clinical condition. The
amount in the bloodstream is expected to rise,
eventually reach peak (maximum) which screens
drug toxicity, and gradually fall to a trough or
minimum level which ensures that the levels within
therapeutic range.
 The timing of collection is important for
aminoglycoside drugs (amikacin, gentamicin, and
tobramycin) which have short half-lives but it is not
critical for phenobarbital and digoxin.
 Appropriate concentrations should be given to
optimize the clinical outcomes while considering the
drug dosage, history of dosage given, the recorded
response of the patient and desired outcome.
MICROBIOLOGY
Fundamentals of Specimen Collection
 If possible, collect the specimen in the acute phase
of the infection and before antibiotics are
administered.
 Select the correct anatomic site for collection of the
specimen.
 Collect the specimen using the proper technique
and supplieswith minimal contamination from
normal biota (normal flora).
 Collect the appropriate quantity of specimen
 Package the specimen in a container or transport
medium designed to maintain the viability of the
 organisms and avoid hazards that result from  Expectorated or induced
leakage.
 Label the specimen accurately with the specific Nasal:
anatomic site and the patient information—patient’s  Insert premoistened swab 1 inch into nares
name and a unique identification number.  Nasopharynx:
 Transport the specimen to the laboratory promptly  Insert flexible swab into the nose
or make provisions to store the specimen in an Throat:
environment that will not degrade the suspected  Swab posterior pharynx, tonsils and inflamed area
organism(s).
 Notify the laboratory in advance if unusual Tissue:
pathogens or agents of bioterrorism are suspected.  Disinfect skin
 Don not allow tissue to dry; moistened with sterile
COLLECTION PROCESURES saline

 sterile containers except for stool specimens, which Urine:

can be collected in clean, leakproof containers  Clean catch midstream
 swabs are not recommended for collection  Clean genital area
 Swabs are appropriate for specimens from the  Collect sample after a few urine have passed:
upper respiratory tract, external ear, eye, and midstream
genital tract.
 The tips of swabs may contain cotton, Dacron, or Urine:
calcium alginate.  Catheter
 Cotton-tipped swabs tend to have excessive fatty  Clean urethral area
acids, which may be toxic to certain bacteria.  Allow 15 mL to pass then collect the remainder
 Wounds/lesions: the exact anatomic site must be urine
provided  Indwelling catheter:
 specimen is collected from the advancing margin of  Disinfect catheter collection port
the lesion  Aspirate 5-10mL with needle and syringe
 collected by needle aspiration rather than by swab.  Suprapubic aspirate:
 Before the specimen is collected, the area should  Disinfect skin
be cleansed to eliminate as much of the  Aspirate with needle and syringe
commensal flora as possible.  Through abdominal wall into full bladder
 should be placed into a sterile tube or transport vial
Patient-Collected Specimens
 provide verbal and written instructions
 Blood culture: alcohol and iodine  The instructions should be written using simple
 Aerobic and anaerobic blood culture set language and pictures to help the patient
 Adults: 20mL per set understand the procedure as it is verbally explained.
 Children: 5-10mL per s  The specimens commonly obtained by the patient
are urine, sputum, and stool.

Labeling and Requisitions

 Specimen label:
 Name
 Identification number
 Room number
 Physician
 Culture site
 Date of collection
 Time of collection

The requisition form should provide the following

information:
Genitalia, cervix, vagina  Patient’s name
 Remove mucus before collection  Patient’s age (or date of birth) and gender
 Swab endocervical canal or vaginal mucosa  Patient’s room number or location
 Physician’s name and address
Urethra:  Specific anatomic site
 Flexible swab inserted 2-4cm for 2-3 secs  Date and time of specimen collection
 Collect discharge  Clinical diagnosis or relevant patient history
 Antimicrobial agents (if the patient is receiving)
Sputum:  Name of individual transcribing orders
 Remove dentures
 Rinse mouth or gargle with water Specimen Storage
 Deep cough  Some specimens, such as urine, stool, sputum,
 For Mycobacteria: 3 separate early morning swabs (not for anaerobes), foreign devices such as
specimens
 catheters, and viral specimens can be maintained
at refrigerator temperature (4° C) for 24 hours.
 Pathogens that are cold sensitive may be found in
other specimens, and those specimens should be
kept at room temperature if culture is to be
performed.
 This includes samples that might contain anaerobic
bacteria as well as most other sterile body fluids,
genital specimens, and ear and eye swabs.
 If cerebrospinal fluid is not processed immediately,
it can be stored in a 35° C incubator for 6 hours.
Preservatives
 Urine and stool.
 Boric acid is used in commercial products to
maintain accurate urine colony counts.
 24 hours
 Stool specimens can be refrigerate
 if the delay is longer than 2 hours, the specimen
can be added to Cary-Blair transport media.
 Stools for Clostridium difficile toxin assay should be
collected without a preservative and can be
refrigerated; if the delay is expected to be longer
than 48 hours, the specimen should be frozen at
−70° C.
 Preservatives for ova and parasite (O & P)
examinations maintain the morphology of
trophozoites and cysts.
Anticoagulants
 Anticoagulants are used to prevent clotting of
specimens, including blood, bone marrow, and
synovial fluid.
 Sodium polyanethol sulfonate (SPS) is the most
common anticoagulant used for microbiology
specimens.
 The concentration must not exceed 0.025% (wt/vol)
because some Neisseria spp. and certain
anaerobes are inhibited by higher concentrations.
 Heparin is often used for viral cultures and for
isolation of Mycobacterium spp. from blood.
 Citrate and ethylenediamine tetraacetic acid (EDTA)
should not be used for microbiology specimens.
Holding or Transport Media
 These media usually contain substances that do
not promote multiplication of microorganisms but
ensure their preservation and are available in swab
collection systems.
 Stuart’s or Amie’s transport medium is commonly
used.
 Specimens for N. gonorrhoeae can be placed
directly onto a commercial transport system such
as the JEMBEC system. This system contains
selective agar and a carbon dioxide (CO2)–
generating tablet.
Unacceptable Specimens and Specimen Rejection
 The information on the requisition does not match
the information on the specimen label.
 The specimen is not submitted in the appropriate
transport container or the container is leaking.
 The quantity of the specimen is inadequate to
perform all tests requested.
 The specimen transport time is more than 2 hours
and the specimen has not been preserved.
 The specimen is received in a fixative such as
formalin; stools for O & P are an exception
 An anaerobic culture is requested on a specimen in
which anaerobes are indigenous.
 Microbiology processing of a particular specimen
results in questionable data (e.g., Foley catheter
tip).
 Specimen is dried up.
 More than one specimen from the same source
was submitted from the same patient on the same
day; blood cultures are an exception.
 ONE swab was submitted with multiple requests for
various organisms.
 Gram stain of expectorated sputum reveals less
than 25 white blood cells (WBCs) and more than 10
epithelial cells per lowpower field and mixed
bacterial flora.
Specimen Processing
Macroscopic Observation
 Notations from the macroscopic observation should
include the following:
 Swab or aspirate
 Stool consistency (formed or liquid)
 Blood or mucus present
 Volume of specimen
 Fluid—clear or cloudy
 determine the adequacy of the specimen and the
need for special processing
 Anaerobic cultures may be indicated if gas, foul
smell, or sulfur granules are present
Microscopic Observation
 Microscopic observation serves several purposes:
• (1) It can be used to determine the quality of the
specimen.
• (2) It can give the microbiology technologist and the
physician an indication of the infectious process
involved.
• (3) The routine culture workup can be guided by the
results of the smear
• (4) It can dictate the need for nonroutine or additional
testing
PRIMARY INOCULATION
Types of Culture Media
 Nonselective media support the growth of most
nonfastidious microbes.
 Sheep blood agar is the standard nonselective
medium
 Selective media support the growth of one type
or group of microbes but not another.
 A selective medium may contain inhibitory
substances such as antimicrobials, dyes, or alcohol.
 MacConkey agar is selective for enteric gram-
negative bacilli, and CNA (Columbia agar with
colistin and nalidixicacid) is selective for gram-
positive organisms.
 Differential media allow grouping of microbes
based on different characteristics demonstrated on
the medium.
 Enriched media contain growth enhancers that
are added to nonselective agar to allow fastidious
organisms to flourish.
 Chocolate agar is an enriched medium
 Enrichment broth is a liquid medium designed to
encourage the growth of small numbers of a
particular organism while suppressing other flora
present.
 Lim broth (Todd Hewitt with CNA) is used to
enhance the growth of group B streptococci.
 Broth media can be used as a supplement to
agar plates to detect small numbers of most
aerobes, anaerobes, and microaerophiles.
 Thioglycollate broth (THIO) is an example of a
supplemental broth media.
The routine primary plating media include the following
items:
 Nonselective agar plate
 Enriched medium for fastidious organisms for
normally sterile body fluids or a site in which
fastidious organisms are expected
 Selective and differential medium for enteric gram-
negative bacilli for most routine bacterial cultures
 Selective medium for gram-positive organisms for
specimens in which mixed gram-positive and gram-
negative bacteria are found
 Additional selective media or enrichment broths for
specific pathogens as needed
 Broth medium may be used as a supplement with
specimens from sterile body fluids, tissues, lesions,
wounds, and abscesses
Isolation Techniques
 The specimen is applied by rolling the swab or
placing a drop of liquid specimen onto a small area
at the edge of the plate.
 The inoculating loop is sterilized and allowed to
cool thoroughly before streaking the agar.
 The general-purpose isolation streak is useful for
most specimens.
 The relative number of organisms can be estimated
based on the extent of growth beyond the original
area of inoculum.
 Growth in the first quadrant can be graded as 1+, or
light growth; growth in the second or third quadrant
can be graded as 2+ to 3+, or moderate growth;
and growth in the third or fourth quadrant can be
graded as 4+, or heavy growth.
Incubation
 Most bacteria cultures are incubated at 35° C to
37°C.
 Aerobes grow in ambient air, whereas anaerobes
cannot grow in the presence of oxygen and require
an anaerobic atmosphere.
 Some bacteria are capnophiles and require an
increased concentration of CO2; this can be
achieved by a candle jar, a CO2 incubator, jar, or
bag.
 Microaerophiles grow with reduced oxygen and
increased CO2 and can be isolated using jars or
bags.
 Most routine bacterial cultures are held for 48 to 72
hours.
 Cultures for anaerobes and broth cultures may be
held for 5 to 7 days.
PREPARATION OF SAMPLES
Smears from Swabs
 Smears should not be prepared from a swab after it
has been used to inoculate culture media.
 Smears from swabs are prepared by rolling the
swab back and forth over contiguous areas of the
glass slide to deposit a thin layer of sample material
Smears from Thick Liquids or Semisolids
 The swab is immersed in the specimen for several
seconds and used to prepare a thin spread of
material on the glass slide for staining and viewing.
Smears from Thick, Granular, or Mucoid Materials
 It is most desirable to have both thick and thin
areas.
 Granules within the material must be crushed so
that their makeup can be assessed.
 Granules that are too hard to crush between two
glass slides probably do not represent infectious
materials.
Smears from Thin Fluids
 “Thin” specimens of fluids such as urine,
cerebrospinal fluid (CSF), and transudates should
be dropped but not spread on the slide.
 Cytocentrifugation is preferred for this type of
specimen, if available.
 The cytocentrifugation process deposits cellular
elements and microorganisms from the specimen
onto the surface of a glass slide as a monolayer.
GRAM STAIN
PRINCIPLES
 was developed empirically by the Danish
bacteriologist Christian Gram in 1884.
 crystal violet (hexamethyl-p-rosanaline chloride) to
color all cells and background material a deep blue
 Gram’s iodine to provide the larger iodine element
to replace the smaller chloride in the stain molecule.
 Bacteria with thick cell walls containing teichoic acid
retain the crystal violet–iodine complex dye after
decolorization and appear deep blue; they are
gram-positive bacteria.
 Other bacteria with thinner walls containing
lipopolysaccharides do not retain the dye complex;
they are gram-negative bacteria.
 The alcohol-acetone decolorizer damages these
thin lipid walls and allows the stain complex to wash
out.
 All unstained elements are subsequently
counterstained red by safranin dye.

Bacilli
 Gram-positive bacilli
 Small: Listeria monocytogenes, Corynebacterium
spp.
 Medium: Lactobacillus, anaerobic bacilli
 Large: Clostridium, Bacillus spp.
 Diphtheroid: Corynebacterium, Propionibacterium,
Rothia spp.
 Pleomorphic, gram-variable: Gardnerella vaginalis
 Beaded: Mycobacteria, antibiotic-affected
lactobacilli, and corynebacteria
 Filamentous: Anaerobic morphotypes, antibiotic-
affected cells
 Filamentous, beaded, branched: Actinomycetes,
Nocardia, Nocardiopsis, Streptomyces, Rothia spp.
 Bifid or V forms: Bifidobacterium spp., brevibacteria
 Gram-negative coccobacilli: Bordetella,
Haemophilus spp. (pleomorphic)
 Masses: Veillonella spp.
 Chains: Prevotella, Veillonella spp.

Gram-negative bacilli
 Small: Haemophilus, Legionella (thin with filaments),
Actinobacillus, Bordetella, Brucella, Francisella,
Pasteurella, Capnocytophaga, Prevotella, Eikenella
spp.
 Bipolar: Klebsiella pneumoniae, Pasteurella spp.,
Bacteroides spp.
 Medium: Enterics, pseudomonads
 Large Devitalized: clostridia or bacilli
 Curved: Vibrio, Campylobacter spp.
 Spiral: Campylobacter, Helicobacter,
Gastrobacillum,
 Borrelia, Leptospira, Treponema spp.
 Fusiform: Fusobacterium nucleatum
 Filaments: Fusobacterium necrophorum
(pleomorphic)

RESULTS
• Fluorescent Stain
 Mycobacteria stain bright orange. Count the
number of acid-fast bacilli seen on the smear and
report as follows:
 No. Acid-Fast Bacilli Report
 1-20 Number seen
 21-80 Few
 81-300 Moderate
 >300 Numerous
 Kinyoun and Ziehl-Neelsen Stains
 Mycobacteria stain red, whereas the background
material and non– acid-fast bacteria stain blue.