SPECIMEN PROCESSING AND POINT OF CARE TESTING

SPECIMEN PROCESSING
Blood is the most common sample processed in a clinical laboratory

CENTRIFUGATION:
 Is a process in which centrifugal force is used to separate solid matter from a liquid suspension which is
achieved by spinning the tubes inside the vessel at a high speed.
 Centrifuges are generally used to separate serum or plasma from the blood cells as the blood samples
are being processed.

The speed is expressed in :

1. Revolution per minute (RPM)
 simply a measurement of how fast the centrifuge rotor does a full rotation in one minute.

2.) Relative Centrifugal Force (RCF) or gravities (g)

 is a measurement of the gravitational force that a sample is subject to.

2000- 2500 RPM or 700 RCF or 2000 x g

A centrifuge needs to be balanced based on equalizing both the volume and distribution across the
centrifuge head.

PLASMA PREPARATION
 Collect the specimen
 Ensure the integrity of the Sample
Make sure that the tube is properly labeled with the correct information.
Do not forget to invert the tubes in order to properly mix the blood with the anticoagulant
Check signs for possible rejections.
 Centrifuge the specimen at 2500 RPM for 5 minutes
 Pipette the Plasma into a clean test tube

SERUM PREPARATION
 Collect the specimen
 Ensure the integrity of the Sample
 Allow the sample to clot completely for 30-60 mins
 You can check the completeness of the clot by gently inverting the tube

WHY IS ADEQUATE CLOTTING IMPORTANT IN SERUM PREPARATION?

 In order to make sure that no fibrin clot is left in the serum which can cause interferences in testing
causing false and erroneous results
 Centrifuge the specimen at 2000- 2500 RPM for 5 minutes
 Check if the serum is completely clear. If not, rim the contents of the tube using an applicator is
stick and centrifuge
 Pipette the Serum into a clean test tube
ALIQUOTING
 An aliquot (portion of a specimen) is often created when multiple tests are ordered on a single specimen.
 An aliquot is prepared by transferring a portion of the specimen into one or more tubes labeled with the
same ID information as the specimen tube.

POINT OF CARE TESTING :

 Also called alternate site testing, bedside testing, rapid diagnostics.
 Is any analytical test that is done outside the centralized laboratory and near the site where the patient
receives treatment.
 The testing ranges from a simple procedure such as glucose meter to an automated tests using portable
analyzers.
 Other health professionals such as nurses and physicians can also perform POCT.

ADVANTAGES
 Tests are simple and convenient
 Shortens turnaround time (TAT)
 Improved patient morbidity and mortality

DISADVANTAGES
 Increase in administrative work with training of operators
 Increased risk of errors
 Not as accurate and precise as proper analyzers

MODIFIED ALLEN TEST

ARTERIAL PUNCTURE
Used to collect blood specimen for arterial blood gas (ABG) analysis to manage pulmonary disorders and
maintain the acid-base balance of the body.

ARTERIAL BLOOD GAS

A test that measures the acidity, or pH, and the levels of oxygen and carbon dioxide from an artery.

Performed to diagnose respiratory disorders.

ANALYTE DESCRIPTION
pH
A measure of acidity or alkalinity of blood (acidosis and alkalosis)
PaO2
Partial pressure of O2 dissolved in arterial blood
PaCo2
Partial pressure of CO2 dissolved in arterial blood
HCO2
A measure bicarbonate in blood
O2 saturation
Percent O2 bound to hemoglobin
Base excess
A calculation of non-respiratory part of acid-base balance
Sites and Criteria Used for Arterial Puncture
Collateral circulation
Accessible and large
Low risk of injury
Free

RADIAL ARTERY
BRACHIAL ARTERY
FEMORAL ARTERY

MODIFIED ALLEN TEST

Performed to ascertain collateral blood flow to the hand prior to the radial arterial blood gas (ABG).
Usually done at the bedside by the phlebotomist to patients who will undergo the ABG test.

PROCEDURE:
 Properly identify the patient.
 Orient the patient on the procedure to be performed.
 Wash your hands thoroughly with warm, soapy water, and put on gloves.
 Ask the patient make a tight fist.
 Using the middle and index fingers of both hands, apply pressure on the patient’s wrist,
compressing both the radial and ulnar arteries at the same time.
 While maintaining pressure, have the patient open his/her hand slowly. The hand should appear
blanched or drained of color.
 Lower the patient’s hand and release pressure on the ulnar artery only.

POSITIVE ALLEN TEST

 The hand flushes pink or returns to normal color within 15 seconds, indicating the return of blood to
the hand via the ulnar artery and the presence of collateral circulation.
 If the allen test is positive, proceed to the arterial blood gas collection.

NEGATIVE ALLEN TEST

 The hand does not flush pink or return to normal color within 15 seconds, indicating the inability of
the ulnar artery to adequately supply blood to the hand, leading to the absence of collateral
circulation.
 If the Allen Test is negative, the radial artery should not be used and another site must be selected.

ABG SPECIMEN COLLECTION

ABG test result includes the current body temperature,
respiratory rate, ventilation status, fraction of inspired oxygen(FIO2), and prescribed flow rate in liters per
minute.
CRITERIA FOR REJECTION OF ABG SPECIMEN
 Air bubbles are found in the specimen.
 The specimen is clotted.
 The specimen has hemolyzed.
 The submitted specimen did not comply with the proper labeling requirement.
 The prescribed transportation temperature for the specimen was not met.
 The specimen did not meet the required volume.
 It took so much time for the specimen to reach the laboratory.
 The wrong type of syringe was used.

COLLECTION AND HANDLING OF URINE SPECIMEN

 Standard precautions must always observed
 Urine is a biohazardous material  ALL SPECIMEN MUST BE LABELED PROPERLY
 REQUISITION FORM MUST ACCOMPANY SPECIMENS delivered in the laboratory and the time the
specimen is received should be recorded on the form.

I. SPECIMEN CONTAINER
-clean, dry leak proof containers made up of clear materials and with a wide mouth and a wide, flat bottom.
-disposable containers are recommended
- recommended capacity: 50mL

II. SPECIMEN REJECTION:

 Improperly labeled and collected specimens should be rejected by the laboratory and request for a new
specimen
 Specimen in unlabeled containers  Nonmatching labels and requisition form
 Specimen contaminated with feces and toilet paper  Specimens that have been improperly transported

III. SPECIMEN INTEGRITY

-After collection, specimens must be sent to the laboratory and tested within 2 hours. -Preserve specimen
by refrigeration or use of an appropriate chemical preservative
*SPECIMEN PRESERVATION
 Physical
a. Refrigeration- most routinely used method
-temperature: 2
oC to 8
pC
EFFECTS: - decreases bacterial growth and metabolism
- Increase specific gravity when measured by urinometer
- PRECIPITATION OF AMORPHOUS PHOSPHATE AND URATES
 Chemical - the routine use of preservative is not recommended; may be used when refrigeration is
impossible
- Ideal preservative: bactericidal, can inhibit urease, can preserve formed elements in the sediment
and should not interfere with chemical test
a. BORIC ACID: bacteriostatic at 18g/L; can be used for culture transport
b. FORMALIN: preservative for ADDIS COUNT
c. SACCOMANO FIXATIVE: used for cytology studies
IV. TYPES OF URINE SPECIMEN
 Random
- most commonly received specimen
- collected at any time
- used for routine screening tests  First morning
- collected immediately on arising and delivered to the lab within 2 hours
- IDEAL SCREENING SPECIMEN
- Concentrated and acidic
- for preventing false-negative pregnancy test result - for evaluating orthostatic proteinuria  Fasting (second
morning)
- second voided specimen after period of fasting
- recommended for glucose monitoring
 2-hour Post Prandial - collected shortly before consuming a routine meal and collecting again 2 hours
after eating
- for monitoring INSULIN THERAPY in diabetic patients  Glucose Tolerance Specimen
- collected to correspond with the blood samples drawn during OGTT
- may include fasting, 1 hour, 2-hour and 3-hour specimen
 24 Hour (Time) Specimen
- for analytes that exhibit diurnal variations and are affected by changes brought by daily activities
such as exercise, meals and body metabolism. - PATIENTS MUST BE INSTRUCTED ON THE
PROCEDURE FOR COLLECTING A TIMED SPECIMEN. - must begin and end the collection period with
an empty bladder
- collect ALL urine samples voided during the next 24 hours, including the last sample voided exactly
at the conclusion of the 24-hour period.  Early Afternoon Specimen
- for UROBILINOGEN DETERMINATION
 12 Hour Urine
- for ADDIS COUNT
 Catheterized Urine Specimen
- collected under sterile conditions by passing a catheter through the urethra into the bladder
- for bacterial culture
- may also be used to measure function of individual kidney
 Midstream Clean-Catch Specimen
- an alternative to the catheterized specimen
- safer, less traumatic method of obtaining urine for bacterial culture  Suprapubic Aspiration
- collected by external introduction of a needle through the abdomen into the bladder
- for bacterial culture
- also used for cytologic examination
 Prostatitis Specimen (Three Glass Collection)
- for PROSTATIC INFECTION/ PROSTATITIS determination
 Pediatric Specimen
- soft, clear plastic bags with hypoallergenic skin adhesive to attach to the genital area of the both
boys and girls  Drug Specimen Collection
- Chain of custody
- Volume of Urine specimen: 30 to 45 mL
- Temperature: 32.5
oC to 37.7
oC taken within 4 minutes

PHYSICAL EXAMINATION OF URINE

1. Urine Volume
- main determinant- water intake (body’s state if hydration)
- not routinely performed
 Normal daily output: 600 to 2000 mL/day; average of 1200 to 1500 Ml  VARIATIONS:
a. Oliguria- decrease urine output; <400mL/day in adults
b. Anuria- cessation of urine flow
c. Nocturia- increase in nocturnal excretion of urine
d. Polyuria- increase in daily urine volume; >2.5mL/day in adults

- associated with DIAABETES MILLITUS AND DIABETES INSIPIDUS

2. Color
- normal urine has a wide range of color- mainly determined by its CONCENTRATION
- PALE YELLOW- dilute urine
- DARK YELLOW- concentrated urine  NORMAL URINE COLOR: Colorless, pale yellow, yellow, dark
yellow, amber  URINE PIGMENTS:
- Urochrome – causes yellow color of urine
- Uroerythin- a pink pigment most evident in refrigerated specimens as a result of

amorphous urates precipitation

PMLS 2 LAB NOTES: ROUTINE URINALYSIS Page 4

- Urobilin- oxidation product of urobilinogen and imparts an orange-brown color to urine

that is not fresh

 VARIATIONS IN URINE COLOR AND THEIS CAUSES

a. Colorless
- recent fluid consumption
- polyuria due to diabetes insipidus and diabetes mellitus
b. Dark Yellow/ Amber/ Orange
● Bilirubin- dark yellow to amber
- abnormal pigment from the degradation of hemoglobin
- produces stable yellow foam when shaken
● Urobilin- dark yellow to amber
- oxidation product of urobilinogen
- no yellow foam when shaken
● Biliverdin- dark-yellow green; oxidation product of bilirubin
● Phenazopyridine (Pyridium)- orange
- urinary analgesic
- produces yellow foam when shaken, can be mistake for bilirubin

c. Red/ Pink/ Brown

● Red blood cells- red, pink or brown

- intact cells from bleeding in the urinary tract

● Hemoglobin- red or pink

- intravascular hemolysis
● Red or brown- red or brown

- rhabdomyolysis or skeletal muscle breakdown

● Porphyrins- port wine color (red purple)

● Rifampin- red/ bright orange red
- anti-tuberculosis medication

d. Brown/ Black
● Homogentisic acid- black

- metabolite of phenylalanine
- color develops upon standing in alkaline urine in
ALKAPTONURIA

● Melanin- dark brown to black

- oxidation product of melanogen
- develops upon standing and associated with malignant MELANOMA

PMLS 2 LAB NOTES: ROUTINE URINALYSIS Page 5

e. Blue/ Green
● Pseudomonas- green

- UTI cause by pseudomonas

● Indicans- green
- intestinal tract infection causing increase in indicans
● Clorets- blue
- breath deodorizer
● Azure A- blue
- used in diagnex bue test for HCl

3. Clarity
- refers to the TRANSPARENCY or TURBIDITY of a urine specimen
- assessed at the same time as urine color
- provides a key to the microscopic examination results  NORMAL URINE CLARITY: usually CLEAR
 Reported as: clear, hazy, cloudy, turbid and milky

4. Specific Gravity
-indicator of concentration of dissolved material/ chemicals in the urine
- FUNCTION: used to measure the concentrating and diluting ability of the kidney in its effort

to maintain homeostasis

- affected by both NUMBER and SIZE of particles in the solution

 NORMAL URINE SPECIFIC GRAVITY

RANDOM: 1.002 to 1.035

24-HOUR: 1.015-1.025
*S.G. <1.002- probably not urine

*S.G. >1.035- may be due to radiographic contrast media  METHODS/INSTRUMENT USED TO

MEASURE SPECIFIC GRAVITY

URINOMETER REFRACTOMETER
METHOD Direct Indirect
VOLUME 10 to 15 mL 1-2 drops
CALIBRATION 20

oC 15- 38
oC

CORRECTION/S
NEEDED

Temperature, glucose and

protein correction needed

-Temperature correction: not necessary

- glucose and protein correction needed

CLARITY TERM
CLEAR No visible particulate, transparent
HAZY Few particulates, print easily seen through urine
CLOUDY Many particulates, print blurred through urine
TURBID Print cannot be seen through urine
MILKY Many precipitate or be clotted

PMLS 2 LAB NOTES: ROUTINE URINALYSIS Page 6

5. Odor
- seldom of clinical significance and not part of the routine urinalysis  Normal Odor of freshly voided urine:
FAINT AROMATIC
 Lack of odor: ACUTE TUBULAR NECROSIS in patients with ACUTE RENAL FAILURE

CHEMICAL EXAMINATION OF URINE USING REAGENT STRIP

REAGENT STRIP:
*PRINCIPLES
• Consists of chemical-impregnated absorbent pads attached to a plastic strip
• A color producing chemical reaction takes place when the absorbent pad comes in contact with urine
• Reactions are interpreted by comparing the color produced on the pad with a chart supplied by the
manufacturer. • By careful comparison, results are described as NEGATIVE, TRACE, 1+, 2+, 3+, OR 4+
*ERRORS CAUSED BY IMPROEPR TECHNIQUE
1. Formed elements such as red and white blood cells sink to the bottom of the specimen and will be
undetected in an unmixed specimen. 2. Allowing the strip to remain in the urine for an extended period may
cause leaching of reagents from
the pads. 3. Excess urine remaining on the strip after its removal from the specimen can produce a run-
over
between chemicals on adjacent pads, producing distortion of the colors. To ensure against run-over,
blotting the edge of the strip on absorbent paper and holding the strip horizontally while comparing it
with the color chart is recommended. 4. A good light source is essential for accurate interpretation of color
reactions. *CARE OF THE REAGENT STRIPS
• Reagent strips are packaged in opaque containers with a desiccant to protect them from light and
moisture. • Strips are removed just prior to testing, and the bottle is tightly resealed immediately. • Bottles
should not be opened in the presence of volatile fumes. • Manufacturers recommend that reagent strips be
stored at room temperature below 30°C (but never
refrigerated). • All bottles are stamped with an expiration date that represents the functional life expectancy
of the
chemical pads. Reagent strips must not be used past the expiration date. • Care must be taken not to touch
the chemical pads when removing the strips. • A visual inspection of the strip should be done each time a
strip is used to detect deterioration, even
though the strips may still be within the expiration date

*QUALITY CONTROL
• Test open bottles of reagent strips with known positive and negative controls every 24 hours
• Resolve control results that are out of range by further testing. • Test reagents used in back-up tests with
positive and negative controls.

PMLS 2 LAB NOTES: ROUTINE URINALYSIS Page 7

• Perform positive and negative controls on new reagents and newly opened bottles of reagent strips. •
Record all control results and reagent lot numbers
PARAMETERS:
1. pH
- determines by the concentration of free H+
 CLINICAL SIGNIFICANCE: • acid base balance (Respiratory or Metabolic acidosis/ alkalosis) • Renal
Tubular Acidosis
• Renal Calculi Formation and prevention
• Treatment of UTI • Precipitation/Identification of Crystals
• Determination of Unsatisfactory Specimen
• pH of >8.5 or 9.0= improperly preserved specimen
 URINE pH range
- RANDOM: 4.5 to 8.0
- FIRST MORNING: 5.0 to 6.0
- With normal protein diet: 4.5 to 6.5
- Urine pH <4.5 or >8.0 is physiologically impossible; investigate for causes of such.  REAGENT STRIP
PRINCIPLE: DOUBLE INDICATOR SYSTEM
 Reaction
a. red-orange yellow (pH 4 to 6)
b. green blue (pH 6 to 9)
 Read at: 60 secs
2. Protein
- Most indicative of renal disease; first indicator of renal disease
- Proteins normally found in urine: • Albumin- major serum protein found in urine
• TAMM- HORSFALL PROTEIN (uromodulin/ uromucoid)- a mucoprotein produced by
the renal tubules and forms matrix of all types of casts.  NORMAL URINE PROTEIN: <10mg/dl or
100mg/24 hours
(150mg/24 hours – Henry)

 CLINICAL SIGNIFICANCE: CLINICAL PROTEINURIA (PRERENAL, RENAL, POSTRENAL)

 REAGENT STRIP PRINCIPLE: PROTEIN ERROR OF INDICATOR
 Reaction: yellow to blue-green
 Read at: 60 secs
3. Glucose
- Most frequently performed chemical analysis on urine

PMLS 2 LAB NOTES: ROUTINE URINALYSIS Page 8

• RENAL THRESHOLD FOR GLUCOSE: 160 to 180 mg/dL

• CLINICAL SIGNIFICANCE- detection and monitoring of DIABETES MELLITUS
• Glucosuria/ glycosuria- presence of detectable levels of glucose in the urine
• NORMAL URINE GLUCOSE: 15mg/dL

Fasting: 2 to 20 mg/dL per 100ml urine

• REAGENT STRIP PRINCIPLE: DOUBLE SEQUENTIAL ENZYME REACTION

• Reaction:
a. Multistix: green to brown
b. Chemstrip: yellow to green
• Read at: 30/60 secs
4. Ketones
-Ketone bodies represent intermediate products of fat metabolism/products of incomplete fat
metabolism
• Acetone (20%) • Acetoacetic acid (20%) • Beta-hydroxybutyric acid (70%)  NORMAL URINE KETONE:
NORMALLY NOT IN URINE
 REAGENT STRIP PRINCIPLE: SODIUM NITROPRUSSIDE REACTION
 Read at: 40/60 secs
5. Blood
- May be present in the urine either in the form of hematuria or hemoglobinuria/ myoglobinuria
• Hematuria- relatively common; presence of 3 or more RBCs in urine
• Hemoglobinuria- uncommon; presence of free hemoglobin in solution in urine
• Myoglobinuria- rare; presence of myoglobin in urine
• May be assessed microscopically or through the use of reagent strip

• NORMALLY, NO BLOOD IN THE FORM OF HEMATURIA, HEMOGLOBINURIA

OR MYOGLOBINURIA SHOULD BE DETECTED IN THE URINE
• Presence of >5rbcs/uL is clinically significant (Microscopic hematuria)
• REAGENT STRIP PRINCIPLE: PSEUDOPEROXIDASE ACTIVITY OF HEMOGLOBIN
• Reaction: color change from orange to green to dark blue
• Read at: 60 secs
6. Bilirubin
- Highly pigmented yellow compound from degradation of hemoglobin
• Provides early indication of liver disease since it can be detected long before patients exhibit
jaundice

• CLINICAL SIGNIFICANCE: SCREENING OF ABNORMAL HEPATOBILLARY FUNCTION

- Pre-hepatic jaundice (hemolytic anemias)

PMLS 2 LAB NOTES: ROUTINE URINALYSIS Page 9

- Hepatic jaundice (hepatitis, cirrhosis)

- Post-hepatic jaundice (biliary obstructions, gallstones, carcinoma) • Urine bilirubin is excreted in very
small amount and normally should not be detectable in urine
and only the conjugated form of bilirubin can appear in the urine. • REAGENT STRIP PRINCIPLE: DIAZO
REACTION
• Reaction: color change from tan/pink to violet • Read at: 30 to 60 secs
7. Urobilinogen
- A colorless pigment formed from the breakdown of bilirubin in the intestines. - Urobilinogen excretion
reaches peak levels between 2pm to 4pm
- Same significance as bilirubin
• NORMAL URINE UROBILINOGEN: <1 mg/dL or 1 EHRLICH UNIT
• REAGENT STRIP PRINCIPLE: EHRLICH REACTION
• Read at: 30 to 60 seconds
8. Nitrite
- Rapid (indirect) screening test for the presence of URNINARY TRACT INFECTION (UTI) or
asymptomatic bacteriuria
- Detects bacteria capable of reducing nitrate to nitrite
• CLINICAL SIGNIFICANCE: Cystitis, Pyelonephritis, Evaluation of antibiotics therapy, screening of

urine culture specimens

• REAGENT STRIP PRINCIPLE: GREISS REACTION
• Read at: 30 to 60 secs
9. Luekocyte Esterase
- Indicates pyuria and that an inflammatory process is occurring in the kidney or urinary tract • CLINICAL
SIGNIFICANCE:
- Bacterial and nonbacterial urinary tract infection I
- Inflammation of the urinary tract - Screening of urine culture specimens
- Leukocyturia without bacteriuria may be caused by Trichomonas, Chlamydia, yeast, and inflammation of
renal tissues (i.e., interstitial nephritis) • REAGENT STRIP PRINCIPLE: LUEKOCYTE ESTERASE
• Read at: 120 secs
10. Specific Gravity
• REAGENT STRIP PRINCIPLE: pKa change of polyelectrolyte
• Read at: 45 secs