Evolve.

Adapt.
Overcome.
CEFI is now ready.

MODULE 4 / SPECIMEN COLLECTION

MLS 109: Clinical Chemistry 1

WILLIAM CHRISTOPHER C. SALAZAR, RMT

College of Medical Technology
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

PRE-ANALYTICAL VARIABLES
• Factors that affects specimen before tests are
performed
• Most common cause of error = mislabelling
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

PRE-ANALYTICAL VARIABLES
1. Exercise
 Causes the following
oVolume shifting between intravascular and extravascular
compartment
oVolume loss through sweating
oHormonal changes
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

PRE-ANALYTICAL VARIABLES
1. Exercise
 Increases the following
oTransient – LDH, fatty acid, ammonia
oL o n g t e r m – C r e a t i n e k i n a s e , A s p a r t a t e
aminotransferase, aldolase
oProtein in urine
oVigorous hand exercise (f is t clenching) – increases
potassium, lactate and phosphate
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

PRE-ANALYTICAL VARIABLES
2. Fasting
 Required for some test like FBS, glucose tolerance
tests, lipid profile, gastrin and insulin
 Overfasting causes
oIncreased serum bilirubin (48 hours)
oIncrease plasma triglyceride (72 hours)
oIncreased plasma glucose (72 hours)
 Basal state collection
oBlood is collected early in the morning, 12 hours after
the last ingestion of food
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

PRE-ANALYTICAL VARIABLES
3. Diet
 High protein – increased urea
 Low carbohydrate – increased ketone in the urine
 Caffeine – increased glucose and cathecolamines
 Increased fat – increased potassium, triglycerides
and 5-HIAA
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

PRE-ANALYTICAL VARIABLES
4. Position
 Preferred: upright or supine
Ideally, the patient must be supine/seated for at least 15
- 2 0 m i n u te s b e fore b l ood c ol l e c ti on to p rev e n t
hemodilution or hemoconcentration
 Supine  seating position = constriction of blood
vessels  reduction of plasma volume =
hemoconcentration (albumin, enzymes, and calcium)
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

PRE-ANALYTICAL VARIABLES
4. Position
 Sitting  supine = water and electrolytes shift into
tissue = hemoconcentration (proteins, lipids, BUN,
iron and calcium
 Standing  supine = extravascular water to
transfer to the vascular system = hemodilution
(cholesterol, triglycerides, and lipoproteins
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

PRE-ANALYTICAL VARIABLES
5. Torniquet/ tourniquet application
 Must be applied no longer than 1 minute
 Prolonged tourniquet application results to
oHemoconcentration
o Increased levels of Potassium, proteins (albumin), enzymes,
lactate, cholesterol, and ammonia
oAnaerobiasis
 It should not be applied for ammonia determination
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

PRE-ANALYTICAL VARIABLES
6. Tabacco smoking (nicotine)
 Increases plasma cathecolamines and cortisol
 Increased in glucose, growth hormone, cholesterol,
triglycerides, ammonia, urea, lactate, insulin, and
urinary 5-HIAA, neutrophils and monocytes
 Fo r a m m o n i a de t e rm i n a t i o n – pa t i e n t a n d
phlebotomist are not allowed to smoke
 Decreased plasma levels of vitamin B12
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

PRE-ANALYTICAL VARIABLES
7. Alcohol intake
 Increases in the following
oUrates
oTriglycerides
oGamma Glutamyl Transferase (GGT)
 Causes hypoglycema (chronic alcoholism)
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

PRE-ANALYTICAL VARIABLES
8. Stress (anxiety)
 Affects adrenal hormone secretion
 Increases the following
oCathecolamines, cortisol, ACTH, prolactin, insulin, albumin,
glucose, and lactate
 Total cholesterol has been reported to increase with
mild stress; HDL cholesterol to decrease by as much
as 15%
 It also result in hyperventilation which in turn affects
acid-base balance
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

PRE-ANALYTICAL VARIABLES
9. Drugs
 Medications affecting plasma volume can affect
protein, BUN, iron and calcium concentrations
 Therapeutic Drug Monitoring (TDM) specimen
collection should be scheduled according to the time
of the last dose
 He patotoxi c drugs can e l evate l i ve r functi on
enzymes
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

PHYSIOLOGIC VARIATION
 Refers to the changes that occur within the body such as
cyclic changes (diurnal or circadian) or those resulting
from exercise, diet, stress, gender, age, drugs, posture or
underlying medical conditions
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

PHYSIOLOGIC VARIATION
 Affected by diurnal variations
Increased in AM
oACTH, aldosterone, cortisol, iron
Increased in PM
oACP, GH, PTH, TSH
 Affected by age (increased levels)
Albumin, ALP, cholesterol, and phosphorus
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

PHYSIOLOGIC VARIATION
 Affected by gender (increased levels
Males – albumin, ALP, creatine, uric acid, cholesterol, BUN
Females – HDL, iron, cholesterol
 Affected by food ingestion
Increased levels – glucose, triglycerides, gastrin, free
Calcium
Decreased levels – electrolytes (Chloride, Potassium,
Phosphorus) alkaline phosphatase, amylase
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

TYPES OF BLOOD SPECIMEN

1. Venous blood
 Obtained by venipuncture
 Deoxygenated blood
 Contains products of metabolic activities of
different organs
 Specimen of choice for most chemistry tests
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

TYPES OF BLOOD SPECIMEN

1. Venous blood
 Plasma
Liquid portion of anticoagulated blood
Used in medical emergencies
 Serum
Liquid portion of a clotted blood
Adequate time for clotting must be allowed to prevent
latent f ib rin formation which may cause clogging in
automated chemistry analyzers
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

TYPES OF BLOOD SPECIMEN

2. Arterial blood
 Obtained by arterial puncture
 Oxygenated blood
 Uniform in composition throughout the body
 Specimen of choice for blood gas analysis
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

TYPES OF BLOOD SPECIMEN

2. Capillary blood
 Obtained by skin puncture.
 Mixture of venous and arterial blood
 May contain tissue fluids
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

TYPES OF BLOOD SPECIMEN

3. Capillary blood
 Suitable in
Pediatric patients
Obese individuals
Patients with thrombotic tendencies
Severe burns
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN COLLECTION - VENIPUNCTURE

 Blood is obtained from a vein
Median cubital
Preferred site for venipuncture
Largest and best anchored
Cephalic
Second choice
Basilic
Third choice
Close to the brachial artery
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN COLLECTION - VENIPUNCTURE

 Precaution
Selection of site
Sites adjacent to IV therapy must be avoided
IV fluid contamination causes the ff
Increase in glucose, chloride, potassium and sodium
10% contamination with 5% dextrose = glucose increases up
to 500 mg/dl
Decrease in urea and creatinine
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN COLLECTION - VENIPUNCTURE

 Precaution
Tourniquet application
Pressure = 60mmHg
Tourniquet too close to the site = vein collapses
M u s t b e a p p l i e d o n l y w i t h i n 1 m i n u t e t o a v o i d
hemoconcentration
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN COLLECTION - VENIPUNCTURE

 Precaution
Disinfection
No traces of alcohol should remain – it may cause hemolysis
and contaminate the specimen
Ethanol testing = use benzalkonium chloride (Zephiran) for
skin disinfection
Blood culture = 70% alcohol followed by iodophor
If allergic to iodophor = use chlorhexidine gluconate
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN COLLECTION - VENIPUNCTURE

 Precaution
Needle
The gauge is inversely related to the size of the needle
21-gauge = standard size, 1-1.5 in
23 gauge = for children / small veins, 1-1.5 in
25 gauge = premature infants, ½ - ¾ in, increases chance of
hemolysis
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN COLLECTION - VENIPUNCTURE

 Precaution
Tubes for blood collection
Plain tube = clotting time is about 60 minutes
Tubes with silica clot activator = clotting time is within 15-30
min
Never transfer blood collected in an additive tube into another
additive tube
Tubes containing gels are not used in immunologic testing
and TDM as gel may interfere with immunologic reactions
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN COLLECTION - VENIPUNCTURE

 Sites to be avoided
IV lines
Burned / scarred areas
Hematoma
Thrombosis
Edematous limb
Arms at the mastectomy side and AV shunt / fistula
Fractured limb
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN COLLECTION - VENIPUNCTURE

 Complications of venipuncture
Infection
Thrombosis
Thrombus develops within a blood vessel
Thrombophlebitis
Inflammation of the vein accompanied by a clot
Caused by trauma to the vessel wall
Hematoma
Pooling of blood under the skin
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN COLLECTION – ARTERIAL

PUNCTURE
 Blood is obtained from an artery
 Used in blood gas analysis and pH measurement
 Sample is collected without a tourniquet
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN COLLECTION – ARTERIAL

PUNCTURE
 Preferred site
Radial artery
Brachial artery
Femoral artery
Scalp artery
Umbilical artery
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN COLLECTION – ARTERIAL

PUNCTURE
 Allen’s Test
Test for patency of the ulnar and radial arteries
Done before performing an arterial puncture
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN COLLECTION – ARTERIAL

PUNCTURE
 Complications
Bleeding
Harder to control compared to venous bleeding
Apply pressure to the puncture site for 3-5 minutes
Thrombosis
Hemorrhage
Infection
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN COLLECTION – SKIN PUNCTURE

 Done by fingerstick technique
 Preferred for children
 Length of lancet : 1.75mm
 Length of incision : < 2 mm (children),
 Cut should be oriented across f in gerprints to
generate a large drop of blood
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

MAJOR CAUSES OF SAMPLE INTERFERENCE

1. Hemolysis
 Destruction of RBC
 Causes
oIn vitro
 Due to underlying disease
 Inherent to the patient
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

MAJOR CAUSES OF SAMPLE INTERFERENCE

oIn vivo
 May be due to RBC fragility, inappropriate venipuncture
technique or specimen contamination.
 Use of a small bore needle
 Pulling the plunger too fast
 Expressing the blood forcefully into the tube
 Vigorous shaking / mixing
 Alcohol contamination
 Bubble formation
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

MAJOR CAUSES OF SAMPLE INTERFERENCE

 Effects
oIncreased levels of intracellular analytes
oDilutional effect on the analytes present in the serum or
plasma
oInterference in color reactions
oInhibition of certain analytes
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

MAJOR CAUSES OF SAMPLE INTERFERENCE

2. Icterus
 Yellowish discoloration of the plasma / serum
 Inherent to the patient
 Caused by increased bilirubin levels (>20 mg/dl)
 Causes optical interference by its high absorbance
at 340 and 500nm
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

MAJOR CAUSES OF SAMPLE INTERFERENCE

3. Lipemia
 Caused by high triglyceride levels (>400 mg/dl)
 Causes turbidity of the serum which eventually
blocks transmission of light
 Can be cleared from the plasma by
ultracentrifugation.
 Inherent to the patient
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

MAJOR CAUSES OF SAMPLE INTERFERENCE

 Inhibits the following
Amylase
Urate
Urea
CK
 Bilirubin
Total protein
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN CONSIDERATIONS

TRANSPORT AND STORAGE
 Separate serum / plasma as soon as possible.
Prevents movement of water into the cells resulting to
hemoconcentration
Standing time
o Plain tube = 60 minutes
o Tubes with silica particles = 30 minutes
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN CONSIDERATIONS

TRANSPORT AND STORAGE
Tests unstable if not removed from clot after 30 min
Potassium
Phosphorus
Glucose
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN CONSIDERATIONS

TRANSPORT AND STORAGE
Tests unstable after 6 hours if serum was not separated
from the clot
Albumin
Bicarbonate
Chloride
C-peptide
HDL and LDL
 Iron
Total protein
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN CONSIDERATIONS

TRANSPORT AND STORAGE
Centrifugation
3000 RCF for 10 minutes
Excessive centrifugation
 Causes red cell lysis
 Elevates LD and K
Undercentrifugation
 Incomplete barrier formation
 Failure of the cellular elements to settle
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN CONSIDERATIONS

TRANSPORT AND STORAGE
 Serum or plasma must be stored at 4-6C if analysis
will be delayed longer than 4 hours.
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN CONSIDERATIONS

TRANSPORT AND STORAGE
 Specimens that require chilling during transport and
storage
Ammonia
Blood gases
Cathecolamines
Gastrin
Lactic acid
Renin
PTH
Pyruvate
Lactic acid
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN CONSIDERATIONS

TRANSPORT AND STORAGE
 Specimens that require protection from light
(photosensitive)
Bilirubin
Beta-carotene
Folate
Porphyrins
 Vitamin A and B6
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN OF CHOICE
 Lactic acid and glucose
S o d i u m f lu o r i d e a n d p o t a s s i u m o x a l a t e a s
anticoagulant
Lactic acid must be kept on ice and separated from
cells as soon as possible
 Acid phosphatase
Serum must be separated as soon as possible and
buffered with citric acid
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN OF CHOICE
 Ammonia
Heparinized plasma
Kept from ice
Separated from cells and perform immediately
 LD isoenzymes
Serum
Store at room temperature
Do not refrigerate / freeze
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

SPECIMEN OF CHOICE
 Ionized calcium
Heparinized whole blood
Must be collected anaerobically and kept on ice
 Blood gases
Heparinized whole blood
Drawn anaerobically and kept on ice
Evolve.
Adapt.
Overcome.
CEFI is now ready.

THANK YOU!!!
MLS 109: Clinical Chemistry 1

WILLIAM CHRISTOPHER C. SALAZAR, RMT

College of Medical Technology
Evolve.
Adapt.
Overcome.
CEFI is now ready.

THANK YOU!!!
MLS 109: Clinical Chemistry 1

WILLIAM CHRISTOPHER C. SALAZAR, RMT

College of Medical Technology