 SPECIMEN COLLECTION AND PROCESSING

The first step in all chemical analysis is the collection of a specimen. It may be a blood specimen, urine, stool,
CSF, etc. Most of the analysis in clinical chemistry is done either on a whole blood, plasma or serum. Blood is by far the
most frequent body fluid used for analytical purposes.

 PHLEBOTOMY: A procedure wherein blood is collected from a blood vessel using a needle for diagnostic,
therapeutic, or blood donation purposes.

 PATIENT IDENTIFICATION
"Proper patient identification is the first step in sample collection” - this is the prime factor in order to attain
accurate results in the clinical laboratory. Likewise, proper techniques in specimen collection must be strictly followed
including the observance on the confidentiality of results.

 PATIENT IDENTIFICATION PROCEDURES:

1. Conscious Inpatients/Hospitalized patients

 Verbally ask their full names including middle names.
 Verify the name using the identification bracelet which includes first and last names, hospital/unit number,
room/bed number and physician's name.

2. Sleeping patients
 They are identified in the same manner as conscious in-patients.
 They must be awakened before blood collection.

3. Unconscious, Mentally Incompetent Patients

 They are identified by asking the attending nurse or relative; ID bracelet.

4. Infants and Children

 A nurse or relative may identify the patient, or by means of an identification bracelet.

5. Outpatient/Ambulatory Patient
 Verbally ask their full names, address or birth date, and countercheck with driver's license, or ID card with photo.
 If the patient has identification card or bracelet, same manner as with hospitalized patients.

 3-Way ID
To avoid misidentification, a phlebotomist may require what is referred to as 3-Way ID, in which the patient is
identified by:
1. The patient's verbal ID statement
2. A check of the ID band
3. A visual comparison of the labeled specimen with the patient's ID band before leaving the bedside
Some facilities are also showing the labeled specimen to the patient to ensure accurate labelling of the tubes.

 PATIENT PREPARATION
Prior to blood collection, patients must be given correct instructions on how to prepare for each laboratory test.
Utmost care must be observed to minimize factors that may influence laboratory results.

 PRE-ANALYTICAL VARIABLES OR FACTORS CONTRIBUTING TO THE VARIATION OF RESULTS:

1. EXERCISE
  Physical activity can have different effects on analyte concentrations – volume shirts between the vascular and
interstitial compartments, volume loss by sweating and changes in hormone concentrations.
 Transient increased: lactate, fatty acid, ammonia
 Long-term increased (skeletal muscle enzymes): CPK, AST, LD and aldolase

 Increased in hormones such as prolactin and growth hormone, while decreased plasma levels are seen in follicule
stimulating hormone, luteinizing hormone, estrogen and testosterone.
 Vigorous hand exercise (fist clenching) increases potassiurn, lactate and phosphate.
 Elevated levels of proteins in urine (proteinuria) are observed.

2. FASTING
 Fasting requirement is between 8 to 12 hours.
 Fasting specimen: FBS, GTT, lipids, lipoproteins, gastrin and insulin .
 Fasting for 48 hours may increase serum bilirubin.
 Fasting for 72 hours may result to increase of plasma triglyceride in males while glucose decreases in healthy
women to 45 mg/dL.
 Basal state collection is early morning blood collection, 12 hours after the last ingestion of food.
 Basal state collection includes glucose, lipids, lipoproteins and electrolytes.
 Basic metabolic panel: glucose, BUN, creatinine, sodium, potassium, chloride, CO2 and calcium

3. DIET
 Metabolic products of food can increase in venous blood.
 High protein diet can increase plasma levels of urea and uric acid.
 Atkins diet (high protein-low carbo diet) greatly increased plasma urea and urine ketones.
 Glucose, lipids and catecholamines may show variation because of post-absorptive hormonal effects.
 Caffeine increases concentration of glucose through the release of catecholamines from the adrenal medulla and
brain tissue.
 Increased in obese persons: glucose, cortisol, TAG and LD

4. POSTURE OR POSITION
 Preferred position during phlebotomy: upright position or supine (lying)
 Recommendation: patient should be seated/supine for at least 15 minutes to 20 minutes before blood collection to
prevent hemodilution or hemoconcentration.
 Changing from a supine to sitting or standing position causes constriction of the blood vessels and reduction of
plasma volume: increased levels of albumin, enzymes and calcium
 Changing from sitting to supine causes shifting of water and electrolytes into tissue causing hemoconcentration:
increased levels of proteins, lipids, BUN, iron and calcium
 Changing from standing to supine causes extravascular water to transfer to the vascular system and dilutes
nondiffusable plasma constituents: decreased levels of cholesterol, triglyceride and lipoproteins
 Significant elevation of potassium potassium after 30 minutes of standing is due to the release from muscles.
 Prolonged bedrest results to decreased plasma albumin due to fluid retention.
 Renin plasma level is higher when standing than supine.
 Drugs bound to proteins are affected by postural changes.

5. TOURNIQUET APPLICATION
 One-minute application of tourniquet is recommended.
 Effects of prolonged tourniquet application: hemoconcentration (venous stasis) and anaerobiosis
  Increased levels due to prolonged tourniquet application: potassium, proteins (albumin), enzymes, lactate,
cholesterol, and ammonia
 The pressure from the tourniquet causes biological analytes to leak from the tissue cells into the blood.
 Prolonged use of a tourniquet with fist exercises can increase the serum potassium level byl mmol/L.
 For accurate measurement of lactate, tourniquet should not be applied, and the patient should not clench his fist
at the time of the blood draw.
 Tourniquet application and or muscular activity may decrease venous pO2 and pH.

6. TOBACCO SMOKING (NICOTINE)

 It can cause elevated hormone levels such as the catecholamines and cortisol. Increased levels: glucose, growth
hormone, cholesterol, triglyceride, ammonia, urea, lactate, insulin and urinary 5-HIAA
 Decreased plasma levels of vitamin B12 is also observed.

7. ALCOHOL INGESTION
 It can cause increased plasma levels of urate, lactate, triglyceride and gamma glutamyl transferase (GGT).
 It causes hypoglycemia among patients with chronic alcoholism.

8. STRESS
 Increased: catecholamines, cortisol, ACTH, prolactin, albumin, glucose and latate
 Total cholesterol is increased with mild stress, while HDL cholesterol declines by almost 15% (Dufour, 2003 cited
by McPherson and Pincus, 2017).

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.
 Hepatotoxic drugs can elevate liver function enzymes.
 Diuretics can cause decreased plasma sodium and potassium
 Opiates cause increases in liver and pancreatic enzymes.
 Analytic methods that are based on oxidation-reduction reactions may be influenced positively or negatively by
ingested substances such as ascorbic acid (vitamin C).

10. PHYSIOLOGIC VARIATION

 It refers to 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.
 Affected by age (increased levels): albumin, ALP, cholesterol and phosphorus
 Affected by gender (increased levels): Male - albumin, ALP, creatine, uric acid, choleste
Female - HDL, iron and cholesterol .
 Affected by recent food ingestion (increased levels): glucose, TAG, gastrin, free Ca+
(decreased levels): electrolytes (Cl-, K+, P+), ALP and AMS

 LABORATORY TESTS AFFECTED BY DIURNAL VARIATION

ANALYTE EFFECT
Cortisol Peaks 4am-6am; lowest 8pm-12am; 50% lower at 8pm than at 8am;
increased with stress
Adrenocorticotropic hormone (ACTH) Lower at night; increased with stress
Plasma renin activity Lower at night; higher standing than supine
Aldosterone Lower at night
Insulin Lower at night
Growth hormone Higher in afternoon and evening
Acid phosphatase (ACP) Higher in afternoon and evening
Thyroxine Increases with exercise
Prolactin Higher with stress; higher levels at 4am and 8am and at 8pm and 10pm
Iron Peaks early to late morning; decreases up Iron to 30% during the day
Calcium 4% decrease supine

 GENERAL METHODS OF BLOOD COLLECTION

A. SKIN PUNCTURE
 also known as capillary puncture or dermal puncture; it is used to collect microsamples of blood using lancets.
It is divided into two types:

1. FINGER STICK
 for blood collection from older children or adults including:
a. Burned or scarred patients
b. Patients receiving chemotherapy who require frequent tests and whose veins must be reserved for
therapy
c. Patients with thrombotic tendencies
d. Geriatric or other patients with very fragile veins
e. Patients with inaccessible veins
f. Obese patients
g. Apprehensive patients
h. Patients requiring home glucose monitoring and point-of-care tests
 Site selection: palmar surface of distal phalanx, ring or middle finger
 Procedure: massage finger from hand to fingertips (3x); warm with moistened towel if fingers are cold;
decontaminate; puncture site with lancet perpendicular to fingerprints

2. HEEL STICK
 performed when collecting blood from infants
 Site selection: most lateral or medial portion, plantar surface, big toe
 Procedure:
a. Apply warm moist towel on site;
b. Grasp infant’s foot - forefinger over foot arch, thumb below puncture site, remaining fingers on top of
foot;
c. Decontaminate with 70% alcohol;
d. Position lancet and puncture

B. VENIPUNCTURE
 most frequently performed procedure in phlebotomy, it is the act of obtaining a blood sample from a vein
using a needle attached to a syringe or a stoppered evacuated tube.
 The preferred site for venipuncture is the antecubital fossa located anterior and below the bend of the elbow.
Three major veins are located in this area and, in most patients, at least one of these veins can be easily
located:
1. MEDIAN CUBITAL VEIN: is the vein of choice because it is large and does not tend to move when the
needle is inserted. It is often closer to the surface of the skin, more isolated from underlying structures,
and the least painful to puncture as there are fewer nerve endings in this area.
 2. CEPHALIC VEIN: located on the thumb side of the arm is usually more difficult to locate, except possibly
in larger patients, and has more tendencies to move. The cephalic vein should be the second choice if the
median cubital is inaccessible in both arms.
3. BASILIC VEIN: located on the inner edge of the antecubital fossa near the median nerve and brachial
artery. The basilic vein is the least firmly anchored; therefore, it has a tendency to “roll” and hematoma
formation is more likely.
 Venipuncture can be done in 3 ways:
1. EVACUATED TUBE SYSTEM – a system that uses a:
a. multi-sample two-way needle
b. tube holder or adapter
c. evacuated tube
 more preferred than syringe system
2. SYRINGE SYSTEM – most commonly-used system; used for small, fragile and damaged veins (easily
collapsible)
3. BUTTERFLY INFUSION SYSTEM – used for pediatric patients, difficult veins are also for collections
requiring more than one syringe. Standard is gauge 23.
 In this procedure, the following equipment must be conveniently available in the collection area:
1. TOURNIQUET: used during venipuncture to make it easier to locate patients’ veins. They do this by
impeding venous but not arterial blood flow in the area just below where the tourniquet is applied. The
distended vein then becomes more visible or palpable.
2. NEEDLES: All needles used in venipuncture are sterile, disposable, and are used only once. Needle size
varies by both length and gauge (diameter). For routine venipuncture, 1-inch and 1.5-inch lengths are
used. Gauge and bore size are inversely proportional.
3. COLOR-CODED EVACUATED TUBES

C. ARTERIAL PUNCTURE
 A process by which blood is obtained from a patient's artery
 Arterial blood is the oxygenated blood with a bright red color.
 Use: for blood gas analysis and pH measurement
 Sites: radial artery, brachial artery, femoral artery, scalp artery and umbilical artery
 Blood sample is collected without a tourniquet.
 Before blood is collected from the radial artery, modified Allen test should determine whether the ulnar
artery can provide collateral circulation to the hand after the radial artery puncture.
 The femoral artery is relatively large and easy to puncture, but extra care must be given to older individuals
because the femoral artery can bleed more than the radial or brachial.
 Arterial bleeding is the hardest to control and usually requires special attention.
 Major complications: thrombosis, hemorrhage, and possible infection
 Unacceptable sites: irritated, edematous, near a wound, or in an area of an arteriovenous(AV) shunt or fistula

 ANTICOAGULANT ADDITIVES OF EVACUATED TUBES

1. OXALATE
 It combines with calcium to form an insoluble salt.
 it interferes with Na+, K+, and most BUN (urease) measurements.
 Concentration: 1-2 mg/mL of blood.

2. CITRATE
  It combines with calcium in a non-ionized form.
 Concentration: 3.2% or 3.8% (0.105 M or 0.129 M) in a ratio of 1 part to 9 parts of blood
 An insufficient blood volume (short draw) leads to falsely increase clotting time.

3. ETHYLENEDIAMINE TETRAACETIC ACID (EDTA)

 It combines with calcium in a process called chelation.
 Preparation: di-potassium (K2EDTA) in plastic tubes and tri-potassium (K2EDTA) in glass tubes.
 Concentration: 1-2 mg/mL of blood.
 Use: Carcinoembryonic antigen (CEA), TDM and lead
 K2EDTA is the spray-dried form while the K2EDTA is the liquid form.
 Excess EDTA, which results when tubes are under filled, can cause red blood cells to shrink and thus, change
blood count results.
 Blood specimens for nucleic acid testing used EDTA to inhibit enzymes that might lyze it.

4. FLOURIDE
 It forms weakly dissociated calcium components.
 It interferes with the measurements of Na+, K+, and BUN (urease method).
 Concentration: 10mg/ml of blood

5. HEPARIN (MUCOITIN POLYSULFURIC ACID)

 It acts as antithrombin and antithromboplastin; anti-Factor X; ideal universal anticoagulant.
 It accelerates the action of antithrombin III, neutralizing thrombin and preventing the formation of fibrin.
 Preparation: Sodium, lithium, potassium and ammonium salts.
 Concentration: 0.2 mg/mL of blood
 Use: Tests for K+, NH3, carboxy/methemoglobin, pH and blood gas and cytogenetic studies
 Lithium heparin: measurements of glucose, BUN, ionized calcium, electrolyte and creatinine
 Lithium heparin may be used for most chemistry tests.
 Sodium heparin is the injectable form used for anticoagulant therapy.
 Lithium and ammonium heparin are the preferred anticoagulants for microcollection tubes.
 Heparinized plasma is preferred over serum for potassium tests because when blood clots, potassium is released
from platelets into the serum and can false increase results.
 Heparin is not the choice for nucleic acid testing because it can be coextracted with DNA and inhibits DNA
polymerase in polymerase chain reaction.
 Hospitalized patients are likely to be receiving heparin (especially undercritical care), which can delay clotting in
blood collection tubes even with activators and lead to fibrin strands that can clog up aspiration probes on
instrumentation.

 TUBE COLOR OF EVACUATED TUBES

STOPPER COLOR ANTICOAGULANT/ ADDITIVE SPECIMEN TYPE/USE MECHANISM OF ACTION
Red (glass) None Serum/chemistry and N/A
serology
Red (plastic/Hemogard Clot activator Serum/chemistry and Silica clot activator
serology
Lavender (glass) K3EDTA in liquid form Whole blood/hematology Chelates (binds) calcium
Lavender (plastic) K2EDTA/spray-dried Whole blood/hematology Chelates (binds) calcium
Pink Spray-dried K2EDTA Whole blood/blood bank Chelates (binds) calcium
and molecular
 diagnostics

White EDTA and gel Plasma/molecular Chelates (binds) calcium

diagnostics
Light blue Sodium citrate Plasma/coagulation Chelates (binds calcium
Light blue Thrombin and soybean trypsin Plasma/coagulation Fibrin degradation products
Black Sodium citrate Plasma/sedimentation Chelates (binds) calcium
rates – hematology
Light green/black Lithium heparin and gel Plasma/chemistry Inhibits thrombin formation
Green Sodium heparin, lithium Plasma/chemistry Inhibits thrombin formation
heparin
Royal blue Sodium heparin, K2EDTA Plasma/chemistry and Heparin inhibits thrombin
toxicology formation; Na2EDTA binds
calcium
Gray sodium fluoride/potassium Plasma/glucose testing Inhibits glycolysis
oxalate
Yellow Sterile containing sodium Serum/microbiology Aids in bacterial recovery by
polyanetholesulfonate culture inhibiting complement,
phagocytes and certain
antibiotics
Yellow Acid citrate dextrose Plasma/blood bank, HLA WBC preservatives
phenotyping, and
paternity testing
Tan (glass) Sodium heparin Plasma/lead testing Inhibits thrombin formation
Tan (plastic) K2EDTA Plasma/lead testing Chelates (binds) calcium
Yellow/gray and orange Thrombin Serum/chemistry Clot activator
Red/gray and gold Clot activator separation gel Serum/chemistry Silica clot activator

 ORDER OF DRAW OF FILLING EVACUATED TUBES

PROCEDURE ORDER
Drawing evacuated tubes Sterile, red, light blue, green, lavender, gray
Filling evacuated tubes from syringe Sterile, light blue, lavender, green, gray, red
Filling microcontainers from finger-/heel-stick Lavender, green, red

 INTERFERING CONDITIONS IN THE MEASUREMENT OF ANALYTES

1. HEMOLYSIS
 Severe hemolysis causes a slight dilutional effect on the analytes present in serum or place.

2. ICTERIC SAMPLE
 Serum bilirubin reaching 25.2mg/L (430 mmol/L) which means icteric specimen, interferes with the measurement
of total protein, albumin, cholesterol and glucose.
 Bilirubin in a specimen is not readily removed and so may cause spectral interference through its high
absorbance at wavelengths between 340 and 500 nm.

3. LIPEMIA
 It occurs when serum triglyceride exceeds 4.6 mmol/L (400 mg/dL).
 It scatters light and eventually blocks transmission of light.
 It can potentially be cleared from a serum or plasma specimen by ultracentrifugation.
 Lipemia inhibits amylase, urate, urea, CK, bilirubin, and total protein.
 Corrective measures for artifactual absorbance (lipemia): blanking technique and dualwavelength reading
 STORAGE AND TRANSPORT OF SPECIMENS
 During storage (ambient temperature, refrigeration on freezing), the concentration of a blood constituent in the
specimen may change as a result of various processes, including adsorption to glass or plastic tubes, protein
denaturation, evaporation of volatile compounds, water movement into cells resulting in hemoconcentration of serum
and plasma, and continuing metabolic activities of leukocytes and erythrocytes.
 The ice crystals formed during storage cause disruptive effects to molecular structure particularly to large protein
molecules.
 Serum or plasma must be stored at 4°C to 6° C if analysis is to be delayed for longer than 4 hours.
 LDH 4 and 5 Isoenzymes (decrease) and alkaline phosphatase (increase) are affected by low temperature storage
prior to testing

 SPECIMEN CONSIDERATIONS
 Specimens that require chilling (4°C) during transport and storage of specimens: ammonia, blood gases,
cathecholamines, gastrin, lactic acid, renin, PTH and pyruvate
 Photosensitive analytes: bilirubin, beta-carotene, folate, porphyrins and vitamin A and B6
 Plasma may be used in medical emergencies because samples do not have to clot before centrifugation.
 Increased of substances (proteins and urea) in unseparated serum or plasma is also due to movement of water into
cells resulting to hemoconcentration.
 Rimming the tube should be avoided because it may cause hemolysis and aerosol infection.
 Normally platelets release potassium during clotting, so serum has a slightly higher value of potassium than plasma
from the same individual; this difference is accentuated when the platelet count is extremely elevated.
 Excessive centrifugation (>3000 RCF for tubes without gel separator) may cause cell lysis and slight elevation in LD
and potassium, however, insufficient centrifugation (<1000 RCF or < 10 minutes) may cause incomplete barrier
formation in gel tubes or cell contamination of the specimen.
 Whole blood or plasma transfusion may cause increase plasma proteins, bilirubin, LD and potassium but decrease
sodium and chloride.
 Electrolytes are affected by evaporation of specimen prior to testing.

 COLLECTION OF OTHER BODY FLUIDS

1. CEREBROSPINAL FLUID (CSF)
 Most common method of collection Lumbar puncture (between the third and fourth lumbar vertebrae, or between
the fourth and fifth lumbar vertebrae)
 Other methods of collection: cisternal puncture and lateral cervical puncture
 Purpose of collection: to establish a diagnosis of Infection (bacterial, fungal, mycobacterial, or amebic meningitis),
malignancy, subarachnoid hemorrhage, multiple sclerosis, or demyelinating disorders
 Required pressure before collection: between 90 and 180 mm Hg
 Volume that can be collected: 20 mL of CSF (not more than 2 mL can be removed when the pressure is greater
than 200 mm Hg)
 Purpose of the CSF Vials/Tubes: Tube #1 goes to chemistry for glucose and protein analysis, or to
immunology/serology; Tube #2 goes to microbiology for culture and Gram stain; Tube #3 goes to hematology for
cell counts (tube #3 is the least likely to be contaminated by a bloody tap at collection)
 Complications of Lumbar Tap: cerebellar tonsillar herniation in patients with elevated intracranial pressure,
asphyxiation in infants, paresthesia; headache; and, rarely, hematomas
2. URINE
 Random specimens may be collected at any time, but a first-morning-voided aliquot is optimal for constituent
concentration, as it is usually the most concentrated and has a lower pH caused by decreased respiration during
sleep.
 For 24-hour urine collection, the first morning specimen should be discarded, record the time, and collect the
succeeding voiding for the next 24 hours - overcollection occurs if the first morning specimen is included in this
routine.
 24-hour urine creatinine - to assess the completeness of the specimen
 Sodium fluoride can be added to 24-hour urine for glucose determinations to inhibit bacterial growth and cell
glycolysis.
 Pediatric collections require special attention to avoid stool contamination.

3. SYNOVIAL FLUID, PLEURAL FLUID, PERICARDIAL FLUID, AND PERITONEAL FLUID

 Synovial fluid is collected by arthrocentesis, an aspiration of the joint using a syringe, moistened with an
anticoagulant, usually 25 units of sodium heparin per mL of synovial fluid.
 Synovial fluid differs from the other serous fluids in that it contains hyaluronic acid (mucin) and may contain
crystals.
 Sodium heparin is the preferred anticoagulant for synovial fluid.
 Some hospitals transfer synovial fluid to aerobic and anaerobic blood culture bottles for microbiologic culture.
 Thoracentesis is a surgical procedure to drain fluid (effusions) from the thoracic cavity and is helpful in diagnosing
inflammation or neoplastic disease in the lung or pleura.
 Pericardiocentesis and peritoneocentesis refer to the collection of fluid from the pericardium (effusion) and the
peritoneal cavities (ascites), respectively. These cavities normally contain less than 50 mL of fluid.
 Pleural fluid is an ultrafiltrate of the blood plasma. It is formed continuously in the pleural cavity.

 COMMON ERRORS IN SPECIMEN COLLECTION

1. Misidentification of patient
2. Mislabeling of specimen
3. Short draws/wrong anticoagulant/blood ratio
4. Mixing problems/clots
5. Wrong tubes/wrong anticoagulant
6. Hemolysis/lipemia
7. Hemoconcentration from prolonged tourniquet time
8. Exposure to light/extreme temperatures
9. Improperly timed specimens/delayed delivery to laboratory
10. Processing errors: Incomplete centrifugation, incorrect log-in, improper storage

 REASONS FOR SPECIMEN REJECTION

1. Hemolysis/Lipemia
2. Clots in an anticoagulated tube
3. Nonfasting specimen (if required)
4. Wrong blood collection tube
5. Short draws
6. Improper transport (temperature)
7. Discrepancies between requisition and specimen label
8. Unlabeled or mislabeled specimen
9. Contaminated specimen/leaking container

 Douglas Wilfred N. Rimando, RMT, MSMT