UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY & MOLECULAR DIAGNOSTICS | PHLEBOTOMY

PATIENT IDENTIFICATION

 Use two to three identifiers

 Name, medical record number, date of birth
 Special Situations such as in paternity testing and medicolegal procedures – chain of custody for
the specimen
 Active process
 Patient stating his/her name
 Phlebotomist will verify information via wrist band of patient or through the test requisition form
or computer order
 In case of pediatric patients – parent/guardian should be present and be asked to actively
provide the information
 Submitted samples must be labelled properly by the patinet himself.

BLOOD SPECIMEN

 Types of Blood Sample

 Venous Blood – from vein; usually the specimen of choice
 70 mg/L (0.39 mmol/L) less glucose than capillary blood [about 1.4%)
 0.9% less potassium than capillary blood
 PCO2 is 6-7 mmHg (0.9 kPa) greater than capillary blood.
 Higher levels of bilirubin (5%), Calcium (4.6%), Chloride (1.8%), Sodium (2.3%), and
Total Protein (3.3%) compared to capillary blood content.
 Purplish red
 Arterial Blood
 Bright red
 Capillary Blood
 Mixture of arterial and venous blood
 Plasma
 From anticoagualted blood
 Higher LDH and Total Protein compared to serum values.
 Lower glucose (5.1%), Phosphorous (7.0%), and potassium (8.4%) compared to serum
values.
 Bilirubin, cholesterol, and creatinine values have no significant difference whether from
serum or plasma.
 If used in electrophoresis of proteins, extra band in B region will appear owing to
fibrinogen.
 Serum
 From clotted blood; lacks fibrinogen group

Capillary > Venous Capillary = Venous Capaillary < Venous

Bilirubin 5.0%
Calcium 4.6%
Glucose 1.4% Phosphate
TP 3.3%
Potassium 0.9% Urea
Sodium 2.3%
Chloride 1.8%

Serum > Plasma Serum = Plasma Serum < Plasma

Albumin
ALP
AST
Bicarbonate
Calcium
CK Bilirubin
Chloride
Glucose Cholesterol
LD
Phosphorus Creatinine
Total Protein
Potassium
Sodium
Urea
Uric Acid

 Blood Colelction Tubes, Additives, and Preservatives

 Heparin (Green Tube)
 Most widely used anticoagulant for chemistry testing
 Prevents coagulation by accelarating the action of antithrombin II which neutralizes
thrombin
 Naturally occurring anticoagulant
 Sodium Heparin – used in toxicology, lead and trace elements analysis
 Lithium Heparin – for electrolytes; incompatible with folate and lithium assay

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 UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY
CLINICAL CHEMISTRY & MOLECULAR DIAGNOSTICS | PHLEBOTOMY
 Ammonium Heparin
 Ethylenediaminetetraacetic acid (Lavender Tube)
 Commonly used in Hematology testing
 Chelates divalent atoms such as Calcium and Magnesium
 Used in measurement of intracellualr drugs (immunosuppresants), HbA1c analysis,
isolation of genomic DNA, and in qualitative and quantitative virus determination by
molecular techniques
 Inhibits Creatine Kinase and Alkaline Phosphatase
 Lavender-top tube EDTA
• K3EDTA – liquid; will dilute sample by 1-2%
• K2EDTA – spray dried on tube walls
 Pink-top tube EDTA
• Spray dried K2EDTA
• Used in immunohematology for ABO grouping, Rh typing, and antibody screening
 White-top tube EDTA
• EDTA and gel
• Often used for molecular diagnostic testing of plasma
 Sodium Fluoride with Potassium Oxalate (Gray Tube)
 NaF is a weak anticoagulant and is mainly a preservative for glucose and lactate. The
addition of Potassium Oxalate compensates for the anticogulant activity.
 Lactate and Glucose analysis
 2.5 g/L concentration (2.5 mg per mL of blood)
 Effect is not immediate, glycolysis continues for the first one hour after collection.
• Normally, glucose declines in serum samples at a rate of 70-100 mg/L (0.56
mmol/L) per hour at 25 degrees Celsius.
 Prevents glycolytic enzymes for up to 72 hours
 Oxalates
 Forms insoluble complexes with Calcium to prevent coagulation
 Sodium Citrate
 For coagualtion studies – Light Blue Tube (9 parts blood : 1 part citrate)
• Used in hematology when platelet sattelism is suspected
 0.105 M (3.2%) or 0.129 (3.8%) Sodium Citrate
 Proper ratio must be strictly observed because anticogualant activity will be reversed by
the calcium reagent in coagualtion assays and cause falsely elevated clotting times.
• Similar effect produced by polycythemia = falsely elevated results due to small
plasma content.
 Inhibits aminotransferases and ALP
 ACP will be stimulated when the substrate used is phenylphosphate
 For westergren ESR – Black Tube ; 4 parts blood : 1 part buffered sodium citrate
 Sodium Iodoacetate or Lithium Iodoacetate
 0.5 g/L concentration (0.5 mg per mL of bood)
 Inhibition of glycolysis by acting on glutaraldehyde-3-phosphate dehydrogenase
 Incorporated with Heparin
 Other formulations : Sodium Monoiodine acetate
 Acid-Citrate Dextrose
 For cellular preservations
 Employed in molecular diagnostics and cytogenetics
 ACD A
 ACD B
 Red Tubes
 Glass Red Tubes do not contain anything
 Plastic Red Tubes contain clot activators
 Serum Separator Tubes (Golden Yellow)
 Contains thixotropic gel that separates red cells from serum
 Serum / Plasma Separation
 Preferably done within 1 hour
 When separating serum make sure that sample has clotted first.
 Separate via centrifugation. (3000 RCF for 10 mins)
 Purpose
 Prevent glycolysis
 Prevent fluid exchange or movement into the cells
 Prevent electrolyte shift
 Prevent hemolysis

VENIPUNCTURE

 Equipments

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 UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY
CLINICAL CHEMISTRY & MOLECULAR DIAGNOSTICS | PHLEBOTOMY
 Antiseptic Cleaning Solution
 70% Alcohol
 Benzalkonium chloride solution
 2% Iodine / Iodophor
 10% Povidone-Iodine
 Chlorhexidine Glucuronate – recommended for infants 2 months and above and patients
with iodine sensitivity.
 Bandages and Gauze Pads
 Collection Tubes
 Gloves
 Markers
 Needle Disposal Containers
 Needle Holders
 Needles
 Most commonly used sizes for adults are 19-22 gauge.
 Usually gauge 20 for adults with normal-sized veins
 In pediatric patients gauge 23-25 needles are used.
 Gauge 23 preferred for pediatric patients.
 The larger the gauge number, the smaller the bore.
 1.5 inches long (3.7 cm)
 Syringes with transfer device
 Tourniquets
 Winged infusion sets
 1 inch needle (2.5 cm) length
 Locating Veins
 Apply the tourniquet 3 to 4 inches above the intended puncture site. (Venous Occlusion)
 Some apply the torniquet 4-6 inches (10-15 cm) above the puncture site.
 When a blood cuff is used it is usually inflated to approximately 60 mmHg (8.0 kPa).
 If dorsal vein is being accessed, no tourniquet is used.
 Do not apply tourniquet over an open sore.
 The torniquet should not be left on longer than 1 minute (slight changes in blood composition).
Marked changes have been observed if tourniquet is applied for 3 mins.
 Hemoconcentration – increase in ratio of formed elements to plasma caused by leaving
the tourniquet on too long or too tight which may alter some test results.
 Hemolysis can occur if the tourniquet is too tight or is left on too long
 Petechiae – small red spots on the skin as a result of bleeding
 Sites
 Antecubital Fossa Area
• Median Cubital Vein – preferred site
• Cephalic Vein – same side of the arm as the thumb
• Basilic Vein – same side of the arm as the pinky finger
 Wrist and Hand Veins
• Use of Winged infusion set is recommended but the draw is slower and there is
increased risk of hemolysis.
 Palpate Veins using index finger.
 Disinfecting the site
 Use 70% alcohol. Clean site in a circular motion going outwards.
 If drawig for blood culutre clean area with 70% alcohol followed by 2% iodine or an iodophor /
10% povidone-iodine.
 Alcohol must be air-dried before applying iodine or iodophor (about 30 seconds)
 Iodine or iodophor contact time must be 60 seconds / 1 minute to be effective
 Timing
 Important for blood constituents that undergo marked diurnal variation, those that require
fasting, and those used to monitor drug therapy.
 Important as well in relation to specimens for alcohol or drug measurements in association with
medicolegal considerations.
 Blood Collection
 Puncture the site with the needle (beveled up) angled at 15 degrees.
 Proper Order of Draw must be observed if collecting multiple samples.
 Yellow for Blood Culture – 8 inversions
 Royal Blue without additive
 Clear without additive; discard if no royal blue is used
 Light blue with Sodium citrate for coagualtion – 3-4 inversions
 Gold/Red Serum Sepator Tube – 5 inversions
 Red Serum Tube with/without clot activator – 5 inversions
 Green Heparin Tube – 8 inversions
 Tan (glass) with Sodium heparin – 8 inversions
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 UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY
CLINICAL CHEMISTRY & MOLECULAR DIAGNOSTICS | PHLEBOTOMY
 Royal Blue with Sodium Heparin/Sodium EDTA – 8 inversions
 Lavender/Pearl/Pink with EDTA for CBC or Moelcular Studies – 8 inversions
 Gray with Sodium Fluoride/Potassium Oxalate – 8 inversions
 Yellow Glass with ACD for Molecualr Studies and Vell culture – 8 inversions
 Usual Order of Draw
Yellow for blood culture  Red (glass)  Light Blue  Red Plastic / Yellow SST  Green 
Lavender  Gray  Black for ESR  ACD
 Post Venipuncture Care
 Apply pressure on the site of puncture for 1 minute. If patient is taking blood thinner apply
pressure for 10 minutes.
 Complications
 Syncope
 Ecchymosis
 Thrombosis
 Thrombophlebitis
 Hematoma

PREANALYTICAL CONSIDERATIONS

 Composition of first drawn blood is most representative of the composition of circulating blood and the
least affected by fluid shifts where protein-bound components and other large molecules will be
concentrated.
 First-drawn specimen should be used for calcium analysis and other analytes that are both protein bound
and pertinent to critical medical decisions.
 Pumping of fist before venipuncture must be avoided
• Increases Potassium, Phosphate and Lactate
• Lactate elevations would cause pH to decrease causing the Calcium ions to increase.
 Stress increases cortisol, thyroid-stimulating hormone, and growth hormone.
• In children, may also cause increased plasma glucose concentrations due to adrenal stimulation
and increased activity of serum enzymes of skeleatal muscle origin for chidlren held in physical
restraint.
 Posture
• Upright position or supine
• Patient must be seated or in supine for 15 minutes before collecting blood
• Supine to Sitting/Standing  vasoconstriction  High Albumin, Enzymes, Calcium
• Sitting to Supine  Water moves into tissues  High CHON, Lipids, BUN, Iron, Calcium
 Diet and Lifestyle
• Fasting – for glucose and triglycerides, as well as for GTT, Insulin, Gastrin, Growth Hormone and
IGF-3
 8 to 12 hours
 48 hrs fasting increases bilirubin
• Caffeine intake – increases glucose levels
• Protein intake – increases BUN and urinary ketones
• Alcohol intake – increases Urate, lactate, TAG, and GGT
 Chronic Alcoholism – hypoglycemia
 Diurnal Variations
• Morning Peaks
 Cortisol (8 am)
 Prolactin (4 am to 8 am) also at 8 pm to 10 pm
 Iron
• Afternoon Peaks
 GH
 ALP
• Lower at Night
 ACTH
 Renin
 Aldosterone
 Insulin
 Cortisol (very low at 8 pm to 12 am)
 Hemolysis
• Interference in enzyme and electrolyte analysis
• Evident hemolysis if hemoglobin in plasma or serum reached 50 mg/dL
 Icteresia
• Dark yellow to orange serum samples accounting to hyperbilirubinemia (evident at concentration
of 25 mg/L)
• Interference in Total Protein, Albumin, Cholesterol, and Glucose
• Spectral Interference at wavelengths between 340 nm to 500 nm

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 UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY
CLINICAL CHEMISTRY & MOLECULAR DIAGNOSTICS | PHLEBOTOMY
 Lipemia
• Milky turbid samples accounting to hypertriglyceridemia (evident at concentrations exceeding 4.6
mmol/L or 400 mg/dL)
• Potentially cleared via ultracentrifugation
• Corrective measures : Blanking Technique or Bichromatic Analysis
 Photosensitivity
• Bilirubin, B-carotene, Folate, Porphyrins, Vitamin A and B6
 Cold Temperature Requirement (4 degrees Celsius)
• Ammonia
• Blood Gases
• Catecholamines
• Gastrin
• Lactic Acid
• Renin – Iced EDTA Tube
• PTH
• Pyruvate
 DO NOT REFRIGERATE
• Whole Blood Samples for Electrolyte Analysis  Hemolysis  Pseudohyperkalemia
• Samples for LDH  LDH 4 decreases
• Samples for ALP  increased activity
 Blood Gas Samples
• Left Uncapped  increased pO2, decreased CO2, increased pH
• Capped, prolonged standing  decreased pO2, increased CO2, decreased pH
 Before doing arterial puncture, patient must test positive in the modified allen procedure
 In capillary puncture, always wipe or discard the first drop of blood (contaminated with tissue fluids). The
order of draw for capillary collection is as follows
• Blood gases tube  EDTA tube  Heparin Tube  Other tubes with additives  Non-additive
tube
 Skin puncture depth must only be 2.0 mm to 2.5 mm deep.