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BLOOD SPECIMEN
Bilirubin 5.0%
Calcium 4.6%
Glucose 1.4% Phosphate
TP 3.3%
Potassium 0.9% Urea
Sodium 2.3%
Chloride 1.8%
Albumin
ALP
AST
Bicarbonate
Calcium
CK Bilirubin
Chloride
Glucose Cholesterol
LD
Phosphorus Creatinine
Total Protein
Potassium
Sodium
Urea
Uric Acid
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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
2
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
3
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
4
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.