Clinical Chemistry 2.

Reference Materials
 A basic science that utilizes the specialty of chemistry to a) Primary standard
study human beings  Substance of exact known concentration
 An applied science when analyses are performed on body and purity
fluids or tissues for diagnosis or treatment of disease
b) Secondary standard
Malfunctions:  Substance of lower purity with
 Trauma or by invasive agents concentration determined by comparison
 Genetics deficiency of a vital enzyme with a 1 deg. Standard
 Insufficient supply of essential nutrients
 Insufficient blood and oxygen supply 3. Water Specifications
 Malignancy a) Distilled water
 Accumulation of waste products  Purified by distillation
 Defect in the cellular recognition of signals
b) Deionized water
 Water purified by ion exchange
Role of the Clinical Chemistry Laboratory
 Remove dissolved ionized solids and
 Measure chemical changes in the body for diagnosis,
gases
therapy and prognosis of disease
 Measure the concentration of a particular constituent (the c) Reverse Osmosis (RO) Water
analyte) in body fluids  Use pressure to force water through a semi
permeable membrane
Role of the MT
 Must understand the tools: d) Ultra filtration and nanofiltered water
 Equipment  Ultrafiltration and nanofiltered, UV
 Reagents oxidation, sterilization or ozone treatment
 Principle of the testing methods
 Knowledge of the medical uses of the determinations e) Reagent grade water
 Obtained by initial filter, followed by RO,
Basic Principles and Practices deionization and a 0.2 mm filter
o Type I water
A. Units of Measure  Trace metal analysis by AAS
 Quantitative measurement is expressed in defined  Gas, pH, enzyme and
units electrolyte analysis
 Components of quantitative laboratory results o Type II water
 Number  For analytical preparations
 Unit  Reagents, QC and standard
o Based on the SI system preparation
 Quantitative laboratory results o Type III/autoclave wash water
 Substance concentration  Glassware washing
o e.g: moles
 Derived units C. Clinical Laboratory Supplies
o e.g: mg/dL, g/dL, g/L, mEq/L, IU 1. Thermometers
 Analytical reactions occurs at an optimal
B. Reagents temperature (circulating water or
 Commercially prepared reagents heating/cooling metal blocks)
 In House prepared reagents
a) Liquid in glass
1. Chemicals  Colored liquid or mercury encased in
a) Analytical Grade (AR) plastic/glass material with a bulb at one
 Suitable for most analytical procedures end a graduated stem
 Carry designations as AR or ACS and For  Total Immersion
Laboratory Use or ACS standard-Grade  Partial Immersion
Reference materials  Surface Thermometer
b) Electronic thermometer / Thermistor probe
b) Ultrapure Reagent c) Digital thermometer
 Suitable for techniques that require
2. Glassware and Plasticware
extremely pure chemicals
Glassware
 AAS –
 Borosilicate (KImax/Pyrex)
 EIA –
 Aluminosilicate (Corex)
 MDx –
 High silica
 Carry designations of HPLC or
 Acid/alkali resistant (Vycor)
chromatographic
 Amber colored (Low actinic)
c) Chemically Pure (CP)  Lime soda (Flint glass)
 Impurity limitations are not stated Plasticware
 Preparation is not uniform  Polystyrene
 Not recommended for clinical laboratories  Polyethylene
 Polypropylene
d) United States Pharmacopeia (USP) and National
 Tygon
Formulary (NF) Grade
 Telon
 Used for manufacture drugs
 Based on the criterion of not being injurious a) Laboratory vessels
to man I. Class A volumetric flask
 Calibrated to hold one exact of
e) Technical or Commercial Grade liquid (TC)
 Used for manufacturing II. Erlenmeyer flasks and Griffin beaker
 Hold different volume
  Used in reagent preparation
III. Graduated cylinder
 Used to measure volumes of 3. Desiccators and desiccants
liquid  Uses hygroscopic substance that take up
water/moisture on expose to air
b) Pipets
 Glass or plastic utensils used to transfer 4. Balances
liquids  Must be level and vibration-free
 Classification  Avoid air currents
o Design (To contain/To deliver)  Kept clean
o Calibration marks or drainage (Blow
out/Self draining) I. Electronic Top-loading balance
o Type (Measuring or II. Analytical Balance
Graduated/Transfer)
 Top loading balance
 Design o For knowing the mass of substances
 To contain (TC) (greater quantity)
 Used for viscous samples o Used for preparative experiments
 Uses mercury as calibrating medium
 Proper use requires rinsing technique  Analytical balance
 To deliver (TD) o For preparation of primary standards
 Used for non-viscous samples o With sliding transparent doors
 Uses distilled water as calibrating medium o Measure exact mass but with lower
capacities (operating ranges 0.01 mg
 Calibration Marks or Draining characteristics to 160g)
 To Deliver (TD)
 Blowout pipet (Serologic, Ostwald Folin) D. Basic Separation Techniques
o With etched ring/two small continuous rings I. Centrifugation
 Self-draining (Mohr, volumetric)  A process in which a centrifugal force is used to
o Without marking, drains completely separate solid matter from a liquid suspension
o Floor model
 Type / According to Use o Bench top
I. Measuring or graduated pipets
 Graduated uniformly along its length  Consist of head/rotor (attached to the shaft of
 Designed to deliver any amount within its the motor), carrier and shields
capacity o Swinging Bucket / Swing type rotor
o Serologic pipet o Angled
 Has graduation marks to the tip
 Blow out pipet  The speed / centrifugal force is expresses by:
o Revolution per minute (RPM)
o Mohr pipet o Relative centrifugal force (RCF) or gravities (g)
 No graduation marks to the tip  Centrifuged must be properly balanced and free from
 Self draining excessive vibrations

 Correct operation of glass pipet II. Filtration

 Adjust the meniscus, read meniscus at  Paper, cellulose, polyester fibers & column
the bottom of the curved lgtiquid materials

 Type / According to Use E. Phlebotomy & Specimen Considerations

 Transfer pipet – delivers an exact volume
o Oswald – Folin pipet – for viscous Phlebotomy
fluids (blow out pipet)  The process of collecting blood “to cut a vein”
o Volumetric pipet – for aqueous  Two main phlebotomy procedures:
solutions (self draining)  Venipuncture
 Capillary puncture
Volumetric Pipet Ostwald-Folin Pipet
Size Larger Smaller 1. Professionalism
Location of the bulb Located at the center Located closer to the  Appearance, Attitude, Communication Skills, Bedside
(symmetrical) delivery tip Manner
Design To deliver To deliver and To 2. Patient Consent (implied consent)
contains 3. Legal Issues
4. Infection Control (PPE, hand hygiene, isolation)
 Type / According to Use
 Mechanical or Automatic I. THE VASCULAR SYSTEM
o Micropipet – deliver amount < 1ml  Network of arteries, veins and capillaries
o Macropipet – deliver amount > 1ml  Arteries and veins are comprised of three layers of
o Dilutor/dispenser tissue:
 Tunica intima – innermost, smooth layer
o Positive displacement
 Tunica media – middle, thickest layer
o Air displacement
 Tunica adventitia – outer covering
 Capillaries comprise only one layer of tissue
c) Burets
 For dispensing liquid during titration  Arterial Blood
 Has larger concentration of oxygen than carbon dioxide
d) Syringes
 Pumped by the heart to the body cells
 Used to transfer small volumes in blood
 Venous Blood
gas analysis, chromatography or
 Has a larger concentration of carbon dioxide
electrophoresis
 Pumped by the heart to the lungs
  21 gauge - standard for routine venipuncture

i. Arteries
 Elastic, muscular and thick walled Three Basic Methods
 Arterial blood is bright red (oxygenated blood) 1) Evacuated Tube System
ii. Veins a) Multisample needle
 Thinner walls b) Tube holder (barrel/adapter)
 Venous blood is dark red (deoxygenated blood) c) Evacuated tubes
iii. Capillaries
 Smallest blood vessels  Screw the needle into the tube adapter
 One cell thick to allow for gas and nutrient exchange  Insert the first tube to be collected into the tube adapter
 Push the tube up to the adapter guideline
II. PHLEBOTOMY SITES
 Median Cubital Vein 2) Needle and Syringe
 Cephalic Vein  Syringe needles
 Basilica Vein  Syringe
 Median Antebrachial Vein  Transfer device

The most commonly used veins for venipuncture are located in the 3) Winged Infusion Set (Butterfly)
antecubital fossa. a) Luer fitting for syringe
 Which vein is BEST for venipuncture? b) Luer adapter for ETS
 1st choice – median cubital vein
 2nd choice – cephalic vein  Used to collect blood from people with small, fragile veins,
 3rd choice – basilic vein such as the elderly and children
 Provides greater control with non-stable patients
When the antecubital veins are not accessible, the hand veins may
be used for venipuncture. COMMON STOPPER COLORS AND ADDITIVES
 Metacarpal plexus
 Dorsal venous arch  Tube Additives
 Hand veins are smaller and less anchored A. Anticoagulant
 This can be very painful for the patient  Light Blue (citrate)
 Green (heparin)
III. TYPES OF BLOOD SPECIMENS  Lavender (EDTA)
 Whole blood B. Antiglycolytic Agents
 Contains the liquid portion of the blood (plasma) and  Gray (sodium fluoride)
the cellular components C. Clot Activator
 Arterial  Red (silica)
 Oxygenated blood with bright red color  Orange (thrombin)
 Venous D. Thixotropic gel
 Deoxygenated blood with a dark red color  Gold (serum)
 Plasma  Light Green (plasma)
 The liquid portion of an unclotted or anticoagulated
blood V. ORDER OF DRAW
 Contains fibrinogen  Reduce the risk of specimen contamination by
 Serum microorganisms and additive carry-over
 The liquid portion of clotted blood
 Plasma minus the fibrinogen Order of Draw Tube Stopper Color
1. Blood culture tubes Yellow SPS
PLASMA – 55% of Total Blood Volume 2. Coagulation tubes Light blue
 91% Water 3. Glass non additive tube Red
 7% Blood proteins (fibrinogen, albumin, globulin) 4. Plastic color activator tube Red
 2% Nutrients (amino acids, sugars, lipids) Serum separator tubes (SST) Gold Plastic / Red-gray rubber
Hormones (erythropoietin, insulin, etc.) 5. Plasma separator tubes (PST) Light green / Green-gray rubber
Electrolytes (sodium, potassium, calcium, etc.) Heparin tubes Green
6. EDTA tubes Lavender / Pink
CELLULAR COMPONENTS – 45% of Total Blood Volume Plasma preparation tubes Pearl Top
 Buffy Coat Oxalate / fluoride tubes Gray
 White Blood Cells (7000-9000 per mm^3 of blood)
 Platelets (250,000 per mm^3 of blood) EVACUATED TUBES
 Red Blood Cells (RBCs)
 About 5,000,000 per mm^3 of blood Additive Department
Sodium Polyanethol Microbiology (Blood Culture)
IV. VENIPUNCTURE EQUIPMENT Yellow SPS Sulfonate (SPS)
1) Evacuated Tube System
2) Needle and Syringe Light blue Sodium Citrate Hematology (Coagulation –
3) Winged Infusion Set (Butterfly) PT (prothrombin time),PTT
(partial thromboplastin
i. Tourniquet time),APTT (activated partial
 Made of pliable rubber or a strip with Velcro thromboplastin time))
 Used to locate the patients vein Red / Red Glass Non additive chemistry, Blood Bank and
 Nonlatex strap Rubber tube Serology
 Velcro
 Applied to a patient’s arm during venipuncture Red clot activator tubes Chemistry
 Must not be left on longer than 1 minute
Gold Plastic / Serum Separator Chemistry
ii. Needle Red and Gray Tubes
 Size - gauge and bore are inversely related Tubes
 8. Prepare equipment and put on gloves

Additive Department
Light green Lithium heparin or Sodium Chemistry  Cleansing the Site
(Plasma barrier heparin with Gel Separator  Cleaning the site with an antiseptic (70% isopropyl
tubes) / Green- alcohol)
gray rubber  Cleanse the site using concentric circles
Green Lithium heparin or Sodium Chemistry  Allow alcohol to dry completely or use a gauze
heparin
 NEVER blow on the site
Lavender / Pink EDTA (Ethylenediamine Hematology (CBC)
tetraacetic acid) 9. Reapply tourniquet, uncap and inspect needle
Gray Sodium fluoride (with Chemistry (Blood 10. Ask patient to remake a fist, anchor vein & insert
potassium oxalate) glucose and alcohol) needle
Orange Thrombin Chemistry 11. Establish blood flow, release tourniquet and ask
Yellow ACD (acid citrate Blood Bank patient to open fist
dextrose) (maintain RBC’s 12. Fill, remove and mix tubes in order of draw
viability)
 Anchor the vein below the puncture site
 Insert the needle at a 15 deg to 30 deg angle
Order of Draw  Insert the evacuated tube and allow it to fill completely
 Sterile Specimens (Blood Culture Tubes)
 Coagulation Tubes (Light Blue) 13. Place gauze, withdraw needle, active safety
 Non additive (Red) features and apply pressure
 Light Green 14. Discard needle in holder units
 Green 15. Label tubes
 Lavender 16. Observe special handling instructions
 Gray 17. Check patients arm and apply bandage
 SST (Gold or Red) 18. Thank the patient, remove gloves and sanitize
hands
Apply Your Knowledge 19. Dispose used materials
 Which tube system is the most widely used for blood 20. Transport specimen to the lab
collection?
 Evacuated tube system Apply Your Knowledge
 A lavender-topped tube is primarily used for what types of  At what angle should the needle be inserted in performing a
studies? venipuncture
 Hematology studies (CBC and differential)  15 deg to 30 deg angle
 Which color tube is used for glucose analysis and blood
alcohol levels? Must know
 Gray-topped tubes  A tourniquet is place 3-4 inches above the antecubital are
 After removal from the holder, additive tubes must be
VI. VENIPUNCTURE PROCEDURES inverted gently 3-8 times
 Supplies for venipuncture  Tourniquet must not be left on the arm longer than 1 minute

 Routine ETS Venipuncture Trouble Shooting

1. Review Test Request 1. Tube position
 Manual requisition 2. Lost
 Computer requisition printing at a terminal in the 3. Bevel against the vein wall
laboratory 4. Needle to deep
 Patient’s name, DOB, medical record 5. Needle not deep enough
number 6. Needle beside the vein
 Ordering physician’s name 7. Collapsed vein
 Date and time test is to be done 8. Undetermined needle position
 Type of test ordered
 Test status (timed, fasting, STAT) VII. PEDIATRIC VENIPUNCTURE
 Patient’s location (if inpatient) A. Interacting with a child
 Initials of phlebotomist B. Immobilizing a child
2. Approach Identify and prepare patient’s a) Wrapped in a blanket
 Determine the correct spelling of the b) Restained while sitting on parent’s lap
patients last name C. Pediatric venipuncture
 Requisition form a) 23 gauge butterfly and tube holder
 ID band b) Use of microtubes
 Specimen label
 Example of warning sign is no blood VIII. GERIATRIC VENIPUNCTURE
pressure or venipuncture in the right arm A. Challenges
3. Verify Diet Restrictions and Latex Sensitivity i. Arthritis
4. Sanitize hands ii. Coagulation problems
5. Position Patient, apply tourniquet and ask patient to iii. Hearing loss
make fist iv. Skin and veins are less elastic
6. Select vein, release tourniquet and ask to open fist v. Alzheimers, cataracts, Parkinsons and stroke

 Selecting the venipuncture site PREANALYTIC CONSIDERATIONS

 Always examine the antecubital area first
 Ask the patient to hold the arm still and make a fist  Problem Sites
 Palpate the vein using the tip of your index finger  Burns, Scars and Tattoos
 Select a vein that is large and does not roll  Damages Veins
 Edema
7. Clean and air dry site  Hematoma
  Mastectomy  Chilled (using crushed ice)
 Wrapped on foil
o E.g. bilirubin decrease by 50% after 1
hour of light exposure
 Certain specimens must remain cool, so they are
 Vascular Access Devices placed in an ice-water mixture
 Heparin or saline lock  Specimens to be kept warm and protected from
 Intravenous line light can be wrapped in aluminum foil
 Central vascular access device (CVAD)
 Arterial line 5. Specimen Suitability
 Arteriovenous (AV) shunt or fistula i. Hemolyzed
ii. Collection in the wrong tube
 Procedural Error Risk iii. Failure to follow timing and handling
 Hematoma requirements
iv. Quantity not sufficient (QNS)
 Iatrogenic anemia
v. Clotting in WB or plasma specimen
 Inadvertent arterial puncture
 Infection of the site 6. Centrifugation
 Nerve injury i. Specimens must be completely clotted (30-
 Reflux 60 minutes at room temperature) before
 Vein Damage centrifugation
ii. Visually check
 Patients Conditions and Complication a) Lipemic
 Allergies to supplies and equipment b) Hemolyzed
 Excessive bleeding c) Icteric
 Fainting (syncope)
 Nausea or vomiting 7. Stopper Removal
 Obese patients  Stopper removal devices
 Pain  Face shield
 Petechiae  Splash shield
 Seizures / Convulsion
8. Aliquot Preparation
Capillary Specimen Collection  For multiple tests in a single specimen
 The blood is collected from a skin puncture made with a  Stored at 4 deg C – 20 deg C for 8 hours
lancet or similar device
 Collect site Apply Your Knowledge
 Outer area of the bottom of the foot  Where should the needle and adapter be discarded after a
 The fleshy part of the last phalanx of the third / fourth venipuncture procedure?
finger  Biohazard container
 Fleshy portion of the earlobe
 Material: UNACCEPTABLE BLOOD SPECIMENS
1. Alcohol, gauze, bandages a) Lipemic
2. Lancets  A cloudy turbid appearance, presence of lipid,
3. Warming devices (not to exceed 42 deg C) indicates a non-fasting specimen
4. Microcollection tubes  Interferes with colorimetric analysis in chemistry
5. Microhematocrit tubes b) Hemolyzed
6. Sealants  Destruction of RBC results in plasma or serum
7. Capillary order of tubes appearing red to pink due to hemoglobin release
 EDTA specimens  Affects potassium and enzyme testing
 Other additive specimen c) Icteric
 Serum  Specimen with a yellowish appearance due to
8. Indication for capillary puncture increased bilirubin content
 No accessible veins
 Veins are fragile
 Thrombotic tendencies Question and Answer
 POCT (glucose monitoring)
 Small blood volume 1. This is a type of pipette that contains double rings on the upper
 Prevent injury by restraining end and should be blown out using a rubber bulb (to contain
 New born screening pipette)
2. Lab glassware that is used to dispense a specific volume of
Neonatal Screening liquid. There are usually volume marking along the side and a
 Collected by heel puncture and placed within printed circles stop cock to control the flow (buret/burette)
on filter paper 3. This refers to laboratory glassware that is intended to measure
 First drop of blood is wiped away a specific volume with a high level of accuracy. They
considered precision glassware and typical sizes may include
IX. SPECIMEN PROCESSING 100ml, 250ml, 500ml, 1000ml (volumetric flask)
 Routine handling
1. Mixing tubes True or False
 Mix tubes by inverting 3 to 8 times
2. Transporting specimens 1. Safety begins with the recognition of hazards and is
 Plastic bag with a biohazard logo, liquid tight achieved through the application of common sense, a safety-
closure and slip pocket for paper work focused attitude and good housekeeping (True)
3. Delivery time limits 2. Agencies that have regulations that affects laboratories are
 45 mins of collection Department of Transportation, Medical waste tracking Act
 Centrifuged within 1 hour or up to 2 hours and Nuclear Regulatory commission (True)
o Minimize glycolysis (glucose are 3. Ecological solid waste management of 2000 is Republic Act
lowered by 200 mg/ml per hour) 6969 (False RA 9003)
4. Special handling 4. Philippine Clean Air Act of 1999 id Republic Act 9275 (False
 Maintained at 37 deg C (heat block) RA 8749)
5. Contact lenses should be encouraged inside the laboratory
(False)
6. Biosafety cabinet are used when chemical reagents may
produce hazardous flame (True)
7. Biological safety cabinets contains HEPA filters (high
efficiency particulate air) (True)
8. There are two classes of biological safety cabinet (False)
9. Any blood, body fluid, or any other potentially infectious
material spill must be cleaned up and the area or equipment
disinfected immediately (True)
10. The disinfectant in the laboratory should be a 10% bleach
solution, which is made fresh every 24 hours (True)