Electroanalytical or Electrochemical

Separative Techniques
Techniques
CHROMATOGRAPHY
• measurement of:
• electric potential (millivolts), (potentiometry) eg. • chrom = color
blood ph, pco2 determinations • graph = to write
• electric current (amperes) (amperometry,
polarography, conductometry) eg. glucose and urea
• separation of components in a sample based on:
determinations o physical properties: molecular weight,
• electric charge (coulombs) (coulometry) eg. charge
chloridometer: cl- determinations o polarity
• Potentiometry = measurement of electric • mechanisms of separation:
potential (millivolts) produced by the reaction o adsorption
between ions and electrodes in the electrochemical o partition
cell (electrodes in solution) o size exclusion/stearic exclusion/gel
o for blood ph and pco2 determinations permeation/molecular seive
o ph 1= 59.16 mv
o 2 electrodes • Two phases:
1) stationary (support medium)
• 1.) reference/standard = fixed electric potential. o silica (acidic)
(eg. ag /agcl electrode, hg/hg2cl2 electrode or o alumina (basic)
saturated calomel electrode). o charcoal (neutral)
• 2.) indicator electrode = zero electric potential.
o paper (neutral)
(Produces electric potential after the entry of h+).
o gel (neutral)
POTENTIOMETRY 2) mobile (solvent)
o gas: hexane, octane, etc.
• also used in the determination of electrolytes: na+, o liquid: alcohol, ether, etc.
k+, cl, ca++ (ion selective electrode/ise) STATIONARY MOBILE TYPE OF
• na+ = borosilicate glass PHASE PHASE CHROMATOGRAPHY
• k+ = valinomycin gel
liquid liquid liquid-liquid/partition
• cl = polyvinyl chloride/plastic
• ca++ = glass Solid chrom/lc liquid solid-liquid/adsorption/
• reference electrodes: ag/agcl, hg/hg2cl2 liquid

AMPEROMETRY solid gas solid-gas/gas chrom/gc

• measurement of electric current (amperes)

produced by the reaction between ions and • specific types of chromatography:
electrodes in the electrochemical cell using a single • liquid: amino acids, fatty cids, bile acids
applied electric potential. • simple sugars, therapeutic drugs, lipoproteins,
• 2 electrodes vitamins, etc.
o Anode • high performance liquid
o Cathode chromatography/hplc
• amperemeter measures the electric current • column chromatography
produced. • gas: abused drugs, pesticides, insecticides, poisons
POLAROGRAPY
DETECTORS USED IN CHROMATOGRAPHY
• same with amperometry but uses an increasing electric • Thermal Conductivity Detector = measures heat
potential eg. glucose and po2 determinations. conduction.
• Flame Ionization Detector = light emitted by ions
CONDUCTOMETRY
exposed in flame.
• Electron Capture Detector = loss of cell current
• measurement of electric current (amperes)
when atoms capture low energy electrons.
produced by the reaction between ions and • Mass Spectrometer = mass and charge of fragmented
electrodes in the electrochemical cell (electrodes in ions.
solution) using non-polarized electrodes eg. urea other detectors used in chromatographyby
determination. • Amperometric Detector = electric current
produced by ions in the analytical cell (electrodes in
COULOMETRY solution).
• Coulometric Detector = charge produced by ions in
• measurement of electric charge (coulombs) eg. the analytical cell (electrodes in solution).
chloridometer (cl- determination).
 • Fluorometric/Spectrophotometric Detector = color 2.2 spectrophotometric = narrow
intensity produced by the reaction between analytes wavelenth eg. atomic absorption
and reagents. spectrophotometry (aas.)
• measurement of analytes.
• (ions, molecules, compounds) SPECTROPHOTOMETRY
• Densitometer • light source = provides radiant energy (light).
• light travels in a wave-like manner.
TYPES OF MASS SPECTROMETERS • wavelength is measured in nanometers/nm or
1) quadruple = 4 rods (passage of ions in parallel) millimicra/mu
2) ion trap = 4 rods in 3-d spheres • ultra violet/uv = <299nm
3) tandem = 3 quadruple in series • visible light = 300-699 nm
• separation of proteins and hemoglobin based on the • infra red/ir = > 700 nm
charge using external electrodes (anode and cathode) • tungsten filament lamp/incandescent lamp (360-950
types: nm)
o ionto = small ions • tungsten iodide lamp (220-360 nm)
o zone = medium ions • tungsten halide lamp (220-360 nm)
• support media • hydrogen discharge lamp (220-360 nm)
o planar (paper, agarose, dextran, • deuterium discharge lamp (220-360 nm)
polyacrylamide gel): uses densitometer • mercury vapor lamp (313-546 nm)
stained with fast red b, sudan black b, • monochromator /wavelength selector = isolates
amido black, ponceu s specific wavelength
o capillary: uses spectrophotometer. • interference filters = for efp
(Separates b1 and b2 globulin). • prism
• diffraction
ELECTROPHORESIS • grating
• used for the separation and quantitation of • entrance slit = focuses light on the monochromator
serum/plasma proteins & glycohemoglobin. • exit slit = determines the band width of light to be
dispersed in the spectrum
OTHER TYPES OF SEPARATIVE TECHNIQUES • cuvette/analytical cell/photo cell = holds the solution
• Isoelectric Focusing = modification of to be read.
electrophoresis which uses ph gradient for better • glass, quartz or plastic (automated).
separation of proteins. • galvanometer/read-out device = shows absorbance
• Electroendosmosis = modification of electrophoresis or transmittance.
which uses movement of buffer ions relative to the • detector = converts light to electricity.
fixed support.
• 2-d electrophoresis
BEER LAMBERT’S LAW
1) isoelectric point (+ &- charges)
• controls spectrophotometry.
2) molecular weight
• concentration = absorbance/optical density/od
• logarithm of percent transmittance
MASS SPECTROMETER
• a = abc or a = ebc or a = elc
• sample introduction in mass spectrometry:
• a= absorbance
1) Electron Ionization (EI) = filament with
• a/e = absorptivity
electric potential, gc.
• b/l = pathl ength of solution/surface of cuvette
2) Atmospheric Pressure Ionization (API)
• c = concentration
with high pressure, lc…
o Electrospray Ionization (ESI) = droplets
DETECTORS USED
o atmospheric pressure chemical ionization
(APCI) = vapor • BARRIER LAYER CELL/PHOTOTUBE
3) Matric Assisted Laser Desorption • light ----> cathode ----> electron
Ionization (MALDI) = with sample
preparation. amperes
4) Surface Enhanced Laser (galvanometer)
Desorption Ionization (SELDI) =
no sample

Colorimetry/Spectral Techniques
• measurement of color intensity produced by the
reaction between analytes in a sample and reagents
(chromogen/color developer)
1) visual = detected by the eyes, eg.
reagent strips for urinalysis.
2) photoelectric = detector converts light
to electricity
2.1 photometric = multiple wavelength
eg. emission flame photometry (efp)
 o parametric (bell-shaped curve)
o non-parametric (skewed to the right or to
the left)
• Based on quantity
o interval (numbers: eg. pt, aptt, etc.)
o ordinal/ranked: eg. rare, few,
moderate, abundant
o nominal/categorical: positive (+),
negative (0)
o statistical tools: chi-square (goodness
of fit, mac nemar test), anova, ancova,
fisher exact test

• measurement of reemitted light (fluorescence Descriptive Statistics of Single Observation

1) Frequency Distribution = the number of times a
number appears in a group of data.
2) Cumulative Frequency Distribution = summation of
frequencies
3) Tests for central tendencies
absorbed by the atoms in the sample). 3.1 mean/average = summation x/n
3.2 mode = the most frequently occurring value
TYPES OF FLUOROMETRY 3.3 median (p50) = the central value
1) Fluorescence Polarization (Rotation of Light) 4) Tests for dispersion (spread of data from its
2) Chemiluminescence (No Light Source) = for antibodies location)
(abs), antigens (ags), drugs & hormones (uses rhodamine- 4.1 standard deviation/sd/s= √ € (x-x)/n-1
b, fluorescein. avidin, biotin dyes) 4.2 coefficient of variation/cv = sd/mean x 100
3) Reflectometry/Reflectance Spectrophotometry = 4.3 standard error of the mean (standard
measurement of light reflected from the flat surface deviation of the mean) = s/ √ n
(slide) with colored product (analyte and reagent). no
4.4 mean absolute deviation (average deviation)
cuvette used (replaced by slide); used in vitros analyzer.
= € (x-x)/n
4) Turbidimetry = measurement of light blocked by
particles in solution Descriptive Statistics of Paired Observation
5) Nephelometry = measurement of light scattered at right 1) Linear Regression: if y = x, perfect agreement of
angles by particles in solution; for bacterial studies, results (relationship or association)
globulin determination – comparison of methods/com (2 methods
6) Refractometry = measurement of bent light which passes of the same test)
from one medium to another; for determination of 2) Correlation: r = correlation coefficient
specific gravity of urine. – (-1 to + 1) (strength of relationship)
7) Flow Cytometry = counting of cells in blood based on – if r = +1, increase of one test will cause an
the scattering of light as it strikes a blood cell. increase of another test (eg. high alt leads
8) Radioimmunoassay = measurement of radioactivity to high crp)
produced by radionuclide which reacts with analytes in – if r = -1, increase of one test will cause a
the sample. decrease of another test (eg. high alt leads
to low bun)
• pearson correlation: interval vs. interval (eg. protime
ENZYME LINKED IMMUNOSORBENT
vs. platelet count)
ASSAY/ELISA
1) non-competitive/sandwich assay • spearman correlation: interval vs. ordinal (eg.
o sample (ab) + ag = ab-enzyme) = ab- concentration of antibiotic vs. fungal growth)
• point biserial correlation: interval vs. nominal (eg.
ag-ab-enzyme + substrate + chromogen
= product: spectrophotometer concentration of tannin vs. bacterial growth)
2) competitive assay
o sample (ag) + ab + ab-enzyme = ag-ab- Inferential Statistics (Relationship of Variables)
enzyme + substrate = product: 1) T-test = statistical difference between 2 groups of
spectrophotometer. data.
2) F-test = statistical difference between the sd of 2
groups of data.
Method Evaluation 3) Analysis of variance/anova = multiple data either
nominal or interval.
Two Types of Statistics 4) Chi-test /chi-square = nominal/categorical data.
1) Descriptive = summarize important features of a:
Method Evaluation
1.1 single observation
1.2 paired observations • Ways of verifying the accuracy and reliability of
2) Inferential = compare the important features of two laboratory test results.
or more groups of data. • Regulatory agencies:
• American association for clinical chemistry/aacc
(guidelines and methods evaluation)
Types of Data • National academy of clinical biochemistry (guidelines
• Based on distribution and methods evaluation)
• College of american pathology/cap (accreditation of o Anova
laboratories) o Cv
• Clinical laboratory improvement amendment/clsi o Sd
(regulated by cms)
• Center for medicine, Medicaid services/cms Estimation of inaccuracy
(government agency for lab testing methods) 1) Recovery studies
• Food and drug administration/fda (lab instruments • comparison of analyte with sample matrix
and reagents) (spiked/modified) sample matrix without
• The joint commission/tjc formerly the joint analyte(unmodified)
commission on accreditation of healthcare
organizations/jcaho (performance of method
evaluation)
• Office of in vitro diagnostic device evaluation and
safety/oivd (regulates diagnostic tests)
• Office of in vitro diagnostic device evaluation and
safety/oivd
• Categorizes lab tests into:
o Waived (simple and cleared by fda)
Eg. Dipstix for urinalysis, glucometer
o Moderate complexity/nonwaived 2) Interference study (measurement of compounds
eg. Automated methods which affect the results)
o High complexity/nonwaived • Hemoglobin (hemolysis), bilirubin (icteric
eg. Manual methods sample), turbidity (lipemic sample),
• Non-waived fda approved tests anticoagulants (inhibit enzymes)
• Demonstrate test performance comparable to that
established by the manufacturer
• Must have:
o Accuracy (closeness of results to the true
value)
o Precision (agreement of results)
o Reportable range (test results)
• Verify reference values appropriate for patient population
• Non-waived fda approved (modified or developed by
the laboratory)
• Performance standards for common laboratory
• Determine: tests (allowable % error by clinical laboratory
o Accuracy improvement amendment/clia)
o Precision o Total calcium = +/- 1 mg/dl
o Analytic sensitivity (ability of the method to o Chloride =+/- 5%
detect small quantities of the analyte) o Total cholesterol =+/-10%
o Analytic specificity (ability of the method to o Hdl = +/-30%
detect the desired analyte in the sample) o Glucose = +/- 6-10%
• Determine: o Potassium = +/-.5 mmol/l
o Reportable range o Sodium = +/-4 mmol/l
o Reference range o Total protein = +/- 10%
o Other performance characteristics o Triglyceride = +/-25%
o Calibration and control procedures o Bun = +/- 2-9%
o Method selection (costs, quality results, o Uric acid = +/-17%
client satisfaction, efficiency) 3) Comparison of method studies
o T-test and linear regression analysis
• Method selection (costs, quality results, client o Test method is compared with the
satisfaction, efficiency) reference method (gold standard method)
o Eg. Hexokinase (glucose), creatininase
(creatinine),
• Method validation (using gold standard/reference o 40-100 specimens in 8-20 days (within 4
methods) hours)
• Eg. Hexokinase (glucose), creatininase (creatinine), o 4-5 patients in 8 days
Uricase (uric acid) o Plotted in the y-axis (reference) and x-axis
(test)

QUALITY ASSURANCE
• A broad spectrum of plans, policies and procedures
that together provide an administrative structure for
laboratory’s effort to achieve quality goals.
• Measurement of imprecision • Qa has 3 elements:
• Repeated analysis study o Commitment
• 2 controls/day for 10-20 days o Facilities and resources
• Tested by: o Technical competence
 o Standard/calibrator = contains
QUALITY CONTROL/QC known amount of the analyte
• Techniques and procedures of systematic o Test = contains the desired analyte
monitoring of analytical process to detect analytical to be measured
errors
• Errors occur before, during and after analysis
o Blank = sets spectro at o absorbance
• Also checks reagent quality and machine stability or 100 % transmittance
o Control = commercial or non-
Types of errors commercial sera used to monitor
1) Systematic errors
• One direction of the graph/histogram o (non-commercial sera or pooled sera must be
• Errors due to the quality of reagents and/or stability clear, non-icteric, non-lipemic, non-
of the machine hemolyzed)
1.1 Constant = the same direction 3) Post-analytical
1.2 Proportional = magnitude changes with the o Data from patients (clinical correlation)
analyte delta checks (previous)
2) Random = varies with samples, instrument, limit checks (reference values)
temperature, reagent, handling, operator. test duplication
3) Total = random plus systematic (replication and 4) Qc charts/histograms
comparison). 4.1 bell-shaped curve
4.2 cusum graph
3 stages of qc 4.3 sheuhart levey-jennings chart/levey-jennings
1) Establishing allowable limits of variation for each control chart/jennings chart/ dot chart/sl/j chart
analytic method
How to make and use sl/j chart…
2) Using the limits as criteria for evaluating the data
• Gather all control values of all tests (20-31 days).
3) Taking action to remedy errors
o Cause • Determine the mean or average
o Action • Determine the standard deviation
o Control and patient’s data • Determine the confidence limits
o +/- 1sd (99%), +/-2sd (95%), +/-3sd
Indications of quality control (the68%)
1) Accuracy (tested by recovery and interference • Plot control values on the chart
studies) • Interpret the chart
2) Precision: cv = sd/mean x 100%
3) Sensitivity = tp / tp + fn x 100%
4) Specificity = tn /tn + fp x 100%
5) Reliability = accuracy and precision

Sl/j chart interpretation

• Sl/j rules
• In control:
o All control values fall within the confidence
limit (+/-2sd = 95%)
o 1 outlier in 20 days (value above or below
the mean)
• Not in control:
o Outliers in 20 days
o Trend: continuous increase or decrease of
6 consecutive values (which cross the
mean).
o Shift: 6 consecutive values which are
Variables of QA and QC below or under the mean (without
1) Pre-analytical crossing the mean).
o Patient id and preparation
o Specimen collection, labelling, handling, External quality control/external quality assurance
transport, and processing system/equas
o Turnaround time • Cap
• Clia
o Test utilization • Cdc
2) Analytical (technical competence, inventory and • Tjc
input control of equipment and materials) • Api/american proficiency institute
 Ist/international serolimos proficiency testing
•
scheme
Phlebotomy
• Pcqacl /phil council for quality assurance in clinical
laboratories • To cut a vein
• Ritm/research institute for tropical medicine (bacte) • Types:
• Nkti/national kidney and transplant institute (hema) • Venipuncture (venous blood) arterial
• Lung center of the philippines (chem) puncture (arterial blood),
Proficiency test • Capillary/arteriolar puncture
• Comparison of results of a single test in several (capillary/arteriolar blood)
laboratories 1) Venous blood (dark red, unoxygenated)
• Eg. Human chorionic gonadotropin • Blood chemistry
• Cap value: 6.4% 2) Arterial and capillary blood (bright red, oxygenated)
• Sd = 4.8% • Blood gases, ph, hematology
• Evaluation criteria = 3sd 3) 55% fluid, 45% blood cells
• Acceptable range = 65.6-85.2%
• Testing laboratory value = 71.54 % (acceptable) Types of blood specimens
4) Whole blood:
Requirements for proficiency testing • Hematology: (completye blood count/cbc:
• Incorporate sample as part of the specimens received rbc, wbc, platelet counts, hemoglobin,
hematocrit, smear for malarial
daily
• Samples must not be shared by other laboratories parasite/smp)
• Clinical chemistry (capillary blood
• Samples are tested by staff/med. Tech.
glucose/cbg, arterial blood gases /abg and
• Completion of test within the usual time (no
blood ph)
duplication)
• Blood bank/immunohematology
(compatibility testing or cross-matching,
Applications of reference intervals
antibody screening and identification
• Diagnosis of a disease/condition 5) Plasma: (liquid portion of unclotted blood with
• Monitoring a physiologic condition (age, pregnancy)
fibrinogen)
• Monitoring therapeutic drugs
• Clear, light yellow to slightly hazy.
Criteria for evaluation of reference intervals • Selected blood chemistry and hematology
• New analyte studies
• New method o Fluoride (glucose)
• Change in patient population o Citrate (coagulation
studies)
Steps in determining reference interval o Heparin (blood ph, gases)
• Define biological variables and analytic interferences 6) Serum: (liquid portion of clotted blood without
• Choose selection and partition criteria (age and fibrinogen)
gender) • Routine and special blood chemistry
• Complete a written consent (creatinine, bun, uric acid, glucose,
• Categorize the potential reference intervals cholesterol, hormones, drugs, antigens,
• Exclude some individuals (drugs, illness, pregnancy, antibodies)
genetic factors) • Fasting (8-10 hours for glucose) = clear,
• Define the number of reference individuals pale yellow
• Standardize collection and analysis (12-14 Ours for lipid profile)
• Inspect data and prepare histogram
(12-15 Non-fasting: lipemic (turbid or
• Identify possible errors or outliers
• Document all steps and procedures milky) due to chylomicrons (95 %
triglyceride or triacyl glycerol/tag)
Diagnostic efficiency (12-16 Icteric: dark yellow (high bilirubin
• Determines how good a lab test can detect or due to liver diseases, etc.)
predict disease
• True positive/tp = patients with the disease or Public relarions and client interactions
condition and correctly classified by the test 1) Professionalism
• True negative /tn= patients without the disease or • Appearance: lab coats, hair, nails, gloves, closed
condition and correctly classified by the test shoes (center for disease control and
• False positive/fp = patients without the disease or prevention/cdc)
condition and incorrectly classified by the test Attitude:
• False negative /fn= patients with the disease or o Integrity/honesty (clsi standards),
condition and in correctly classified by the test compassion reassurance),
o Motivation (goal),
Method evaluation o Dependability and work ethics
1) Sensitivity = tp / tp + fn x 100% (enthusiasm, reliability, punctuality),
2) Specificity = tn /tn + fp x 100% o Diplomacy (communication skills),
3) Positive predictive value = tp/tp + fp o Ethical behavior (patient’s welfare)
4) Negative predictive value = tn/tn + fn 2) Patient’s consent
o Informed (voluntary)
o Expressed (verbally or written)
 o Implied (action) Venipuncture methods
o Hiv (patient’s informations) • Evacuated tube system
o Consent for minors (parent/guardian) • Multisampling needle/two-way needle/double-
o Refusal of consent (right) pointed needle, tube holder/adapter, evacuated
• Confidentiality and the health insurance portability tubes, tourniquet, antiseptic set
and accountability act (hipaa) • Syringe: needle (gauge 21-23), 1-1 2/3 inches long,
o Protected health information (phi) reheating feature (transfer device/needle guard)
o Fines, imprisonment syringe parts:
Infection control
• Personal protective equipment/ppe: lab coats or
gowns (disposable), gloves (1 patient)
• Hand hygiene: washing, alcohol-based antiseptic hand
cleaners (cdc/health care infection control practices
advisory committee/hicpac)
• Isolation: additional precautions: (n-95masks, etc. (tb,
meningococcimia, sars, etc.)

Circulatory System Anticoagulants

Vascular Anatomy
• Antecubital Fossa (Arm In Front Of The Elbow)
• H Pattern (70% Of Population)
– Median Cubital (Center: Large, Stationary,
Less Nerves) • Ethelenediaminetetraacetic acid/edta
– Median Cephalic (Lateral To Median o Chelates or sequesters calcium
Cubital, 2nd Choice, For Obese) o Disodium edta or dipotassium edta
– Median Basilic (Medial To Median Cubital, • Fluoride (sodium fuoride: anti-glycolytic agent)
3rd Choice) • Citrate: sodium citrate for (prothrombin time and
activated partial thromboplastin time) and citrate
phospate dextrose adenine/cpd-a (for blood bags)
• Oxalate (potassium oxalate)
o Combines with calcium (clotting factor 4)

Order of draw for evacuated tube system

• Stop = sterile = yellow (sodium polyanethol
sulfonate/sps/ acid citrate dextrose/acd) bacterial
culture, paternity testing, prevents complement
activation which kills bacteria
• Red = no additive routine blood chemistry, blood
• M Pattern (30% Of Population) bank, serology, immunology
• Other Veins: Back Of The Hand, Wrist, Leg, Ankle, • Light = light blue (citrate) coagulation studies
Foot • Green = heparin blood gases, electrolytes, nh3
• Arteries • Light = lavender (edta) hematology
o Brachial, Radial, Ulnar • Ready = red with gray (clot activator) routine blood
o Umbilical Artery (Catheterized) For Infants chemistry
o No Tourniquet At 45-90 Degrees • Go = gray (fluoride/oxalate) glucose, lactic acid,
o Allen Test May Be Done alcohol
o Arterial Blood Must Be Placed In Ice Phlebotomy essentials, 4th edition
(Prevent Escape Of Gases)
Mccall and tankersley, 2010
Allen test • Yellow
1) The patient rests his/her hand on the bed or bedside • Light blue
table with wrist up • Red (glass)
2) The patient then clenches the fist. • Red (plastic) with silica (clot activator)
3) Using the index or ring finger of each hand, press the • Red with gray (with serum separator gel and
radial, ulnar or brachial artery. Silica as clot activator)
4) While continuing to hold the pressure, have the • Red with black (with silica as clot activator)
patient to unclench the fist. • Gold (plastic) (with silica as clot activator)
5) The obstructed flow, causes the blanching of the • Light green
palm. Release the pressure on the artery. • Green
6) The palm and fingers become pink after the release. • Green and gray (plasma separator) for potassium
7) The pink coloration indicates the artery is providing • Lavender
circulation to the hand and is refilling the capillary • Pink
bed. • Pearl (edta) for polymerase chain reaction
• Gray
Other evacuated tubes o Apply pressure on the puncture site for 5-
• Orange/gray/yellow = thrombin = blood chemistry 15 minutes using gauze pad and a non-latex
o Activates clotting of blood (procoagulant) elastic wrap
• Tan = heparin/edta = lead analysis Collection of a blood sample with an iv line
• Royal blue = heparin/edta = toxicology, trace metals, 1) Try to avoid the arm if at all possible (use the other
nutritional analysis arm, foot, finger)
• Black = citrate/oxalate = erythrocyte sedimentation 2) If not, ask the nurse to shut off the iv line for 3
rate minutes.
3) Apply the tourniquets below the iv to avoid
Venipuncture methods disruption.
• Butterfly system (winged infusion set) 4) Select the vein other than the vein with the iv.
o Gauge 23 5) Collect 5 ml of blood and discard (to clear any
o Small and delicate veins backed-up iv fluid).
6) Collect the required blood sample/s.
7) Put bandage on the punctured site.
8) Ask the nurse to restart the iv line and say thank you.
Label the tube/s with a notation that the sample was
collected from the arm with an iv line.

Causes of failed venipuncture

• Tube position
• Vacuum
Complications of venipunctunre • Bevel against the vein wall
1) Local immediate • Needle too deep
1.1 hemoconcentration • Needle not deep enough
False increased of solid or chemical • Needle beside the vein
components of blood • Collapsed vein
(tourniquet > 1 minute) • Undetermind needle position
1.2 hematoma • Only two attempts are allowable or else notify the nurse
Bluish discoloration of the site due to
trauma or embroidery or anticoagulant therapy Causes of hemolysis
of the patient • The alcohol used to clean the skin was not allowed
1.3 Failed venipuncture to dry.
Piercing the outer wall of the vein, • The finger or heel was squeezed vigorously in an
collapsed vein, hitting thru and thru attempt to produce greater blood flow.
Turn the needle clockwise, re apply • Newborn infants have increased red cell fragility and
tourniquet high red cell volume.
2) Local delayed • Blood was scraped off instead of allowing it to flow
2.1 thrombosis (clotting in the site due to trauma) into the micro collection container.
2.2 thrombophlebitis (inflammation of the site due to • Needle was not removed when the sample was
trauma) delivered from the syringe to the tube
2.3 syncope/fainting
lack of oxygen to the brain Pediatric venipuncture
let the patient lie down with feet up
• Antecubital fossa forearm only
3) General delayed
• May lead to anemia, shock and cardiac arrest (10% of
3.1 Skin allergies
total volume collected)
o Latex, iodine (bloodletting)
• Determine degree anxiety or fear
o Use hypoallergenic gloves and ask before
• Talk (child’s level) and explain the procedure
collection
• It may hurt a little (don’t say in will not hurt)
3.2 Nerve damage
• Bravery as reward
o Use veins of the antecubital fossa, inform
the dr. • Crying is ok
3.3 Nausea • For violent children (blankets, parents)
o Apply cold compress to the patient’s • Butterfly needle (gauge 23)
forehead • Use microtubes (containers)
3.4 Anemia (due to frequent
o Collection, inform consultant) Geriatric venipuncture
3.5 Diabetic shock • Alzheimer’s disease
o Due to fasting (give candy, soft drinks, etc.) • Arthritis
3.6 Convulsions or cardiac arrest • Coagulation problems (hematoma, prolonged
bleeding)
o Due to fever
• Cataracts
o Call physician or nurse (cardio-pulmonary
• Hearing loss
resuscitation/cpr) • Less elastic, narrowed, fragile veins (collapse)
Move tables, chairs, etc away • Parkinson’s disease
3.7 Continued bleeding • Slower nerve conduction (pain, reaction)
o Due to clotting factor deficiency or
anticoagulant therapy
Quality assurance in phlebotomy Indications:
Problem sites: • Small blood sample (infants, newborn screening)
1) Burns, scars and tatoos (impaired circulation) • Unsuccessful venipuncture
2) Damaged veins: sclerosed (hardened), thrombosed o No accessible veins
(clotted)= occluded and lack resiliency o Fragile veins
3) Edema (fluids) = false decreased o Poct (glucose)
4) Mastectomy (stoppage of lymph flow: swelling) o Thrombotic tendencies
5) Hematoma (swelling or mass of blood in skin due to
wrong puncture)
o Fragile veins Special handling and processing
o Large needle • Bilirubin: hemolysis, light (false decrease)
o Hitting through and through • Neonatal screening: hanging and stacking (erroneous
o Partial insertion of the needle results)
o Excessive blind probing • Tubes: incomplete mixing (clots), vigorous mixing
o Removing needle with tourniquet (hemolysis)
o Insufficient pressure after collection • Transport: plastic bag with biohazard logo (clinical
o Arterial line (bp) laboratory and standards institute/clsi or
o Arteriovenous shunt/fistula (fuses artery occupational health and safety act/ohsa)
and vein for dialysis) • Delivery: 45 mins. (lab), 60 mins. (centrifugation):
o Heparin/saline lock (catheter for prevent glycolysis (200mg/l/hour)
medications) • Temperature: 37 degrees celcius or lower
o Intravenous line/iv (collect below iv line) • Chain of costudy: legal matters
o Central vascular access device/cavd • Others: specimen adequacy/sufficiency, correct
(central venous catheter, implanted port, labelling, aliquoting
peripherally inserted central
catheter/picc)
Factors affecting the laboratory tests
1) Exercise
Procedural error risks (complications), stop the • Icala (increased ck-mb, ast ld, alt)
venipuncture • Iclaf (increased creatinine lactate, ammonia,
• Hematoma: bluish discoloration of the skin fatty acids)
• Iatrogenic anemia: repeated collection (infants) • Du (decrease urate/uric acid)
• Inadvertent arterial pressure: accidental puncture 2) Fasting
• Infection/thrombophlebitis: trauma • Igbt
• Clotting/thrombosis: trauma • (Increased glucose (10 hours), bilirubin (48
• Nerve injury: poor site collection (extreme pain) hours) tag (72 hours)
• Reflux: backflow 3. Diet
• Vein damage: repeated punctures • Piucs5h
• Patient conditions and complications • (Protein increases urea, creatinine (canned
• (stop collection) meats) serotonin and 5 hydroxyindole
o Allergies (latex, iodine, etc.) acetic acid
o Excessive bleeding (notify physicians,
• Cinc (caffeine increases nefa,
nurses)
cathecholamines)
o Fainting (syncope): perspiration, pale
• Tlit (turbidity/lactascence increases tag
color, hyperventilation
o Nausea and vomiting (cold compress on • Lobnu
forehead) • (legumes organs, beans, nuts increases uric
o Pain (nerve injury) acid)
o Petechiae (dhf, endothelial damage) 4. Posture/position
o Seizures/convulsions (hold pressure over • Change leads t0 efflux of water
the site) • Icccetlea (increased creatinine, calcium,
cholesterol, tag, lipopoprotein enzymes,
CAPILLARY BLOOD COLLECTION albumin)
• 70% isopropanol 5. Tourniquet application
• gauze • (i minute only, if prolonged)
• Towel/diaper dampened with warm water (increase • hemoconcentration (increased cell
blood flow) counts)
• Micro collection tubes/microtubes/bullets (color- • anaerobiosis (decreased po2, so2, spo2)
coded) • Ipleck increased proteins, lactate,
• Microhematocrit tubes enzymes, cholesterol, potassium/k
• Sealants (claylike for hematocrit) 6. Alcohol intake
• Aituga (alcohol increased tag, urric acid,
ammonia)
Order of draw
• Adg (alcohol decreased glucose)
• Edta: lavender: hematology 7. Smoking (tobacco)
• Plasma: with additives
• Sincccatugg (smoking increased nefa,
• Serum: no additives
cholesterol, catecholamines, cortisol,
ammonia, tag, urea, gh, glucose)
 -- derived from arginine and lysine
8. Stress -- ammonia = .2 % (derived from amino
• Sicoc (stress increased cortisol, oxygen, acids)
catecholamines)
9. Drugs • Located retroperitoneally
• Iicl (intramuscular injection increased ck, • Functions include:
ld) o Elimination of waste products of
• Ddspc (diuretics decreased sodium, metabolism through: filtration,
potassium, chloride) reabsorption and secretion
• Dic (diuretics increased calcium) o Filtration occurs in the glomerulus of the
• Cibccdttt (creatinine increased by nephron
cephalosphorine, cemetidine, dopamine, o Regulates erythropoiesis (erythropoietin)
trimetoprim, testosterone) o Regulates acid-base balance
• Aibbad (ammonia increased by -- hco3- is reabsorbed if acidosis
barbiturates, analgesics, diuretics) -- hco3- is excreted if alkalosis
o Regulates blood pressure through the
• Adbdll (ammonia decreased by
renin- angiotensin and aldosterone system
dephenhydramine, lactoluse, levadopa)
(counteracted by atrial natriuretic peptide.
• Uaibbbppst (uric acid increased by
barbiturates, benzothiazide, probenecid,
Compounds processed in the nephron
phenylbutazone, sulfinpyrazone, thiazide)
10. Other factors
• Pdb (pregnancy decreased bun)
• Bddb (bacterial decomposition decreased
bun)
• Adb (age decreased bun)
• Cibgaua (creatinine increased by glucose
ascorbic acid, uric acid, alpha keto acids)
• Dvibpatagaic (diurnal variation increased
by prolactin, acth, thyroxine, acp, gh,
aldosterone, iron, cortisol) • Compounds reabsorbed and secreted in the renal
tubules
Kidney Function Tests • Pct/proximal convoluted tubule= reabsorption of
glucose, h2o, amino acids, organic acids, na+ and
hco3-
KIDNEYS • Loh/loop of Henle = reabsorption of h2o and na+
• Located retroperitoneally • Dct/distal convoluted tubule = reabsorption of na+
• Functions include and h2o (facilitated by adh/avp)
o Elimination of waste products of • Secretion of h+ and na+
metabolism through: filtration, • Ct/collecting tubule = reabsorption of water and
reabsorption and secretion urea
o Filtration occurs in the glomerulus of the • Secretion of h+ and na+
nephron
o Glomerulus has 3 layers of tissues:
endothelium, basement membrane and
epithelium
• Only compounds with more than 50,000 kda are
filtered.
• All proteins, lipids and enzymes are not filtered
except beta amylase

• Located retroperitoneally
• Functions include
o Elimination of waste products of
metabolism through: filtration,
reabsorption and secretion
o Filtration occurs in the glomerulus of the
nephron
o Glucose, electrolytes and non-protein
nitrogens or npn’s are filtered
o Npn’s include:
-- bun/urea = 50% (end product of protein
metabolism
-- amino acids = 25 % (building blocks of
protein)
-- uric acid = 10 % (end product of purine
metabolism)
-- creatinine = 5 % (end product of muscle
metabolism)