SEMR1

SEMR1 | M1 | Clin Chem

Ex. of Patient Variables That May Affect Chem

OUTLINE Values
VARIABLE ANALYTES
AFFECTED
Diurnal Variation  am: ACTH, cortisol,
iron
 in pm: growth
hormone, PTH, TSH
Day-to-Day Variation 20% for ALT, bili, CK,
steroid hormones,
triglycerides
Common Preanalytical Errors Recent food  glucose, insulin,
ingestion gastrin, triglycerides,
Na+, uric acid, iron,
LD, Ca2+
 Chloride, phosphate,
K+
Fasting required:
Fasting glucose,
triglycerides, lipid
profile
Alcohol  glucose, 
Triglycerides, GTT
Posture  albumin, cholesterol,
Ca2+ when standing
Activity  in ambulatory
patients: creatinine
kinase (CK)
 with exercise: K+,
Phosphate, lactic acid,
Pre-Collection Variables creatinine, protein,
• Physiologic Factors: CK, AST, LD
 Diurnal Variation Stress  ACTH, Cortisol,
 Exercise Catecholamines
 Diet Age, gender, race, Various
 Stress drugs
 Posture
 Age Common Interferences
 Gender • IN VIVO:
 Tobacco Smoking
• IN VITRO:
 COLLECTION ASSOCIATED
VARIABLES:
• HEMOLYSIS
• HEMOCONCENTRATION
• HEMODILUTION
• ICTERIC AND LIPEMIC SPECIMENS
− Hemolyzed - Rupture of RBC’s so, HB
released from RBC’s
− Icteric - Serum appears yellow due to high
bilirubin
− Lipemic - Serum appears milky/turbid due to
high lipid

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Specimen Collection
SEPARATION TECHNIQUES (BISHOPS)
• TEST ORDER
• Centrifugation
• TIME OF COLLECTION
 Is a process in which centrifugal force is
• SPECIMEN ACCEPTABILITY AND used to separate solid matter from a liquid
IDENTIFICATION ISSUES solution. *10 min centrifugation ang ating
• ANTICOAGULANTS AND ADDITIVES blood
• Filtration
 Can be used instead of centrifugation for
the separation of solids from liquids.
However, paper filtration is only
occasionally used in today’s laboratory.
• Dialysis
 Another method for separating
macromolecules from a solvent or smaller
SPECIMEN CONSIDERATIONS substances. It became popular when used
Require Fasting FBS, in conjunction with the Technicon Auto
ALDOSTERONE/RENIN, Analyzer system in the 1970’s.
TAG, GTT, LIPID
PANEL, INSULIN, Quality Control
GASTRIN • Gaussian Distribution Curve:
Require Chilling LACTIC ACID,  68-95-99 Rule:
AMMONIA, BLOOD GAS  Summarizes the relationships between the
AFFECTED BY VITA, BILIRUBIN, area under the Gaussian distribution curve
LIGHT CAROTENE, VIT B6, VIT and the SD. Given any data in the Gaussian
B12, FOLATE, curve, 68.3% of the data will be fall between
PORPHYRINS +/-1SD from the mean. 95.4% the data will
AFFECTED BY CK, POTASSIUM, fall between +/- 2SD from the mean. 99.7%
HEMOYSIS AST,ALT, PHOSPHATE, will fall between +/- 3SD from the mean.
ALP,  The reference range is usually set at 95%
CATECHOLAMINES, or sometimes referred as 95% limits.
MAGNESIUM, IRON, LD,
AMMONIA

• Calculation of Control Limits:

 FOR +1SD: MEAN + (SD) (1)
 FOR -1SD: MEAN - (SD) (1)
 FOR +2SD: MEAN + (SD) (2)
 FOR -2SD: MEAN - (SD) (2)
 FOR +3SD: MEAN + (SD) (3)
 FOR - 3SD: MEAN - (SD) (3)

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 PARASITOLOGY M3

• Plotting in the Levey-Jennings Charts • Define - Problems & Objectives

 A common method to assess the • Measure - What do we need to improve?
determination of control materials over time • Analyze - Process & Factors of influence
is by use of a Levey- Jennings control • Improve - Implement improvement
chart/Shewhart plot. • Control - Assure that improvement will sustain
 The generally accepted minimum protocols
for target value assignment is to use the
mean value from assaying a QC material a
minimum of 20 times on 20 different days.
West Gard rules
• 12s: One control observation exceeding the
mean +/-2s. A warning rule that initiates testing
of control data by other rule.
• 13s: One control observation exceeding the
mean +/-3s. Allows high sensitivity to random
error.
• 22s: Two control observation exceeding the
mean +/- 2s or -2s. Allows high sensitivity to
systematic error.
• R4s: One control exceeding the +2s and other
exceeding -2s. Allows detection of random
error.
• 41S: Four consecutive control observations
exceeding the +1s or -1s. This allows
detection of systematic error.
Principles of Instrumentation
• 10x: Ten consecutive control observation falling
• Spectrophotometry:
on one side or the other of the mean (no
 Beer’s Law: States that the concentration of
requirement for SD size). This allows the
unknows is directly proportional to its
detection of systematic error.
absorbance and indirectly proportional to
• SHIFT - One side of the mean it’s transmittance.
• TREND - Away from the mean  SAMPLE = ABS = %T
Random error 12s (Warning rule)
− Acceptable - 13s
3SD R4s
− Unacceptable
- outside
+3SD
Systematic error 22s
41s
10x
Lean Six Sigma
• Methodology is the combination of Six Sigma
quality management, developed by Motorola,
with Lean manufacturing strategy, pioneered by
Toyota, to provide tangible metrics for quality
improvement. In its simplest form.
• Six Sigma asks the question, How can this
process be improved? while Lean
manufacturing asks the question does this
process (or step) need to exist? Together as
Lean Six Sigma, They are being increasingly
used to reduce error (Six Sigma) and waste
(Lean) within the health-care system.

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PARASITOLOGY M3

Photometric Methods

Protein Electrophoresis
Rate of Migration Depends on size, &
charge of molecule
Support Medium Cellulose, acetate or
agarose
Buffer Barbital buffer, ph 8.6
Stains Ponceau S, amido
blue, bromphenol
blue, Coomassie
brilliant blue
Charge At ph 8.6, proteins are
Principles of Instrumentations negatively charged &
move toward anode.
Order of Migration Albumin, alpha-1-
(fastest - slowest) globbulin, alpha-2-
globulin, beta globulin,
gamma globulin.
Largest fraction Albumin
Electroendosmosis Buffer flow toward
cathode. Causes
gamma region to be
cathodic to point of
application.
Urine Must be concentrated
first because of low
protein concentration.
Bence Jones proteins
migrate to gamma
region in urine
electrophoresis.
CSF Must be concentrated
first because of low
protein concentration.
CSF has prealbumin
band.

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 PARASITOLOGY M3

Common Serum Protein Electrophoresis Laboratory Automation and Computer

Patterns Systems
Normal 1. Albumin, 2. Introduction to Automation
Alpha-1, 3.  Clinical chemistry continuous to be one of
Alpha-2, 4. Beta, the most rapidly advancing areas of
5. Gamma laboratory medicine.
Acute inflammation  alpha-1 &  CLINICAL CHEMISTRY - is a polyglot
alpha-2 discipline combining chemistry,
Chronic Infection  alpha-1, alpha- biochemistry, immunochemistry,
2, & gamma endocrinology, toxicology (abused and
Cirrhosis Polyclonal (all therapeutic drug testing), analytical
fractions) in chemistry, engineering, informatics and
gamma with beta doubtless other specialties, to provide the
gamma necessary support to physicians and other
Monoclonal gammopathy Sharp  in 1 healthcare providers to improve the
diagnosis and treatment of patients.
immunoglobulin
(“M spike”).  in
Definition of Terms
other fraction
• Batch analysis - means that multiple samples
Polyclonal gammopathy Diffuse  in
are tested in a ‘run’.
gamma
• Sequential analysis - means samples are
Hypogammaglobulinemia  gamma
tested one after the other and results are
Nephrotic syndrome  albumin,  reported in the same order
alpha-2 • Continuous-flow analysis - is a form of
Alpha-1-antitrypsin  alpha-1 sequential analysis through a continuous
deficiency stream at a constant rate, e.g. the Auto
Hemolyzed specimen  beta or unusual Analyzer.
band between • Discrete analysis - each sample is tested in a
alpha-2 & beta separate cuvette or other reaction chamber
Plasma Extra band with reagents added to each individual sample
(fibrinogen) container.
between beta & • Single-channel analysis - uses an analytical
gamma ‘line’ dedicated to a single test.
• Multiple-channel parallel analysis uses two or
Chromatography more ‘lines’ or channels, each dedicated to a
single test, and analysis occurs
simultaneously.
• Random access analysis - specimens are
tested in or out of sequence with each other,
as reaction vessels are available and without
regard to accessioning order, although testing
of designated specimens, such as stats, may
be given priority.
• Assays - are either end-point tests (reaction is
complete after a fixed time) or continuous
monitoring tests (multiple data points recorded
over a specified time interval).
*Mobile Phase - Is Non Polar
*Stationary Phase - Is Polar Hydrophilic

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Steps in Automated Analysis
Step Comments
Sample ID Usually by bar code
reader
Test Selection Usually
communicated by LIS
Sampling Usually closed tube
sampling for primary
collection tubes. Some
analyzers have short
sample & clot
detection
Reagent delivery Usually by syringes,
pumps, or pressurized
reagent bottles. Vitros
uses dry slides. Some
offer reagent inventory
Chemical reaction Mixing & incubation
Measurements Visible & UV
spectrophotometry,
ion selective
electrodes,
fluorescence
polarization,
chemiluminescence,
bioluminescence.
Most offer automatic
dilution & restoring
when linearity is
exceeded
Data Handling Concentration derived
from calibration curve
stored in analyzer
Reporting Usually reported to LIS
through interface
Troubleshooting Can be done remotely
by modem on many
analyzers
Traditional Methods
• Atomic absorption - Radiation source - Lens -
Atomized sample (Flame) - Lens -
Monochromator - Output
• Flame emission photometry - Measure the
light emitted by excited atoms (excitation)
• Gasometry
• Potentiometry - Used to determine the
difference between the potential of two
electrodes. The potential of one electrode − the
working or indicator electrode − responds to the
analyte’s activity, and the other electrode − the
counter/reference electrode − has a known,
fixed potential
• Amperometry - Determining the changes in
electric current
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• Spectrophotometry - Measuring the amount
of LIGHT ABSORBED by our analyte.
• Immunophelometry (for competitive and
sandwich immunoassays) - Measures the
amount of light SCATTERED by a small
particle.
• Electrophoresis
Advantages of Automation over Manual
Procedures
• Rapid results
• Increase in the number of tests performed
• Save time and effort
• Eliminates the need for staff (Personnel)
• Economical
• Errors in calculations and transcription are
reduced
• Better precision and accuracy
Early Automated Analysis
• Leonard Skeggs
 Inventor of continuous flow analyzers (e.g.,
Auto Analyzer, SMA 12/60, and SMAC)
 In 1956, Leonard Skeggs developed the
first practical and completely automated
system for measuring urea, glucose, and
calcium, the Auto Analyzer, an instrument
designed to meet the specific needs of the
clinical chemistry laboratory.
• Technicon Company - Fielded commercially
available Technicon single and multichannel
continuous flow autoanalyzer’s, the initial Auto
Analyzer
• Four Main types of Automatic Analyzer for
processing Clinical Chemistry tests:
 Continuous Flow System
− Principle:
• All samples are carried through the
same analysis pathway.
• All samples automatically pass from
one step to another without waiting to
bring the samples to the same stage of
completion.
• The reactions are not necessarily
carried to equilibrium since samples
and standards are treated exactly alike.
• This system has won wide acceptance
in both routine and research
laboratories.
• Examples
1. Technicon Autoanalyzer II -
Capable of running three different tests
at 60-80 samples per hour.
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 PARASITOLOGY
2. Sequential Multiple Analyzer (SMA 6/60) -
capable of running 6 tests at 60 samples per hour.
3. SMA 12/60 - capable of running 12 tests at 60
samples per hour.
4. Sequential Multiple Analyzer with Computer
(SMAC) - capable of running 40 tests at 120
samples per hour.
• Features
− Use of plastic tubes of different diameters
and peristaltic pump for continuous
pumping of samples and reagents. This
maneuver replaces the pipetting steps in
the manual procedures
− Introduction of Air Bubbles:
a. To separate the sample and reagent
streams into segments
b. To separate one sample from the next
c. For continuous scrubbing of tubing
d. Prevents cross contamination or carry
over by the previous specimen
− Removal of proteins by dialysis
− Flow-through cuvettes in an
interferences filter photometer, using a
fixed reference light path
− Recorder read-out
− Modular design permitting interchanging
of major parts
− Apparatus Constituents
The Technicon Auto Analyzer II consists
of the following components:
• Auto-sampler
• Proportioning pump
• Manifold
• Photo colorimeter
• Recorder (Chart recorder)
• Parts of Continuous Flow System
1. Sampler - Holds the cups containing
standards and specimens for analysis
which are introduced into the analytical
system by means of aspiration, in a preset
sequence and at a pre-selective rate.
2. Pumps and Manifold - For continuous and
proportional delivery of samples, reagents
or gases. This analogous to pipetting in
manual techniques.
3. Dialyzer - Employs dialysis through a semi-
permeable membrane to separate proteins
from the analyte, thus eliminating the need
for manual deproteinization techniques.
4. Heating Bath - For heating and incubating
the reaction mixture at fixed temperatures.
5. Detector-Recorder - For continuously
measuring and recording optical density or
absorbency changes
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 Discrete Sampling Analyzers
− Principle:
• Each sample reactions is handled in a
separate compartment and does not
come into contact with another sample.
• The samples and standards are
handled on a batch basis and must be
brought before proceeding to the next
procedure.
• All reactions must be carried out until
equilibrium is reached.
• This system stimulates very closely
manual procedures except that the
various steps are done automatically.
• Robot Chemist by the Research
Specialties Company
• Examples
1. Dupont ACA (Automatic
Clinical Analyzer)
2. Abbott ABA-100
Biochromatic Analyzer, ABA-
200 and VP Analyzer
3. Beckman ASTRA 8 and
ASTRA 4
4. Beckman DSA 560 and DSA
564
5. American Monitor KDA
 Centrifugal Fast Analyzers
− Principle:
• As the rotor is accelerated centrifugal
force moves the reagents and sample
to a mixing chamber and then through
a small channel into the cuvette. As the
filled cuvette rotates past a fixed light
beam, the absorbance of the reaction
is measured spectrophotometrically
• Examples: CentrifiChem: RotoChem
• Developed by Norman Anderson
 Thin-film Analyzers
− Principle:
• A 16 mm square chip which contains
several very thin layers, accepts a
metered drop of serum, spreads it
evenly into a reagent layer, then
confines the colored product to a fixed
area for reflectance spectrophotometry
• Example: Kodak “Ektachem”
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Automated Stand-alone and Integrated
Analyzers
• Mixture of analytical techniques, including
electrophoresis, LC-MS/MS,
colorimetry/spectrophotometry, potentiometry,
and immunoassay (using various
methodologies)
• Integrated system offer the advantage of
consolidation.
• It is preferable to place as many assays on a
single analyzer than to maintain two or more
analyzers because each instrument will require
separate QC, preventive maintenance, record
keeping, etc.
• Stand-alone automation provides most of the
major benefits of TLA without the cost of a track.
• With stand-alone automation, sample transport
is still performed manually by the laboratory
staff, sometimes called the ‘sneaker network’.
• The stand-alone options offer a smaller footprint
(some components are even bench top
modules) and lower cost, and thus may be good
choices for laboratories with limited floor space.
Benefits of Replacing Manual Procedures
with Automation include:
• Eliminating potentially dangerous, error-prone
manual procedures with automated processes
requiring minimal technician involvement.
• Increasing productivity
• Decreasing TAT
• Improve safety
• Minimizing error
• Improving sample handling
• Allowing practical reallocation of laboratory staff
who are freed from manual tasks.
Open System Vs. Closed System
• Open System - reagents from many vendors
may be adopted to an analyzer
 Assay applications for closed systems
are optimized for them and verified by
the manufacturer. Laboratories need to
perform method validation for every
assay they adapt to an open system. It
usually considered desirable for an
analyzer to have an open system
capability
• Closed System - Only the proprietary reagents
from the instrument manufacturer are suitable
for use on the analyzer.
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Radioimmunoassay
• Radioimmunoassay (RIA) was developed by
Yalow and Berson in 1959 and resulted in
the award of the Nobel Prize in Medicine in
1977
• RIA-ARIA AND ARIA II
• RIA was automated by Becton-Dickson with
the ARIA and ARIA-II instruments but these
analyzers were short-lived. EMIT, using non-
isotopic labels, allowed for practical
automation of immunoassay.
Variations of Immunoassay
• Immunoradiometric assay (IRMA)
 Lower limits of detection than RIA and
analyte concentrations directly
proportional to increasing signal
• Enzyme immunoassay (EIA)
• Enzyme multiplied immunoassay
technique (EMIT)
 Patent by the Syva Company
• Enzyme labelled immunosorbent assay
(ELISA)
• Cloned enzyme donor immunoassay
(CEDIA)
• Microparticle enzyme immunoassay (MEIA)
• Fluorescence polarization (FPIA)
 Fluorescent immunoassay (FIA) is
perhaps best recognized as the
automated technique of Fluorescence
Polarization Immunoassay (FPIA)
methodology from Abbott, long used on
the TDX analyzer
Total Laboratory Automation (TLA)
• In the early 1980’s, Dr. Masahide Sasaki at
the Kochi Medical School, Kochi, Japan,
developed conveyor belt systems, robots to
load and unload analyzers, and process
control software, and is credited with the first
attempt at TLA
• TLA combines a wide variety of processes,
including accessioning and sorting
specimens, decapping tubes, centrifugation,
aliquoting, delivery to analyzers, recapping
tubes, and storage and archiving of samples.
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Important Considerations to be Examined

when considering TLA
• Methods (provide for the existing assays and
also allow for open channel applications)
• TAT (aim for achieving desired TAT about 90%
of the time)
• Specimen handling (allow for stats, decrease
manual procedures, check for
hemolysis/icterus/lipemia (HIL))
• Ability to locate, retrieve, and test a sample for
dilution, repeat, or add-on testing: Throughput
(accommodate fluctuating loads through the
day)
• Cost (be affordable and even decrease
operating costs over time)
• Ability to operate with a laboratory information
system (LIS; communicate with the LIS and
provide middleware)
• Environment and safety (Decrease health
hazards)
• Downtime and service (require minimal
preventative maintenance and downtime and
service will be readily available)

Liquid Chromatography-Mass
Spectrometry/Mass Spectrometry (LC-MS/MS
or LC Tandem Mass Spectrometry
• LC allows for separation of analytes and MS
breaks down analytes, identifies the resulting
ion fragments, quantitates them
• It offers advantages specifically for drugs of
abuse testing therapeutic drug monitoring
(TDM), and endocrinology
Basic Principles of Laboratory Mathematics

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