In the clinical laboratories several analytical determinatio 􀁸 Is used to measure concentration by detecting

ns are based  absorption of electromagnetic radiation by

on radiant energy measurements that are absorbed or tr atoms rather than molecules
ansmitted.  􀁸 A powerful optical technique that is used widely
in clinical laboratories for elemental analysis
(calcium, magnesium, lithium, lead, copper, zinc)
Photometers and spectrophotometers are the instrument
s  Light source
for measuring absorbed or transmitted light energy. 􀁸 Usually a HOLLOW-CATHODE LAMP

FLAMELESS ATOMIC ABSORPTION

The basic components of spectrophotometers 􀁸 A modification of AAS, uses an electric furnace
include radiant energy to break chemical bonds (electrothermal
source, wavelength selector, cuvette holder, atomization)
photodetector, signal 􀁸 The sample is placed in a depression on a
processors, and readout devices. carbon rod or graphite furnace in an enclosed
chamber
Rayleigh Scattering o Tantalum or platinum metal may also be used
Scattering by molecules or aggregates of as sample cups
molecules with dimensions significantly 􀁸 The temperature achieved is up to 2700
smaller than the wavelength of the radiation is degrees Celsius, necessary to vaporize heavier
referred to as metals
Rayleigh scattering. 􀁸 More sensitive than the conventional flame
methods and permits determination of trace
The Tyndall effect occurs with particles of metals in small samples of blood or tissue
colloidal dimensions and can
be seen with the naked eye. Analytical Methods
Wavelength Distance bet 2 successive
Raman Scattering peaks (nm)
Raman scatter involves absorption of photons Lower frequency = Longer
producing vibrational excitation. wavelength (Ex. Red)
Higher frequency =
SPECTROMETRY Shorter wavelength (Ex.
Photometry Violet)
􀁸 Is the measurement of the luminous intensity of
Spectrophotometric Meas. light intensity in a
light falling on a surface from such a source
meas. narrower wavelength
Spectrophotometry
Photometric Meas. light intensity w/o
􀁸 Is the measurement of the intensity of light at
measurement consideration of wavelength
selected wavelength
Multiple wavelength (uses
􀁸 Uses photometers or spectrophotometers
filter only)
Multiwavelength Spectrophotmetry LASER Light Amplification by
􀁸 a.k.a Bichromatic Analysis Stimulated Emission of
􀁸 an alternative arrangement found in many high- Radiation
output instrument in which the Light source for
monochromator is placed between the cuvet and spectrophotometry
the detector Visible region Tungsten light bulb
Mercury arc
Double-Beam Spectrophotometer UV Deuterium lamp
􀁸 Also used in correcting inherent color or Mercury arc
turbidity in samples Xenon lamp
􀁸 Splits or chops the monochromatic beam of Hydrogen lamp
radiation into two components, with one beam IR Merst glower
passes through the sample and the other through Globar (Silicone carbide)
a reference solution or blank Stray light Wavelength outside the
band
Reflectance Photometry Most common cause of
􀁸 Measures the quantity of light reflected by a loss of linearity
sample that has been dispensed onto a grainy Diffraction gratings Most commonly used
or fibrous solid support monochromator
Cutting grooves
ATOMIC ABSORPTION SPECTROPHOTEMTER
Prisms Rotatable
 Nickel sulfate Prevents stray light (Ca++ and Mg++)
Cutoff filter Anti-stray light More sensitive than FEP
Bandpass ½ peak transmittance Atomizer (nebulizer) Convert ions  atoms
Alumina silica glass Most commonly used Chopper Modulate the light source
cuvet cuvet Lanthanum/Strontiu Complex with phosphate
Quartz/plastic cuvet UV m chloride Avoid calcium
Borosilicate glass Strong bases interference
cuvet Volumetric Unknown sample is made
Photodetector Converts transmitted light (Titrimetric) to react with a known solution
into photoelectric energy in the presence of an indicator
Barrier layer cell/ Simplest detector Turbidimetry Light blocked
photocell/ photovoltaic No external voltage Meas. abundant large
cell For filter photometers particles (Proteins)
Phototube Contains anode and Depend on specimen
cathode concentration and particle size
Req external voltage Nephelometry Meas. amt of Ag-Ab
Photomultiplier tube Most common type complexes
Most sensitive Scattered light
UV and visible region Depends on wavelength
Galvanometer/Amm Meter or read-out device and particle size
eter Electrophoresis Migration of charged
Absorbance A = abc (a = absorptivity; b particles in an electric field
= length of light (1cm); c = Iontophoresis Migration of small charged
concentration) ions
A = 2 – log%T Zone electrophoresis Migration of charged
Double beam spectro. Splits monochromatic macromolecules
light into two components: Endosmosis Movement of buffer ions
One beam  sample and solvent relative to the
One beam  reference fixed support
soln or blank (corrects for Ex: gamma globulins
variation in light source Cellulose acetate Molecular size
intensity) Agarose gel Electrical charge
Double-beam in 2 photodetectors (sample Polyacrylamide gel Charge and molecular size
space beam and reference beam) 20 fractions (ex.
Double-beam in time 1 photodetector isoenzymes)
Monochromatic light  Electrophoretic Directly proportional to
sample cuvet and reference mobility net charge
cuvet Inversely proportional to
Dydimium filter 600 nm molecular size & viscosity of
Holmium oxide filter 360 nm the supporting medium
Reagent blank Color of reagents Isoelectric focusing Molecules migrate
Sample blank Optical interference (Hgb) through a pH gradient
FEP Meas. light emitted by a pH = pI
single atom burned in a flame For isoenzymes: same
Principle: Excitation size, different charge
Lt. source and cuvette: Densitometry Scan & quantitate
Flame electrophoretic pattern
For excited ions (Na+, K+) Capillary Electro-osmotic flow
Cesium and Lithium Internal standards (FEP) electrophoresis
Correct variations in flame Southern blot DNA
Lithium Preferred internal std Northern blot RNA
Potent antidepressant Western blot Proteins
AAS Meas. light absorbed by Chromatography Separation by specific
atoms dissociated by heat differences in physical-
Principle: Dissociation chemical characteristics of the
(unionized, unexcited, ground different constituents
state) Paper Fractionation of sugar and
Lt. source: Hollow- chromatography amino acid
cathode lamp Sorbent: Whatman paper
For unexcited trace metals TLC Screening: Drugs
 Retention factor (Rf) Relative distance of
value migration from the point of
application
Rf = Distance leading edge
of component moves
Total distance
solvent front moves
Gas chromatography Separation of steroids,
barbiturates, blood, alcohol,
and lipids
Volatile compounds
Specimens  vaporized
Mobile phase: Inert gases
Gas Solid Differences in absorption
chromatography at the solid phase surfaces
Gas Liquid Differences in solute
chromatography partitioning between the
gaseous mobile phase and the
liquid stationary phase
Mass Spectrometry Fragmentation and
ionization
GC-MS Gold standard for drug
testing
MS/MS Detect 20 inborn errors of
metabolism from a single
blood spot
HPLC Most widely used liquid
chromatography
Fractionation of drugs,
hormones, lipids,
carbohydrates and proteins
Hydrophilic gel Gel filtration
Separation of enzymes,
antibodies and proteins
Ex: Dextran and agarose
Hydrophobic gel Gel permeation
Separation of triglyceride
and fatty acid
Ex: Sephadex
Ion exchange Separation depends on the
chromatography sign and ionic charge density
Partition Based on relative
chromatography solubility in an organic solvent
(nonpolar) and an aqueous
solvent (polar)
Affinity For lipoproteins, CHO and
chromatography glycated hemoglobins
Adsorption Based on differences
chromatography between the adsorption and
desorption of solutes at the
surfaces of a solid particle
Fluorometry/Molecu Det. amt. of lt. emitted by a
lar Luminescence molecule after excitation by
Spectro. electromagnetic radiation
Lt. sources: Mercury arc
and Xenon lamp (UV)
Lt. detector:
Photomultiplier tubes
2 monochromators:
Primary filter – selects
wavelength absorbed by the
solution to be measured
Secondary filter –
prevents incident light from
striking the photodetector
Sensitivity: 1000x than
spectro
Quenching Major disadvantage of
fluorometry
pH and temperature
changes, chemical
contaminants, UVL changes
FLAME EMISSION PHOTOMETRY
􀁸 Used in the clinical laboratory to determine
sodium, potassium, and lithium concentrations in
biological fluids
LUMINISCENCE
􀁸 Types: (1) Fluorescence (2) Phosphorescence
(3) Chemiluminescence (4)
Electrochemiluminiscence
􀁸 Most uncharged molecules contain even
numbers of electrons in the ground state. These
electrons fill molecular orbits in pairs with their
spins in opposite directions. No electron
energies can be detected by application of a
magnetic field to such spin patterns, and this
electronic state is called the singlet state.
FLUOROMETRY
􀁸 Principle:
o Based on an energy exchange process that
occurs when certain compounds absorb
electromagnetic radiation, become excited and
return to an energy level lower than or
equal to their energy level. Because
PHOSPHORESCENCE
􀁸 A luminescence produced by certain substances
after they absorb radiant or other types of
energy
􀁸 Distinguished from fluorescence in that it
continues to occur even after the radiation
causing
it has ceased
CHEMILUMINESCENCE
􀁸 The physical event of light emission in
chemiluminescence is similar to fluorescence in
that it
occurs from an excited singlet state and the light
is emitted when the electrons returns to the
ground state
􀁸 Differs from fluorescence and phosphorescence
in that the emission of light is created from
a chemical or electrochemical reaction, and not
from absorption of electromagnetic energy
BIOLUMINESCENCE
􀁸 A special form of chemiluminescence found in Graduated or Serological: w/ graduations to
biological system measuring pipet the tip (blowout)
􀁸 The naturally occurring chemiluminescence Mohr: w/o graduations to the
􀁸 Utilizes enzyme or photoprotein to increase the tip (self-draining)
efficiency of the luminescence reaction Bacteriologic
(luciferase and aequorin) Ball, Kolmer and Kahn
Micropipettes: <1 mL
ELECTROCHEMILUMINESCENCE Micropipettes TC pipets:
􀁸 Based on the formation of an excited state Sahli-Hellige pipet
chemical intermediate that returns to the ground Lang-Levy pipet
state by emitting a photo RBC and WBC pipets
Kirk and Overflow pipets
Light scattering is a physical phenomenon
Air displacement Piston: suction
resulting from the interaction of light with
pipet Disposable tip
particles
Positive Piston  barrel (like a
in solution
displacement pipet hypodermic syringe)
􀁸 Turbidimetry and nephelometry are techniques
used to measure scattered light Dispenser/Diluto Liquid: common reservoir 
􀁸 Light-scattering measurements are applied best r pipet dispense repeatedly
to immunoassays of specific proteins and Distilled H2O Calibrating medium for TD
Haptens pipettes
Mercury Calibrating medium for TC
Nephelometry pipettes
o Detects light that is scattered at various angles; Acid dichromate Cleaning solution for
scattered light yields a small signal (H2SO4 + K2Cr2O4) glasswares
that must be amplified
􀁸 Turbidimetry
o Measures a reduction in light transmission due Laboratory Mathematics
to particle formation; thus it detects a % w/v Grams of solute = %
small decrease in a large signal solution desired x total volume
desired
Borosilicate For heating and sterilization
glasswares Ex: Pyrex and Kimax 100
Boron-free/Soft High resistance to alkali % v/v mL of solute = % solution
glasswares desired x total volume desired
Corex (Corning) Special alumina-silicate glass
Strengthened chemically than 100
thermally % w/w Grams of solute = %
6x stronger than borosilicate solution desired x grams of the
Vycor (Corning) For high thermal, drastic heat total solution
and shock
Can be heated to 900OC
100
Flint glass Soda-lime glass + Calcium,
Molarity M= _grams of
Silicon, Sodium oxides
solute_______
Easy to melt
GMW x volume of
For making disposable
solution
glasswares
Moles Mol = weight (grams)
TD: To deliver Exact amount
GMW
TC: To contain Does not disperse the exact
To prepare a molar Grams of solute = Molarity x
volume
solution GMW of the solute x Volume (L)
Blowout w/ etched rings on top of
desired
pipet
To convert % w/v M = % w/v  10
Self-draining w/ o etched rings
to Molarity GMW
Drain by gravity
Normality N = _Grams of solute_
Transfer pipet Volumetric: for non-viscous
EW x volume (L)
fluid; self-draining
Equivalent weight EW = __MW___
Ostwald folin: for viscous
(EW) valence
fluid; w/ etched ring
To prepare a Grams of solute = Normality
Pasteur: w/o consideration of
normal solution of x EW x Volume (L)
a specific volume
solids
Automatic
macro-/micropipets
 To convert % w/v N = w/v  10 preparation of standard
to Normality EW solutions
Normality N = Molarity x Valence Uses: FEP, AAS, blood gases
Molarity M = Normality and pH, enzyme studies,
valence electrolyte testing, HPLC, trace
Molality m = Grams of solute__ metal and iron studies
MW x kg of solvent Type II Rgt Water For clinical laboratory use
Milliequivalents mEq/L = mg/dL  10  (hematology, microbiology,
valence immunology, chemistry)
MW For prep. of rgts and QC
Millimoles mmol/L = mg/dL  10 materials
MW Type III For washing glasswares
Ratio Ratio = _Volume of solute_ For urinalysis, parasitology
Volume of solvent and histology
Dilution Dilution = __Volume of Distilled water Purified to remove almost
solute__ all organic materials
Volume of Deionized water Free from mineral salts;
solution removed by ion exchange
0.179 Conversion factor for iron processes
(mg/dL  μmol/L) Organic material may still
0.01 Conversion factor for be present
phospholipid (g/dL to g/L) Occupational Safety Req. manuf. to indicate lot
2.27 Conversion factor for folate and Health Act (OSHA) no., physical or biological health
Analytical reagent For qualitative and hazard of the chem.. rgts, and
(AR) grade quantitative analyses precautions for safe use and
For accuracy storage
Established by American
Chemical Society (ACS) College of American Recommends that a lab.
Uses: Trace metal analysis Pathologists (CAP) document culture growth, pH
and preparation of standard and specific water resistance on
solutions reagent grade water
Ultrapure reagents Additional purification Tests for water Microbiological content
steps purity pH
Ex: Spectrograde, Resistivity
nanograde, HPLC grade Chemical oxygen demand
Uses: Chromatography, Ammonia
atomic absorption, Ions
immunoassays Metals
Chemically Pure Indicates that the impurity Detergent- Alkaline pH
(CP) or Pure Grade limitations are not stated contaminated water
Purity is delivered by meas. Hard water Contains calcium, iron and
of melting point or boiling point other dissolved elements
Technical/Commer In manufacturing NCCLS Now: Clinical and
cial grade Never used in clin. lab. Laboratory Standards Institute
testing (CLSI)
United States For human consumption Dilute solution Relatively little solute
Pharmacopoeia (USP) Not applicable for lab. Concentrated Large quantity of solute in
and National Formulery analysis solution solution
(NF) Purpose: For drug Saturated solution Excess of undissolved
manufacturing solute particles
Preparation of Filtration (1st)  Super saturated Greater concentration of
reagent grade water Distillation, Ion exchange, solution undissolved solute particles
Reverse Osmosis than does a saturated solution
Type I Rgt Water Min. interference of the same substance
Max. water purity Primary standard Highly purified
Used immediately (IUPAC) Measured directly to
For ultramicrochemical produce a substance of exact
analyses, measurements of known concentration
nanogram or subnanogram Secondary standard Low purity
concentrations, tissue or cell Concentration is
methods (microscopy) and determined by comparison w/ a
 primary standard Dimension Dade
Beckman ASTRA System (4 & 8)
Hitachi
Chapter 5 Bayer Advia
Roche Cobas Integra 800
The lab test has improved over the last decade  Roche Analytics P Module
to completely automate both routine chemical and hemat Automated Clinical Analyzer (ACA)
ological research. Star (Dade)
Dupont ACA
Abbott ABA-100 Bichromatic
For preanalytic, analytical and postanalytical  Analyzer
laboratory studies, automated solutions are available.  ABA-200
A laboratory's degree of automation should suit its requir VP Analyzer
ements and vary 
American Monitor KDA
from an individual analyser to a workplace to a fully auto
Olympus Demand
mated solution. 
Thin-Film 4 or 5 layers:
Analyzers -Spreading layer
The automation of preanalytics is largely dependent on t (Dry slide -Scavenger layer - Ascorbate
he identification  technology) Oxidase
of the principal collection container (often with a bar code -Reagent layer
).  -Indicator layer
When combined with other automated systems, analyser -Support layer
s can automatically perform several functions, reducing p Colored reaction  Reflectance
ractical  spectrophotometry
time considerably and allowing employees to focus on ex Examples: “KV2(75)”
ception management, problem solving and quality control Kodak Ektachem
. Vitros 750XRC
Vitros 550XRC
Carry over Transport of quantity of analyte or
There have been developed a range of automation appro rgt from one specimen rxn into
aches that fulfill flexible needs and tackle employee defic another, and contaminating a
its; other  subsequent one
key advantages include shorter processing time, fewer m Batch testing All samples loaded at the same
istakes, a cleaner working atmosphere and a more value time
-based testing orientation. Single test is conducted on each
sample
Parallel One specimen
Continuous Common reaction vessel testing More than one test is analyzed
flow analyzer Air bubbles: separates and cleans
Random Any sample
Glass coil: mix
access testing Any test
Examples: “STS” Any sequence
Simultaneous Multiple Analyzer STAT
(SMA) Sequential Multiple tests analyzed one after
Technicon Autoanalyzer II testing another on a given specimen
SMAC Open System other than manufacturer’s
Centrifugal Acceleration and deceleration of reagent system reagents can be utilized for
analyzer the rotor measurement
Advantage: Batch analysis Closed The operator can only use the
Examples: “RICC” reagent system manufacturer’s reagents
Cobas-Bio (Roche) Why did you choose to automate?
IL Monarch Why did you pick your current manufacturer?
CentrifiChem How did your lab function before automation?
RotoChem Do you ever have downtime?
Discrete Most popular How is the service response?
Analyzer Req. vol: 2-6 μL Are there any recurrent problems?
Uses positive-displacement pipets How did the staff react to the new automation?
Run multiple-tests-one-sample-at- What LIS do you have?
a-time What kind of remodeling did your lab perform?
Random access capability (STAT) Are there any expectations that were never met?
Examples: What were your ultimate goals after the
Vitros automation?
Did you realize all your goals? discussed in the fields of hematology, chemistry and micr
Would you buy this product again? obiology.
What is the one idea or “words of wisdom” that
you would give us?
Point-of-Care Testing (POCT)
1 . Definition: Performing diagnostic tests outside the main
laboratory and at or
Chapter 6 near patient care areas
2. Applications: POCT is designed to provide immediate
Point-of-care research refers to the type  laboratory test results
of clinical studies performed in patient care.  for immediate patient assessment and determination of
It involves medical examinations and numerous outside r appropriate treatment.
esearch facilities such  POCT may be used in neonatal intensive care, coronary care,
as the emergency department, operating room and inten intensive care,
sive care unit. or the emergency department.
3. Operators: Only waived laboratory tests can be performed
using pointof-
Simple tests (such as urinary dipsticks and blood glucos care instruments. Clinical laboratory technicians and clinical
e metres) in a doctor's office  laboratory
are exempt from most regulations including staff, qualific scientists must operate instruments that perform complex or
ation checks and strict quality control requirements.  high-complexity
The examinations in the Clinical Laboratory  laboratory tests.
Improvement Act are referred to as "waived."  4. Point-of-care (POC) instrument evaluations: All POC
The studies in hospitals come under the parent hospital  instruments must
or health  be evaluated in accordance with the Clinical Laboratory
care unit laboratory certificate and are usually subject to  Improvement
more strict conditions. Amendments of 1988 (CLIA '88). The values obtained from
POC instruments
must correlate with values obtained from larger laboratory
When a laboratory physician's office carries out moderat
instruments.
e-complexity research, special 
Linearity testing, calculation of control ranges, correlations of
personnel qualifications, including those for the labs dire
sample data,
ctor are required. 
and reference ranges must be done for each instrument.
5. Training: All POC instrument operators must be trained,
and training must
Clear policies and procedures to determine who is respo be documented.
nsible for  6. Quality control: All effective quality control systems must
each part of the program include process testing, training  be set up for each
non-laboratory employees.  POC instrument. The program must use appropriate standards
Oversight of the  and controls,
laboratory is compulsory.  statistical analyses, and a proficiency testing system. This
information must be
documented.
• Select analytical equipment is evaluated and shortcomi
ngs are 

Waived Test - Personnel have specific training and orientation to perform

- Simple test the test.
- CLIA waiver certificate required - Continuing competency must be documented
o CLIA : Clinical Laboratory Improvements Amendments of - Must have a laboratory director or technical consultant or
‘88 clinical consultant
- Personnel have specific training and orientation to perform - Must follow the manufacturer’s instruction to perform the
the test test
- Must follow the manufacturer’s instructions to perform the - At least 2 levels of control material daily
test - Calibration verification every 6 months
- Does not need Quality control - Proficiency testing
- Does not need accreditation - Defined standard procedure manual, corrective action,
- In the Philippines : DOH (for accreditation) record keeping
- Example : Provider-Performed Microscopy (PPM) – QC
Moderate Complexity Test materials do not exist
- Certificate of Accreditation required
High Complexity Tests
- Seldom performed as POCT (many additional stringent
standards, which are difficult to meet outside the clinical
laboratory)
- Requires Quality Control
- Requires a lot of training
- Requires supervision
POCT Device
1. Transportable, portable, handheld instrument
a. Blood glucose meter
b. Nerve conduction study device
2. Test kits
a. CRP
b. HBA1C (glycosylated haemoglobin)
c. HIV salivary assays
3. Small bench analyser or fixed equipment
Chosen Inspecting Certifying Agencies
1. CLIA : Clinical Laboratory Improvements Amendments of
‘88
2. JCAHO : Joint Commission on Accreditation of Healthcare
Organizations
3. CAP : College of American Pathologist
4. COLA : Commission of Office Laboratory Accreditation
5. DOH : Department of Health
Support Staff
1. Director
a. Can be a pathologist, Laboratory Scientist or MDs
b. Responsible for the policy, administrative financial and
technical decisions
2. Point of Care Coordinator (POCC)
a. Facilitates compliance with procedures, policies, and
regulatory requirements
b. Performs onsite review of patient testing, quality control,
maintenance logs and report problems
3. Designated contacts or trainers in non-laboratory
departments
a. Facilitates the efficient POCT program
b. Communication link between the POCC and testing staff
Validation
1. Accuracy : clinically correct result
2. Precision : clinically same result repeatedly
3. Sensitivity and specific, including interfering substance or
the likelihood that a positive test is truly positive and a
negative test is negative
4. Reportable Range or Linearity : upper and lower limits of
the test
5. Reference Range : expected result in a normal individual
6. Split Sample Correlation versus Reference Laboratory
Method : agreement between POCT and methods used in the
laboratory
POC Glucose
- Most frequently used home POCT test
- Principle :
o Reaction occurs between the blood and reagents in the
test strip.
o The meter measures the reaction and converts the reaction
to quantitative results
POC Chemistries and Blood Gases
- Principle:
o Measurement of potentiometric, amperometric or
conductometric changes via sensors (electrodes)
o The instrument receives the outputs from the sensors as
an electrical output and converts the electrical output and
converts to result.
- For multi-specimen analysis in a multi-sample reagent pack,
which includes the sensors.
- Singe-sample disposal cartridge and contains all the system
compartments
- Thin film
o A square chip which contains several 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
absorbance reading
o Reflectance spectrophotometry for absorbance reading
o Example : Kodak Ektachem
o Specimen : serum or plasma
o Employs dropwise dispensing
POC Hematology
- Principle for Hemoglobin Determination
o The cuvet cavity contains reagent deposited on its inner
walls that hemolyzed red cells when the blood sample is
drawn into the cavity by capillary action.
o The released haemoglobin is converted to azide
methemoglobin.
o The cuvet is then placed in the analyser, where absorbance
is measured and the haemoglobin level is measured.
POC Coagulation
- ACT (activated clotting time , the commonly employed POC
test for heparin therapy monitoring
o Prothrombin time
o Activated Partial Thrombin Time
POC Connectivity
- Connectivity is the ability to electronically document testing
- Device segment : upload data computer network to a
centrally located workstation (data manager)
- Interface connectivity : transmittal of test results from the
data manager to the laboratory information system (LIS) or
hospital information system (HIS)
POCT Limitation
- The linear range for the most POCT device is not as broad as
the main chemistry analyser
- Example : Glucometer loses linearity when glucose levels is
above 400 mg/dL of glucose values
- POCT data is less precise and not suited for monitoring
therapy
 nondisasis are calculated, the likelihood ratio increases. 
POCT : Available but Variable
- Cardiac troponins - Drugs or Toxicity
- Heparin - Magnesium • Predictive value defines the risk for good 
- HIV - Lactate or negative outcomes of disease or no disease. 
- Influenza - H. pylori With occurrence of illness, the predictive 
- Transcutaneous bilirubin - Microalbumin/creatinine value of a positive test increases.

CHAPTER 7
Bayes theorem uses information about test
For the empirical accuracy (using delta measures, linearit characteristics (sensitivity
y ranges etc.) and clinical sense for the patient (applying  and specificity) and disease prevalence (pretest
essential values, reference ranges, pre-test and posttest  probability) to obtain
probabilities, etc.), laboratory findings will undergo a post the posttest probability of disease, given a
-analytical two-step examination.  positive test. Similarly, it
can be used to determine the posttest probability
of no disease,
• Intervals of references are typically specified by a set of  given a negative test.
values that 95% of non-diseased persons  • Evidence-based medicine is a process by which
are covered, which means that 5% of non-diseased pers medical decisions can
ons that have test findings beyond the reference range. be made using as many objective tools as
possible; it integrates the
most current and the best medical evidence with
The sensitivity and specificity of the test are defined in  clinical expertise
the capacity of a test to distinguish disease against no di and patient preferences.
sease.  • Clinical practice guidelines (CPG) can be used to
Sensitivity in a person with the disease (truly  decrease practice
positive rate) is the likelihood of a positive outcome.  variability, establish monitoring parameters,
The specificity of a person without illness is  improve health, increase
the likelihood of a negative outcome (true negativity).  safety, and decrease cost.

Systematic reviews of clinical effectiveness are

transparent,
• Tests for screening need high 
sensitivity to avoid a missed case.  scientifically rigorous, and reliable syntheses of
literature that evaluate
specific medical interventions and are often used
to inform groups
To be sure of the 
developing CPGs.
diagnosis, confirmatory tests need high specificity.
CHAPTER 8
Altering a test cutoff has a reciprocal effect on The major purpose of performing analyte
sensitivity and determinations in the clinical
specificity. A cutoff can be lowered to include all laboratory is to aid in the diagnosis and
patients with management of disease and in
disease (100% sensitivity), but this reduces the
specificity (i.e., health assessment. In this regard, the clinical
increases false-positives). pathologist is often called
• Receiver operator characteristic (ROC) curves upon as a consultant to explain abnormal
plot the true-positive laboratory values, especially those
rate (sensitivity) versus the false-positive rate (1- that do not seem to correlate with one another,
specificity) and and to recommend or even
graphically present the range of sensitivities and to order laboratory tests that may lead to the
specificities at all test correct diagnosis in the
cutoffs. If two tests are compared, the more workup of patients for particular medical proble
accurate test is closer to
the upper-left-hand corner of the ROC curve. In a vast majority of cases, correct differential diagnosis c
an 
The probability of a test refers to the combination of the li be made from a systematic analysis of patient laboratory 
kelihood of a given test  profiles. 
outcome in the diagnosis with the likelihood of a similar t
est in the case of the nondisease. 
When the cut-off values for disease and 
• There are essentially four types of anemia: iron deficien contained uniquely in red blood cells. Because
cy, chronic  anemia can cause
disease anemia, hemolytic anemia and anemia with mac tissue hypoxia, it often produces such symptoms
rocytic / nutritional deficiency.  as fainting, fatigue, pallor,
These can be differentiated easily from each other  and difficulty in breathing.
by both the hematological profile and basic laboratory tes Practically, the best indicator for this condition is
ts.  a low red blood cell
count or number of red blood cells per volume of
whole blood. Although
• The causes of hyponatremia and hypernatremia can be  the reference range for the red cell count varies
easily determined by analyzing the urinary sodium, potas with age, sex, and population,
sium and osmolarity. it encompasses values from around 4 to 6 × 106
red blood cells per
cubic millimeter (cu mm) or microliter. This range
Liver function tests can differentiate between six different  may change somewhat
liver diseases: hepatitis,  depending on population.
cirrhosis, biliary disease, space-occupying liver lesions, p
assive inflammation, and hepatic insufficiency.  Microcytic Anemia
Common microcytic anemias include iron
deficiency anemia (IDA) and
• Renal failure can be easily detected by detecting elevat the thalassemias. Some hemoglobinopathies and
ed BUN (urea) and creatinine; the site of  the anemia of chronic
renal failure — glomerular or tubular — can be identified  disease (ACD) may also be microcytic. In our
from the serum to urine osmolality ratio. discussion, we focus on iron
deficiency anemia and the anemia of chronic
disease, a common differential
Tests of blood gas allow determination of the causes of  diagnosis for patients with microcytic anemia.
metabolic versus respiratory acidosis or alkalosis; there i Both anemias appear to
s a crucial relationship between partial  be disorders involving iron metabolism.
oxygen pressure and carbon dioxide, so that high levels  In IDA, there is a primary deficiency of iron
of carbon dioxide obstruct venous blood oxygenation in r available to the red cell
espiratory diseases lead to respiratory crisis.  (usually due to blood loss, but other causes
include dietary deficiency,
malabsorption, and pregnancy); chronic blood
• In the correct clinical sense, elevation of cardiac  loss should always lead to
troponin in serum is a symptom of myocardial infarction.  further investigation because it is commonly
associated with malignancy.
ACD, however, appears to be due to defective
• Serum C-reactive protein  iron utilization/metabolism
elevations signify an inflammatory illness. 
Serum Ferritin Levels
• Elevations of serum amylase and lipase point to acute Normally, there is an equilibrium between
pancreatitis intracellular and extracellular
ferritin. The lower the stored iron becomes, the
There are two forms of  lower the intracellular
endocrine disease covered: thyroid and adrenal.  ferritin is, and, consequently, the lower the
extracellular ferritin becomes.

T4 (or, better, free T4) and thyroid-stimulating hormone ( In addition to ferritin levels, serum iron and serum

TSH) serum levels may be used to diagnose primary or s iron-binding capacity
econdary hypothyroidism  (IBC) can be measured. On average, serum iron
or hyperthyroidism; cortisol and adrenocorticotropic horm is, of course, reduced in
one (ACTH) serum levels may be used to diagnose prim iron deficiency anemia and normal or sometimes
ary or secondary hypoadrenalism or hyperadrenalism. low in the anemia of
chronic disease. The IBC is a direct measure of
the protein transferrin,
Anemia, a common hematologic disorder, is which transports iron from the gut to iron storage
defined pathophysiologically sites in bone marrow.
as a decrease in the oxygen-carrying capacity of In iron deficiency anemia, the serum iron is
the blood. All oxygencarrying reduced, and the IBC increases.
capacity of the blood is due to the binding of
oxygen to hemoglobin Finally, use of automated procedures for
determination of cell counts and
indices enables us to obtain average erythrocyte
sizes and size distributions.
In iron deficiency anemia, there is a marked
dispersion in cell volumes
(sizes) so that the red cell distribution width
(RDW) increases, whereas it
generally remains within normal limits in the
anemia of chronic disease.
Normal RDWs occur in the range of 12% to 17%.
Normocytic Anemia
In these anemias, the red cells show normal MCVs
and MCHCs—that is,
they are normocytic and normochromic. Common
causes of normocytic
anemia include acute hemorrhage, hemolytic
anemia, marrow hypoplasia,
renal disease, and ACD. It may seem paradoxical
that acute hemorrhage
presents as normocytic anemia because it
involves major blood loss that is
associated with loss of iron stores. However, iron
depletion requires time
to develop; acutely, major blood loss presents as
normocytic anemia.
A very useful measurement in determining the
cause of normocytic
anemia is the reticulocyte count. Reticulocytes
are newly formed red blood
cells that have recently lost their nuclei but retain
high levels of cytosolic
mRNA dedicated to the synthesis of hemoglobin.
This RNA reacts with
the dye, methylene blue, to give a bright green
color, making it possible
to perform reticulocyte counts. In addition,
reticulocytes and other red
blood cell precursors react with the Wright stain
to give dark-staining
cytoplasm, in contrast to the cytoplasm of mature
red blood cells. This
darker staining of the cytoplasm of precursors is
termed polychromasia. In
cases, prominently in hemolytic anemia, where
there is a loss of red blood
cells due to peripheral destruction of these cells,
there is an increased
synthesis in bone marrow of red blood cells and
an early release of red
blood cell precursors, especially reticulocytes.
Hyperproliferative Normocytic Anemias
Hyperproliferative normocytic anemias,
associated with an increased reticulocyte
count, include both hemolytic anemia and the
anemia associated
with acute blood loss, while hypoproliferative
anemias, associated with a
decreased reticulocyte count, include such causes
as renal disease, ACD,
and, variably, bone marrow aplasia/hypoplasia.
Renal disease can affect
juxtaglomerular cells that are involved in the
synthesis of erythropoietin,
the growth factor that induces differentiation of
bone marrow hematopoietic
stem cells into the red cell line.
Hemolytic Anemia
Hemolysis is defined as the destruction of the red
cell membrane, causing
hemoglobin release. This may occur slowly as a
normal physiological
process or may be accelerated in pathologic
states. Many different underlying
causes exist for the decrease in survival/increase
in destruction
of red blood cells. These include membrane
defects (e.g., hereditary
spherocytosis), enzyme defects (e.g., glucose-6-
phosphate dehydrogenase
[G6PD] deficiency), hemoglobinopathies (e.g.,
sickle cell disease or
beta-thalassemia), immune destruction (e.g.,
autoimmune hemolytic
anemia or hemolytic transfusion reaction), and
nonimmune destruction.
Immune and nonimmune destruction include
destruction due to infectious
agents, toxic agents/drugs, physical agents,
hypersplenism, and microangiopathic
hemolytic anemias. Microangiopathic hemolytic
anemias are
caused by the mechanical destruction of red
blood cells, mainly in bone
marrow where they are synthesized, from such
factors as fibrin deposition
in the blood vessels of the bone marrow
microvasculature, fibrosis, or
malignancies such as leukemia, lymphoma, and
metastatic cancer. In addition,
mechanical destruction can result from
extramedullary causes such as
prosthetic heart valves.
Hemolytic anemia can be recognized from
hemoglobin in plasma/
serum and breakdown products of heme. Specific
laboratory measurements
that readily confirm the diagnosis of hemolytic
anemia are based on
the natural events that occur subsequent to
hemolysis. After erythrocyte
membrane breakdown, hemoglobin is extruded.
Thus plasma and
urine may contain free hemoglobin or its
degradation products. Free
hemoglobin may be present acutely in the plasma
(hemoglobinemia) or
urine (hemoglobinuria), while hemosiderin may be
present in the urine
(hemosiderinuria) in more chronic episodes of
hemolysis
Microangiopathic Hemolytic Anemia
As previously mentioned, red cell fragments
(schistocytes) may be present
on peripheral blood films due to mechanical (e.g.,
prosthetic heart valve)
or thermal (severe burns) destruction. Mechanical
rupture of red cells
within the microvasculature may also occur by
physical damage to red cells
in the microvasculature of bone marrow. This may
be due to spaceoccupying
lesions, such as metastatic tumors or leukemia or
lymphoma, to
myelofibrosis, or to the intravascular deposition of
fibrin strands upon
endothelial cell surfaces. Because red blood cells
are damaged and
destroyed, this process leads to so-called
microangiopathic (i.e., lesions in
the microvasculature) hemolytic anemia (MHA).
Bone Marrow Hypoplasia/Aplastic Anemia
This is a hypoproliferative anemia, with MCV and
RDW usually within
normal limits, that typically affects all peripheral
blood elements (red cells,
white cells, and platelets; see below). Immature
white cells and red cells
are not usually present on peripheral blood films.
Bone marrow biopsy is
commonly performed to obtain the diagnosis and
typically shows severely
hypoplastic/aplastic marrow with severe depletion
of all hematopoietic
marrow precursors. Aplastic anemia may be
primary/inherited or secondary/
acquired, with the latter due to chemotherapy,
chemical toxins, infection,
radiation, or immune dysfunction.
Myelodysplastic Syndrome
Another less common condition, but nonetheless
an important cause of
hypoproliferative normocytic anemia, is
myelodysplastic syndrome (MDS).
This syndrome, which often presents as a
normocytic anemia, although it
can also on occasion present as a mildly
macrocytic or microcytic anemia,
is refractory to treatment such as transfusions of
packed red blood cells. It
may present simply as a refractory anemia in its
early stages and is thought
to progress then to refractory anemia with ringed
sideroblasts and eventually
to so-called preleukemic stages, in particular
refractory anemia with
an excess of blasts in bone marrow (generally in
the myeloid or lymphoid
Anemia of Renal Failure
Another normocytic, hypoproliferative anemia is
the anemia of chronic
renal failure. Loss of the kidneys’ excretory
function produces an increase
in BUN and creatinine, as discussed below, as well
as a buildup of metabolic
by products. The resulting uremia appears to be
responsible for
changes in red cell shape, with burr cells
(echinocytes) and ellipsoidal cells
commonly present on peripheral blood films.
Identification of burr cells
on peripheral blood films during the course of
illness may signal the
development of renal dysfunction
Macrocytic Anemia
Macrocytic anemia can be diagnosed from the
hemogram from a low red
blood cell count and a high mean corpuscular
volume (MCV), often
exceeding 100 fL. By far the most common cause
of macrocytic anemia is
nutritional deficiency, such as vitamin B12 and/or
folate deficiency. Lack of
either factor is thought to disrupt DNA synthesis
but not RNA synthesis,
such that the nucleus and cytoplasm of the cell no
longer mature in synchrony.
Morphologically, the cell cytoplasm matures,
while the nucleus
remains immature, and the cell appears
megaloblastic. This lack of synchrony
produces hypersegmented neutrophils (five-lobed
nuclei in more
than 5% of the neutrophils or any neutrophil with
six or more lobes) and
large, oval-shaped red cells termed
macroovalocytes, both of which are
present on blood films of patients with
megaloblastic anemia
Infection
Infection is the most common cause of an
elevated WBC. An elevated
WBC of between about 10,000 and 20,000/μL
commonly points to an
infectious/reactive process. In general,
neutrophilia is associated with
infection (bacterial, fungal, viral), inflammatory
states (trauma, surgery),
certain drugs (e.g., corticosteroids), and
myeloproliferative conditions.
Exceptions to the neutrophilia seen in bacterial
infections are tuberculosis;
brucellosis; pertussis, where the predominant
cells are lymphocytes; and
infections, mainly in newborns, with Listeria
monocytogenes, for which a
monocytic response is predominant.
Eosinophilia is commonly associated with allergic
reactions, parasitic
infections, and hematologic malignancies (Brito-
Babapulle, 2003;
Fischbach et al, 2008; Brigden & Graydon, 1997;
Rothenberg, 1998).
Basophilia is also commonly associated with
hematologic malignancies
(e.g., chronic myelogenous leukemia [CML]), but
it may be seen in some
inflammatory states and allergic reactions.
Lymphocytosis is commonly
associated with acute viral infections, such as
infectious mononucleosis
(EBV infection); chronic infections, such as
tuberculosis, brucellosis, and
pertussis; and hematologic disease and immune
stimulation. Monocytosis
is commonly associated with hematologic
disease, such as acute monocytic
leukemia (M5 by the French-American-British, or
FAB, classification
system), acute myelomonocytic leukemia (M4, in
the FAB system), or
chronic myelomonocytic leukemia, as well as with
some infectious processes,
such as tuberculosis, rickettsia, and listeria.
Elevated WBC Due to Leukemoid Reaction
In patients who do not have leukemia, very high
white blood cell counts
(generally >50 × 103/μL) may produce a
peripheral blood film appearance
similar to leukemia. This is termed a leukemoid
reaction. The more common
type of leukemoid reaction is granulocytic,
although lymphocytic reactions
may also occur. The granulocytic type usually
reveals reactive neutrophils
present in the peripheral blood film, with a left
shift in the neutrophil series
(i.e., immature forms such as bands,
metamyelocytes, and myelocytes).
Elevated WBC Due to Chronic Myelogenous
Leukemia
At present, definitive diagnosis of chronic
myelogenous leukemia (CML)
rests on the demonstration of the Philadelphia
chromosome (i.e., the
BCR/c-abl translocation between chromosomes
#9 and #22) by either
cytogenetics or molecular techniques (e.g., see
George & Arber, 2003;
Hughes & Branford, 2003; Sattler & Griffin
Elevated WBC Due to Chronic Lymphocytic
Leukemia
When the lymphocytes appear normal but are
significantly increased in
number in an older individual, the possibility of
chronic lymphocytic
leukemia (CLL) must be considered. Again,
molecular techniques, such as
flow cytometric, immunophenotypic, and
cytogenetic/fluorescence in situ
hybridization analysis (Oscier et al, 2004;
Shanafelt & Call, 2004) can help
establish the diagnosis.
Both acute myeloid and lymphoid leukemias
present often as markedly
elevated white cell counts. In lymphoblastic
leukemia, numerous lymphoblasts
are seen on the peripheral smear. The myeloid
leukemias can present
in a myriad of forms, including myeloblastic,
promyelocytic, monoblastic/
monocytic, myelomonocytic, erythroblastic, and
megakaryoblastic. Again,
flow cytometric, immunophenotypic, and
karyotypic/molecular analysis
can help establish the diagnosis, as well as define
prognosis
Low White Cell Counts
Aplastic Anemia
Low WBCs, if accompanied by marrow hypoplasia
and two out of three
of anemia (with corrected reticulocyte count
<1%), neutropenia (neutrophil
count <500/μL), and thrombocytopenia (platelet
count <20,000/μL)
may be part of generalized pancytopenia
secondary to marrow failure
(Guinan, 1997; Marsh et al, 2003; Marsh, 2005).
Also known as aplastic
anemia, this condition may be primary/inherited
or acquired/secondary.
Known causes of the acquired type include
chemotherapy, drugs/toxins,
infections (including hepatitis), radiation, and
immune dysfunction
(Gordon-Smith et al, 2002). Cytogenetic study
may be utilized to rule out
myelodysplasia; if unsuccessful, molecular
techniques such as fluorescent
in situ hybridization (FISH) may be necessary for
chromosome analysis
(Guinan, 1997). The primary/inherited types may
not always be present
at birth (i.e., congenital), and diagnosis of this
type of marrow failure relies
heavily on clinical assessment in conjunction with
appropriate laboratory
evaluation (Alter, 1999; 2002).
Gram-Negative Sepsis as a Cause of Leukopenia
Leukopenia may also be seen in other conditions,
including gram-negative
sepsis. Interestingly, gram-negative sepsis with a
low WBC is often accompanied
by a cholestatic pattern in the liver (i.e., a mild
rise in bilirubin and
alkaline phosphatase),
factor. This latter factor is needed for platelet
aggregation.
Bleeding Time Measures Platelet Function.
Historically, the bleeding
time (BT) was utilized as a screening test for
platelet function. It should
be noted that the BT is not felt to correlate
accurately with or to predict
bleeding (DeCaterina et al, 1994; Gerwirtz et al,
1996) and is now uncommonly
used to screen for platelet function abnormalities
(Kottke-Marchant
& Corcoran, 2002; Posan et al, 2003).
Major Causes of Increased PT or aPTT. The
anticoagulant heparin,
which accelerates the inactivation of thrombin
and other coagulation
factors (such as factor Xa), preferentially blocks
the intrinsic system,
leading to prolonged APTTs but not significant
elevations in PTs. On
the other hand, the vitamin K antagonist
Coumadin preferentially
blocks factor VII in the extrinsic system, leading to
prolonged PTs but
not APTTs.
International Normalized Ratios for Patients
Being Treated with
Coumadin. The PTs of patients who are being
anticoagulated on
Coumadin must be followed carefully to ensure
that the therapeutic effect
is occurring but that the effective levels of
Coumadin are not sufficiently
elevated as to initiate hemorrhage
The Other Cause of Isolated PT or aPTT Is
Coagulation Factor
Deficiency. If the PT or APTT becomes elevated,
in the absence of treatment
with heparin or Coumadin, and the platelet count
is normal, it is
important to perform mixing studies of the
patient’s plasma with normal
plasma to determine if the coagulation time
normalizes—that is, whether
there is a factor deficiency
The Major Cause of Elevated PT and aPTT Is
DIC. If the platelet
count diminishes and the APTT and PT both rise,
the diagnosis of disseminated
intravascular coagulation (DIC) should be
entertained. This
diagnosis is confirmed by the finding of elevated
D-dimer, discussed in
Chapter 39, the D–D fragment of fibrin that
results from the proteolytic
action of plasmin upon the fibrin clot that forms
during intravascular
coagulation. D-dimer is detected in an assay
utilizing a very specific monoclonal
antibody to this cross-linked fibrin degradation
product
ABNORMALITIES IN CLINICAL CHEMISTRY:
CHEMICAL PATHOLOGY
ELECTROLYTE ABNORMALITIES
Hyponatremia
The four most common causes of hyponatremia
are given in Table 8-2,
together with a fifth, rare, cause: Bartter’s
syndrome. A sixth, metabolic
cause, diabetes mellitus, is also presented in this
table. In all forms of
hyponatremia, the chloride ion concentration is
also generally low because
chloride is the chief counterion for sodium
TABLE 8-2
Common Causes of Hyponatremia and Electrolyte
Patterns in Serum and Urine with Normal Renal
Function*
Cause Serum Na Urine Na (UNa) Urine
Osmolality Serum K 24-Hour UNa
1. Overhydration Low Low Low Normal or low Low
2. Diuretics Low Low Low Low High
3. SIADH† Low High High Normal or low High
4. Adrenal failure Low Mildly elevated Normal
High High
5. Bartter’s syndrome Low Low Low Low High
6. Diabetic hyperosmolarity‡ Low Normal Normal
High normal
Hypernatremia
Table 8-3 summarizes the three basic causes of
hypernatremia. Note that
each cause is the counterpart of a cause for
hyponatremia. These causes
are summarized as follows.
Common Causes of Hypernatremia and
Electrolyte Patterns in Serum and Urine with
Normal Renal Function*
Cause Serum Na Urine Na (UNa) Urine
Osmolality Serum K 24-Hour UNa
1. Dehydration High High High Normal Varies
2. Diabetes insipidus High Low Low Normal Low
3. Cushing’s disease or syndrome High Low
Normal Low Low
*All Na and K values are concentrations, except
for 24-hour UNa, which is the total number of
milliequivalents of Na excreted in 24 hours in the
urine
Pseudohyponatremia
This condition is usually caused by the presence
of excess lipids in serum.
No sodium ions are dissolved in lipids, which can
take up a considerable
volume of serum.
Hypokalemia occurs when serum potassium level is <3.0
mmol/L.
a) Results from decreased dietary intake, hyperaldosteronism,
diuretics, vomiting, diarrhea, laxative abuse, and excess insulin
which causes increased cellular uptake of potassium
2) Hyperkalemia occurs when serum potassium level is >5.0
mmol/L.
a) Results from increased intake, renal failure,
hypoaldosteronism,
metabolic acidosis, increased red blood cell lysis, leukemia,
chemotherapy
RENAL DISEASE
The four analytes that aid in the diagnosis of this
condition are BUN,
creatinine, calcium, and phosphate. It is amazing
that neither BUN
nor creatinine has any inherent relationship to
kidney function, but both
fortuitously are excellent indicators of renal
condition
BUN and Creatinine
BUN is blood urea nitrogen. Urea nitrogen is
generally measured in
plasma or serum, but it has historically been
referred to as BUN. The
formula for urea is H2N—CO—NH2. There are two
moles of nitrogen
per mole of urea. This is the end product of NH3
metabolism in the liver,
as discussed in Chapter 21. Urea is excreted by
the renal tubules at a rate
that is roughly proportional to the glomerular
filtration rate (GFR
Creatinine is secreted but is also reabsorbed to an
approximately equal
extent over a rather wide range for the GFR so
that the net effect is that
the amount filtered is the amount excreted
Hypocalcemia
- Plasma Ca 2+ level of <8.6mg/dL
- Primary hypoparathyroidism: aplasia or destruction of
parathyroid gland
- Hypomagnesemia causes hypocalcemia in 3 ways:
1. Inhibits secretion of PTH
2. Impairs PTH action on receptor site on bone
3. Causes Vitamin D resistance thereby lowering calcium
- Hypoalbuminemia – for every 1g/dL decrease in serum
albumin, 0.8mg/dL decrease in total calcium
- Causes: (CHARD)
1. Calcitonin
2. Hypoparathyroidism
3. Alkalosis
4. Renal failure
5. Vitamin D deficiency (Tetany – hypocalcemia)
Hypercalcemia
- Common in female geriatric patients
- Plasma Ca 2+ level of >10mg/dL
- Main cause: primary hyperparathyroidism (possibly tumor)
- Multiple myeloma: cancer of the plasma cells that lead over
expression of receptor activator of nuclear K B ligand which
promotes bone resorption forming bone lesions
- Causes: (CHIMPS)
1. Cancer
2. Hyperthyroidism
3. Iatrogenic causes
4. Multiple myeloma –indicator MM: Bence-Jones protein
Bone lytic lesions – found in the x-rays lesions of
patients with MM
About half of calcium circulating in blood is bound
to serum protein, mainly
albumin; the remainder is either chelated in tight
complexes with ions such
as citrate and oxalate or is in ionic complexes
with counterions such as
chloride, so-called “free” calcium
The pH of blood can affect the levels of
electrolytes in serum. In acidosis,
besides bicarbonate buffering, red cells also
buffer the excess H+ ions
by exchanging these for intracellular K+ ions, the
net effect being a mild
hyperkalemia. An attendant hypokalemia occurs
in alkalosis. Remember
also that acidosis can cause a mild
hypercalcemia; alkalosis can cause a mild
hypocalcemia.
All sodium ions must be neutralized by
counterions, most of which, in
blood, are constituted by chloride and
bicarbonate ions, and, to a lesser
degree, by phosphate, sulfate, and protein
carboxylate groups. Normal
serum sodium is about 140 mEq/L, chloride is
usually around 100 mEq/L,
and bicarbonate is around 2 mEq/L. The anion gap
is defined as Na+ −
(Cl− + HCO3
−), which, for normal individuals, is around 16.
This 16 mEq/L
really comprises the other counterions that
neutralize sodium but are not
measured in serum.
GLUCOSE ABNORMALITIES
The normal reference interval for fasting serum
glucose is generally
between 70 and 110 mg/dL. Recently there has
been discussion of lowering
the upper limit to 100 mg/dL (Nathan, 2009;
American Diabetes
Association, 2013). As described in Chapter 16,
the two basic abnormalities
that occur with serum glucose levels are
hyperglycemia, almost always
associated with diabetes mellitus, and
hypoglycemia due to iatrogenic
(overdose with insulin in the diabetic patient) or
other underlying causes
(such as reactive hypoglycemia due to
“hypersensitivity
insulinoma, etc.). To establish hyperglycemia, it is
vital to determine
whether the patient has (1) a fasting serum
glucose level greater than or
equal to 126 mg/dL, (2) a random serum glucose
level greater than or
equal to 200 mg/dL, or (3) a 2-hour postload
plasma glucose concentration
greater than or equal to 200 mg/dL during an oral
glucose tolerance test
Glucose reacts covalently with the α-amino group
of the beta chain of
hemoglobin to form a Schiff base that is then
reduced. This covalent reaction
product is called hemoglobin A1c or glycosylated
hemoglobin. Levels
of glycosylated hemoglobin change slowly over
time and therefore constitute
a stable and reliable indicator of serum glucose
levels over the past 2
to 3 months. Glycosylated hemoglobin levels are
measured as a percentage
of total hemoglobin; percentage levels that are
greater than 6.5% are
considered to be indicative of diabetes mellitus
(Nathan, 2009; American
Diabetes Association, 2013), and efficacy of
treatment is gauged by whether
this serum level is reduced to less than 6.5%. Of
all of the methods for
diagnosing, and especially for monitoring
treatment of, diabetes mellitus,
measurement of glycosylated hemoglobin levels
is perhaps the most accurate
and should be measured in conjunction with blood
glucose determinations
(Blincko & Edwards, 2001; Krishnamurti & Steffes,
2001; Kilpatrick,
2004; Nathan, 2009; American Diabetes
Association, 2013).
OTHER ABNORMAL LABORATORY FINDINGS
IN DIABETES MELLITUS
As discussed in the electrolyte section above,
under the influence of insulin,
whenever glucose is transported into the cell, it is
accompanied by potassium.
In diabetes, in the absence of insulin, blood
glucose is elevated, as is
potassium. Due to increased metabolism of fats,
there is a buildup of
acetoacetic acid, leading to metabolic acidosis. In
diabetes where the blood
glucose becomes exceptionally elevated—for
example, over 300 mg/dL—
serum osmolality becomes dangerously high and
can cause nonketotic,
hyperosmolar coma. In this condition, red (and
white) cell water flows
from the cells into the vascular volume, tending
to dilute analytes such as
sodium. Thus the nonketotic, hyperosmolar coma
patient may have a
hyperosmolar serum, hyperglycemia,
hyperkalemia, and hyponatremia. In
ketotic states, the patient will have, additionally,
a metabolic acidosis and
a large anion gap.
Serum glucose levels of higher than 60 mg/dL on
a series of random
fasting serum specimens strongly suggest
hypoglycemia. The low glucose
values should be associated with adrenergic
and/or neuroglycopenic symptoms,
which are relieved upon administration of glucose
(Whipple’s triad).
Glucose tolerance tests show that after an initial
sharp rise of serum
glucose levels, there is an abnormally rapid drop
to levels substantially
below 60 mg/dL. If hypoglycemia is suspected, it
is advisable to give the
patient a 5-hour glucose tolerance test because
the hypoglycemic “dip”
often is not seen until after 3 hours. Glucose
tolerance tests on patients
with suspected hypoglycemia should be
performed with great caution
because the procedure can induce severe
reactive hypoglycemia causing
loss of consciousness and even shock.
LIVER FUNCTION TESTS
Liver function is discussed in depth in Chapter 21.
Here we summarize a
set of principles that will enable the reader to
formulate differential diagnoses
of liver disease from the pattern of the so-called
liver function tests.
The liver may be considered as three systems:
1. A chemical metabolic system
2. A reticuloendothelial system based on Kupffer
cells (macrophages) that
line the sinusoids in the vascular system of the
liver that are concerned
with metabolism of hemoglobin released from red
blood cells
3. A metabolic system for the secretion of
bilirubin, a hemoglobin metabolite,
into the biliary tract
Because the liver is the site for the synthesis of
over 90% of the proteins
in the body, including all of the albumin, levels of
total protein and albumin
also reflect liver function. Unlike with the two
amino transferases, however,
liver pathology is recognized by decreases in the
serum levels of total protein
and albumin. These decreases do not occur
unless 80% or more of liver
tissue is destroyed, as in cirrhosis and fulminant
hepatic failure.
The liver is the only tissue in the body that can
metabolize ammonia,
the end product of amino and nucleic acid
metabolism via the Krebs-
Hennseleit cycle, in which ammonia is converted
to urea. When more than
80% of liver tissue is destroyed, as in the above-
mentioned conditions,
serum ammonia rises to toxic levels
preventing regeneration of liver tissue wherever
this has occurred, and
nodules of regenerating liver tissue, which are the
only source of any kind
of hepatocytic function. Thus in cirrhosis, as
shown in condition 2 in Table
8-5, almost the reverse pattern occurs from the
one seen in condition 1 in
Table 8-5 for hepatitis. Because in panhepatic
cirrhosis there is destruction
of more than 80% of liver tissue, with no
regeneration of damaged liver
tissue, the AST/ALT aminotransferases and LD
levels (all from the regenerating
nodules) tend to be normal or low, or occasionally
mildly elevated.
However, the total protein and albumin are both
abnormally low. Because
the liver is the sole site of ammonia detoxification
via the urea cycle and
because, in this condition, there is loss of
hepatocytic function, serum
ammonia levels are elevated. Because there is
insufficient viable liver tissue
remaining, and because fibrosis destroys the
cholangioles, both unconjugated
(indirect) and conjugated (direct) bilirubin tend to
be elevated.
Acute biliary obstruction caused by stones in
the biliary tree or by
neoplasms that block bile excretion results in
elevations in direct bilirubin
and biliary tract AP, along with the enzymes GGT
and 5′-N (see above).
All other liver function test results are normal. For
simple biliary obstruction,
therefore, the pattern is as shown in condition 3
in Table 8-5.
Space-occupying lesions of the liver are
characterized, for reasons
that are not well understood, by isolated
elevations of the enzymes AP and
LD. This pattern is shown in condition 4 in Table
8-5. The most common
causes of this condition are metastatic carcinoma
to the liver and hepatocellular
carcinoma.
Passive congestion of the liver is characterized
by a mild elevation of
aminotransferases (AST/ALT) and LD and, in more
severe cases, elevations
of total bilirubin and AP. This pattern is also seen
in infectious
mononucleosis, where the rise in bilirubin may be
marked. The general
passive congestion pattern is shown in condition 5
in Table 8-5.
Acute fulminant hepatic failure from a variety
of causes, which
include Reye’s syndrome, hepatitis C (Gill &
Sterling, 2001; Schiodt &
Lee, 2003), pan-hepatitic hepatitis (mainly from
hepatitis B-delta), severe
hypoperfusion states, cirrhosis, and multiple
abdominal surgeries, is discussed
in Chapter 21. This condition is total liver failure.
The overall
pattern (Sunheimer et al, 1994) is shown in
condition 6 in Table 8-5. It
appears as a combination of hepatitis and
cirrhosis.
CARDIAC FUNCTION TESTS
Diagnosis of Myocardial Infarction
and Acute Coronary Syndrome
Myocardial infarction (MI) and acute coronary
syndrome are discussed at
length in Chapter 18. Because acute MI (AMI)
requires rapid and accurate
diagnosis, especially now that new treatment
options with thrombolytic
agents are available, the clinical laboratory has
been called upon to provide
serum diagnostic tests that can make this
diagnosis at an early stage. Until
recently, laboratory diagnosis was based on serial
determinations of the
MB fraction of creatine phosphokinase (CK-MB).
Confirmation of the
diagnosis was provided by the so-called “flipped
ratio” of the isozymes of
lactate dehydrogenase (LD) 24 to 36 hours after
the initial acute event
and/or by observation of the characteristic time
courses for elevations of
the three enzymes: CK, aspartate
aminotransferase (AST), and LD.
These approaches have been replaced mainly by
two other analytes—
cardiac troponin (cTn) (Aviles et al, 2002) and
myoglobin (MY)—that
provide more rapid and specific diagnostic
capabilities (Morrow et al,
2007). MY is an oxygen-binding/transport protein
found in both cardiac
and skeletal muscle. Its relatively small size and
function allow for early
release from irreversibly damaged cells. However,
current methods of
measurement cannot distinguish MY’s tissue of
origin. Therefore, its use
is confined to screening patients for possible AMI;
positive results suggest
further workup for AMI.
Troponin is a regulatory protein complex in
muscle tissue; it comprises
three subunits designated troponin I (TnI),
troponin T (TnT), and troponin
C (TnC). Different genes encode TnI in skeletal
and cardiac muscle,
giving rise to isoforms that differ significantly in
sequence. In addition,
cardiac TnI contains an additional 31 amino acid
residues on its N-terminal.
Rapid, sensitive, and accurate immunoassays for
cardiac TnT and cardiac
TnI have been developed (Thygesen et al, 2012).
The most useful assays
appear to be for TnI because they are specific for
the cardiac form, and
TnT may be elevated in patients with renal failure
without MI (Diris et al,
2004). In AMI, cardiac TnI becomes elevated 4 to
8 hours after onset of
chest pain, reaches a peak at about 12 to 16
hours, and remains elevated
for 5 to 9 days. Values at or above 1.5 ng/mL are
considered to be suggestive
of AMI. However, because different assays (all
immunoassays) measure
different domains of TnT and TnI, reference
ranges differ, depending on
the antibodies used in the assays. Because cTn
levels rise relatively rapidly
and remain elevated for prolonged times,
troponin determinations have
replaced the so-called “flipped ratio” of the two
isozymes of LD, LD1, and
LD2 (LD2 : LD1 ratio rises to >0.75 and often
exceeds 1.0), which occurs
only about 36 hours after the onset of symptoms.
CK-MB is another biomarker that can be used in
diagnosing AMI. This
is an isozyme of creatine phosphokinase (CK) that
has three isozymes
composed of two chains (called the M and B
chains): MM, MB, and BB.
The MB fraction is predominantly found in cardiac
muscle (Roberts,
1997). To diagnose AMI from CK-MB serum levels,
it is important to
show both a rise in the concentration of CK-MB
and in the ratio of
CK-MB to total CK (also called the cardiac index)
(Thompson et al, 1988;
Woo et al, 1992). Because there is a small amount
of CK-MB in skeletal
muscle, diseases of skeletal muscle that cause
the level of CK-MM to rise
to high values will also cause the levels of CK-MB
to rise to high absolute
concentrations in serum, which can cause false-
positive values for CK-MB.
In addition, to increase both the sensitivity and
specificity of CK-MB in
the diagnosis of acute AMI, it has been found
necessary to perform serial
determinations of MB fraction (at 3- to 4-hour
intervals over a 12- to
16-hour period) that show a progressive rise that
reaches a peak, followed
by a fall to low levels.
This pattern is virtually 100% diagnostic of
myocardial
infarction (Lott, 1984; Wu et al, 1999). More
important, CK-MB
generally rises 4 to 6 hours, and sometimes only
2 hours, after th
97
PART 1
occur in the alpha (including alpha-1-antitrypsin,
alpha-2-macroglobulin)
and beta (including ferritin and C-reactive protein)
regions of the serum
protein electrophoretogram, as discussed in
Chapter 19 on serum protein
electrophoresis. In this regard, quantitative
determinations for serum
C-reactive protein (CRP) are very helpful in
recognizing acute inflammatory
states. Recently, specific antibodies to CRP have
allowed for highly
sensitive measurement of CRP (termed high-
sensitivity CRP or hs-CRP)
at lower concentrations than were previously
measurable (Roberts et al,
2000). Currently, elevated levels of hs-CRP
appear to serve as an early
marker of inflammation and may have utility in
assessing cardiovascular
risk for stroke and MI (Abrams, 2003; Ridker,
2003; Libby & Ridker, 2004).
Fibrinogen, also an acute phase reactant, may
additionally increase.
Often, the platelet count tends to rise, and
platelets themselves have been
considered as “acute phase reactants.” In
addition, in both acute and
chronic inflammatory conditions, erythrocytes
exhibit increased mobilities.
Thus there is an increase in the erythrocyte
sedimentation rate (ESR).
Recent studies suggest that the best marker for
inflammation, especially as
a guide to therapeutic efficacy, is CRP (Crowson
et al, 2009).
Finally, one common cause of acute inflammation
is gout—that is,
hyperuricemia, or elevations of uric acid in serum.
Uric acid crystals cause
a severe, acute arthritic condition (gout). Serum
levels of uric acid over
7.5 mg/dL are indicative of this condition. Less
constant findings are the
presence of uric acid crystals in the urinary
sediment (see Chapter 27) or
in joint fluid (see Chapter 28).
ENDOCRINE FUNCTION TESTING
We noted that abnormal functioning of the thyroid
and adrenal cortex can
give rise to important abnormal laboratory
findings. Hypothyroidism, for
example, can give rise to macrocytic anemia,
while hypoadrenalism gives
rise to electrolyte abnormalities—that is,
hyponatremia and hyperkalemia
and an acidosis. Hyperadrenalism produces the
opposite effect—that is,
hypernatremia, hypokalemia, and an alkalosis. It
is therefore important to
confirm that these endocrine glands are
malfunctioning. The important
topic of endocrine function testing is discussed in
Chapter 24. Here we
present simple principles for the laboratory
diagnosis and identification of
the site of origin of thyroid, adrenal, and
parathyroid conditions.
Principle
All endocrine systems involve a stimulating
hormone that is synthesized
and secreted into the blood from one tissue, such
as the pituitary, parathyroid
glands, and so on. These hormones are delivered
to their targets, which
may be the thyroid gland, adrenal glands, or a
nonendocrine target such as
the renal tubules, in the case of the parathyroid
glands. In all cases, for
normally functioning endocrine systems,
elevations of the hormones or, in
the case of the parathyroid glands, elevations in
serum ionized calcium
levels, will result in decreased levels of the
primary hormones. Conversely,
decreased levels of the target, such as thyroxin
(T4) or ionized calcium, will
result in increased levels of the stimulating
hormones. For example, high
levels of circulating thyroxine or T4 will result in
low levels of thyroidstimulating
hormone (TSH), a condition called primary
hyperthyroidism,
and, conversely, low levels of T4 will result in high
levels of TSH, a condition
called primary hypothyroidism. In contrast, if the
pituitary gland
secretes an excess of TSH due to an adenoma or
other pathological condition,
this increase in TSH will result in elevated T4. This
condition is also
referred to as secondary hyperthyroidism. Thus if
both T4 and TSH levels
are elevated, a diagnosis of pituitary disease, —
that is, pituitary adenoma
in which there is hypersecretion of TSH—can be
made. Conversely,
decreases of both TSH and T4 point to pituitary
hyposecretive states, also
referred to as secondary hypothyroidism. These
considerations may be
summed up as the following principle: Changes in
opposite directions of the
stimulating and target hormones (e.g., T4) or
effectors (e.g., ionized calcium) point
to endocrine target (e.g., thyroid gland) disease,
while changes of the two hormones
in the same direction point to stimulating
hormone gland (e.g., pituitary) disease.
Thyroid Function
Thyroid function tests are the most commonly
ordered tests for endocrine
function. It is important to note that in a hospital
population (as opposed
to an ambulatory population), thyroid screening
tests may be diagnostically
misleading. This is due to endocrine stress
responses (as well as medications)
that may affect hormone levels (Van den Berghe,
2003). As discussed
in Chapter 24, the thyroid gland synthesizes
thyroid hormone, tetraiodothyronine,
or T4, with four iodines, which requires iodine
uptake by the
gland. This activity is strongly stimulated by the
peptide hormone TSH
from the pituitary. In the periphery, T4 is
converted to T3 (three iodines).
Over 99% of T4 is bound to serum proteins—that
is, thyroid-binding
of chest pain, and it peaks within 12 hours.
Therefore, MY and CK-MB
were recommended (Wu et al, 1999; Alpert et al,
2000; Fromm & Roberts,
2001; Lewandrowski et al, 2002) for use as early
markers of AMI. However,
elevated TnT and TnI levels are more specific for
cardiac injury than are
elevated CK-MB levels. Like CK-MB, they rise
within 4 to 6 hours, and
sometimes within 2 hours, after the onset of chest
pain.
Current protocols for the laboratory diagnosis of
AMI vary and are
evolving as assays for cTn improve (see Chapter
18). In some medical
centers TnI or TnT is used exclusively, while in
other centers, both troponin
and CK-MB are used together.
Diagnosis of Congestive Heart Failure
Until recently, this condition was diagnosed
strictly on the basis of symptomatology
and/or as a result of procedures such as
echocardiography, but
more recently a biomarker for this condition is the
brain form or B-type
natriuretic peptide (BNP), which has been
approved as a definitive test for
this condition and appears to be an excellent
marker for early heart failure.
This test may also be both diagnostically and
prognostically significant in
patients presenting with acute dyspnea and chest
pain. The differential
diagnosis in these patients includes dyspnea
caused by chronic heart failure
(signs and symptoms of which are typically
nonspecific) versus other causes
of acute dyspnea (e.g., pneumonia, carcinoma,
effusion, asthma). Normal
levels (i.e., a high negative predictive value for
this test) appear useful in
excluding a cardiac etiology in these patients.
Levels of BNP may also be
an independent predictor of arrhythmia, stroke,
and death (Clerico &
Emdin, 2004; Clerico et al, 2012; Ishii et al, 2003;
Mueller et al, 2004;
Prahash & Lynch, 2004; Wang et al, 2004; Winter
& Elin, 2004; Novo
et al, 2009). Studies suggest that serum BNP
levels are useful in evaluating
the efficacy of treatment of congestive heart
failure (Novo et al, 2009).
SKELETAL MUSCLE FUNCTION
As noted in the Cardiac Function Tests section
above, an elevated serum
CK-MB is a useful marker for myocardial disease.
The MB fraction is one
of three possible isozyme dimer states of the M
and B chains. The MM
fraction is by far the most prominent of the three
isozymes; this isozyme
of CK is found in skeletal muscle. Thus diseases
affecting skeletal muscle
are often accompanied by substantial serum
elevations of this CK isozyme.
The most common cause of skeletal muscle
disease is myositis, a general
inflammation of skeletal muscle fibers affecting
the sarcolemma. This
condition has many causes, including viral causes,
especially coxsackie B
viral infections that predominantly affect skeletal
muscle, autoimmune
conditions, and drugs. One of the most common
causes of drug-induced
myositis is the statin class of cholesterol-lowering
drugs such as lovastatin.
Another closely related condition is
rhabdomyolysis, which is the breakdown
of skeletal muscle due to such causes as trauma,
drug abuse (especially
intravenous drug abuse from needle damage to
skeletal muscle),
severe, generalized myositis due to viral
infections, and third-degree burns.
Thus serum CK levels should always be obtained
in patients complaining
of generalized muscle pain. If these levels are
elevated, efficacy of treatment
may be followed by serial levels of total CK.
As noted in the Cardiac Function Tests section
above, both cardiac and
skeletal muscle contain the oxygen transport
protein myoglobin. In conditions
of acute myositis of skeletal muscle, myoglobin is
released from
damaged muscle cells, giving elevated levels of
this protein in serum.
Because this protein has a relative low molecular
mass, it is filtered in the
kidneys and excreted in the urine, causing
myoglobinuria. Because myoglobin
is toxic to renal tubules, high serum
concentrations of myoglobin
can cause renal failure, a potentially serious
complication of myositis and
especially of rhabdomyolysis (Melli et al, 2005;
Ouyang et al, 2009).
PANCREATIC FUNCTION TESTS
Elevations in serum pancreatic amylase and
lipase are definitive markers
for pancreatic disease. The most common cause
for such increases in the
serum levels of these enzymes is pancreatitis. In
acute pancreatitis, both
enzymes are elevated. Because amylase can also
be produced by the salivary
glands, amylase is less specific than lipase as a
marker for pancreatitis.
Elevations in the latter enzyme are definitive for
pancreatic disease.
MARKERS FOR INFLAMMATORY CONDITIONS
As discussed previously in the hematology
section, increases in the white
blood cell count, especially with a predominance
of neutrophils, indicate
acute infection. In most acute inflammatory
conditions, as noted previously,
acute phase reactant proteins are also found to
be increased. These proteins
globulin (TBG) and albumin. However, it is the
free T4 (<1% of total T4)
that exerts all biologic effects. Accurate methods
exist that directly measure
serum free T4. While, most commonly, both total
and free T4 rise and
fall together in serum, there are circumstances in
which total T4 may be
elevated but T4-binding proteins may also be
elevated so that free T4
levels are normal. This results in a euthyroid state
in which TSH levels
are also normal. Of prime importance, high T4
(more correctly, high free
T4) causes inhibition of TSH secretion, while low
T4 (more correctly, low
free T4) causes elevation of TSH. In primary
hypothyroidism (primary
thyroid gland disease), T4 is present at low serum
levels, while TSH
becomes elevated. More important, often, in
subclinical hypothyroidism,
T4 can be within the reference range, but TSH is
elevated. In ambulatory
patients, elevated TSH in the presence of low
levels of T4 is diagnostic of
primary hypothyroidism. In secondary
hypothyroidism, TSH is low due
to pituitary failure, resulting in low T4.
In primary hyperthyroidism (primary thyroid gland
disease), excess T4
is secreted by the thyroid gland, which is the
source of the problem. As a
result, TSH is decreased, and T4 is elevated. If,
however, there is a primary
lesion in the pituitary gland—for example,
hyperplasia or adenoma—then
TSH is oversecreted. Because TSH is elevated, T4
secretion is elevated.
This is secondary hyperthyroidism (secondary to
pituitary disease). These
conditions and the patterns of T4 and TSH levels
that occur in them are
summarized in Table 8-6.
Adrenal Function
As discussed in depth in Chapter 24, the adrenal
gland is divided anatomically
into two endocrine glands: the adrenal cortex and
the adrenal medulla.
Adrenal cortical hormones are steroid hormones
of three basic types:
mineralocorticoids, like aldosterone, that regulate
sodium and potassium
ions in the distal convoluted tubule, as discussed
above; the glucocorticoids,
like cortisol, that are gluconeogenic; and the sex
hormones—that is,
the estrogens and the androgens. The adrenal
medulla is a neuroendocrine
gland that secretes epinephrine and
norepinephrine that act on the sympathetic
nervous system. By far the most commonly
ordered serum analyte
used to measure adrenal cortical function is
cortisol. Cortisol secretion by
the adrenal cortex is stimulated by the pituitary
hormone adrenocorticotropic
hormone (ACTH). ACTH secretion, in turn, is
stimulated by the
hypothalamic hormone corticotropin-releasing
hormone (CRH). Its
serum levels are under diurnal control such that
serum ACTH levels peak
at about 200 pg/mL in the morning hours (about
7:00 am), while they
decline to their lowest values of around 100
pg/mL at midnight. Cortisol
secretion follows ACTH secretion such that its
serum levels are highest at
around 8:00 to 9:00 am. Cortisol inhibits ACTH
secretion by the pituitary
gland both by directly blocking pituitary ACTH
secretion and by inhibiting
CRH secretion by the hypothalamus.
Therefore, if the adrenal cortex hypersecretes
cortisol secondary to
such conditions as adrenal hyperplasia, adenoma,
or carcinoma, serum
cortisol levels increase and block ACTH secretion.
This condition is called
primary hyperadrenalism. Serum cortisol levels
are elevated (Cushing’s
syndrome), but ACTH levels decrease.
On the other hand, as with thyroid hormone
secretion, if ACTH levels
are elevated by an ACTH pituitary tumor
(Cushing’s disease) or as ectopic
ACTH secretion—for example, by a nonpituitary
tumor—cortisol levels
will also rise, producing secondary
hyperadrenalism. Both cortisol and
ACTH are elevated in this condition. Here, notice
that both cortisol and
ACTH change in the same direction (both are
elevated), pointing to pituitary,
not adrenal, disease. In cases of Cushing’s
disease or syndrome, the
diurnal variation of serum ACTH is absent.
In addition to measurements of serum cortisol, it
is often desirable to
measure urinary free cortisol in cases of
hypercortisolemia. Normally,
almost all cortisol is bound to serum protein,
mainly transcortin, while, in
hypercortisol states, cortisol exceeds the capacity
of transcortin to bind to
it and is consequently filtered by the kidneys. The
reference range for
morning serum cortisol is approximately 10 to 25
μg/dL, and for urinary
free cortisol, it is approximately 24 to 108
μg/24hours for healthy males.
In cases of hypercortisolism, especially where
ACTH levels may remain
in the reference range or have “borderline” high
or low values, it is often
desirable to perform the dexamethasone
suppression test. Dexamethasone
is a potent glucocorticoid that strongly suppresses
normal pituitary ACTH
secretion. This can be accomplished with low-
dose dexamethasone. If
low-dose dexamethasone results in diminished
serum cortisol levels and
low values for urinary free cortisol, pituitary
function is most likely normal
while the adrenals are hypersecreting cortisol—
that is, primary hyperadrenalism.
This test may further be used to distinguish the
possible source of
primary hyperadrenalism—that is, hyperplasia
versus adenoma or carcinoma.
High-dose dexamethasone will generally lower
serum cortisol levels
in adrenal hyperplasia, while it will have no effect
in adrenal adenoma or
carcinoma.
Conversely, in pituitary failure, serum ACTH levels
decrease, as do
serum cortisol levels, because ACTH stimulation
of the adrenal gland
decreases. This condition is referred to as
secondary hypoadrenalism. If
the adrenal gland function is compromised
(primary adrenal insufficiency),
serum cortisol levels decrease, resulting in less
inhibition of pituitary secretion
of ACTH and, consequently, in elevated serum
levels of ACTH. This
condition is referred to as primary
hypoadrenalism or Addison’s disease.
These conditions and the patterns of serum
cortisol and ACTH levels in
these conditions, including dexamethasone
suppression test results, where
relevant, are summarized in Table 8-7.
Parathyroid Hormone and Vitamin D
In the renal section above, it was noted that
parathyroid hormone (PTH)
induces elevated serum calcium levels by causing
increased secretion of
phosphate in the renal tubules and by promoting
increased calcium ion
absorption in the gut. Hypocalcemia with elevated
PTH points to causes
having to do with calcium metabolism—for
example, alkalotic states,
decreased albumin, low dietary calcium and/or
lack of absorption of
calcium from the gut, and low vitamin D levels.
Hypocalcemia with low
serum PTH levels points to parathyroid gland
dysfunction. In rare
instances, hypocalcemia that is not responsive to
calcium infusions and
vitamin D supplements can be caused by
secretion of high levels of calcitonin,
a serum calcium–lowering peptide that blocks
osteoclastic resorption
of bone, thereby blocking release of calcium in
the circulation.
Calcitonin is synthesized and secreted from the
medullary cells of the
thyroid. In medullary thyroid carcinoma, high
levels of calcitonin are
sometimes secreted. At high levels of calcitonin,
hypocalcemia will result.
Hypercalcemia with low PTH again points to
primary calcium metabolic
causes, as in the “CHIMPS” mnemonic mentioned
in the renal
section above and acidotic states, while
hypercalcemia with elevated PTH
indicates parathyroid diseases such as adenoma
(most commonly) or carcinoma
(uncommonly). These conditions and the patterns
of serum
calcium and parathyroid hormone levels in serum
in these conditions are
summarized in Table 8-8.
Vitamin D
Vitamin D induces absorption of calcium from the
gut by acting on
vitamin D receptors in the cells lining the gut. It is
produced from
7-dehydrocholesterol
in skin by ultraviolet (UV) light that induces the
synthesis of cholecalciferol. In the liver,
cholecalciferol is converted to 25-
hydroxycholecalciferol, which has a half-life of
several weeks. This form
is further hydroxylated in the kidneys to 1,25-
dihydroxycholecalciferol, the
active form of vitamin D. The half-life of active
vitamin D is on the order
of hours. Thus in serum assays for vitamin D, the
analyte whose serum
level best reflects vitamin D levels is the
precursor, 25-hydroxycholecalciferol
(Zerwekh, 2008). This may give falsely elevated
levels of vitamin D in cases
of renal failure where conversion of the precursor
to active vitamin D is
impaired. However, 25-hydroxycholecalciferol
itself has some effects on
calcium reabsorption, although its affinity for the
vitamin D receptor is
only about one-one thousandth of that for the
fully active form. The reference
range for serum 25-hydroxycholecalciferol, also
called 25-hydroxy
vitamin D, is 30 to 100 ng/mL, although this is
method-dependent
(Yates et al, 2008).
Over the past few years, there has been a large
increase in requests for
serum vitamin D levels, especially in the pediatric
and geriatric populations.
This is due at least in part to the availability of
chromatographic, mass spectroscopic, and
immunochemical methods that
quantitate serum vitamin D levels. In the pediatric
population, vitamin D
levels are determined and followed for the
diagnosis and treatment of
vitamin D–dependent rickets. In the elderly,
vitamin D levels are determined
in the treatment of such diseases as osteoporosis through analysis of variance.
and osteomalcia, • Regression analysis between two continuous
because vitamin D supplements have been found variables is usually done
to diminish the extent by least squares fit to a straight line. Applications
and severity of these diseases. of regression
The discovery that many cells, besides epithelial analysis are common when different analytic
cells of the gut—the methods for validation
principal site of vitamin D activity—have are compared in clinical laboratories.
abundant receptors for vitamin
D suggests that this vitamin has effects that go CHAPTER 10
beyond regulation of
calcium homeostasis (Bilke, 2009). Indeed, it has Practicability Method is easily
been found that vitamin repeated
D induces apoptosis in breast cancer and other Reliability Maintain accuracy and
malignant cells and has precision
been used to treat such diseases as multiple Intralab/Interlab QC Daily monitoring of
sclerosis, heart failure, psoriasis, accuracy and precision
asthma, Crohn’s disease, and many other Interlab/External QC Proficiency testing
conditions. Thus there has (Reference lab)
been an increased demand for vitamin D levels Long-term accuracy
for patients with a wide Difference of >2: not in
variety of conditions. agreement w/ other lab
QC materials Available for a min. of 1
CHAPTER 9 yr
Bovine control Preferred (Human:
Nominal variables may only take on a small number of va materials biohazard)
lues (or  Not for immunochem,
categories) for statistical analysis, while continuous varia
dye-binding and bilirubin
bles are used for quantitative data reporting. 
Matrix effect Improper product
manufacturing
Unpurified analyte
• Input (cause) are considered independent variables,  Altered protein
and output (effect) are considered dependent variables. 
Precision study First step in method
evaluation
Nonlab. personnel 29% of errors (lab
• Continuous data distribution is defined by a central tren results)
d 
SD Dispersion of values
measure (e.g., mean or median) and dispersion measure 
from the mean
(standard deviation). 
CV Index of precision
Gaussian distributions arise from a theorem 
of mathematics and are thus parametric.  Relative magnitude of
A common application of descriptive statistics  variability (%)
is the identification of reference ranges. Variance SD2
Measure of variability
Inferential statistics Compare means or SD of
Statistical tests such as comparisons of different 2 groups of data
groups of data points T-test Means of 2 groups of
may be parametric (i.e., assume Gaussian data
distributions; example is F-test SD of 2 groups of data
Student t-test) or nonparametric (i.e., make no Cumulative Sum V-mask
assumption of Graph (CUSUM) Earliest indication of
distributions; example is test based on rank systematic errors (trend)
order). Youden/Twin Plot Compare results
• Confidence intervals are preferable over point obtained from diff. lab
estimates to express Shewhart Levey- Graphic representation
level of certainty in the calculation of any Jennings Chart of the acceptable limits of
statistical parameter. variation
• Nominal data are conveniently analyzed with Trend Gradual loss of reliability
proportions using the Cause: Deterioration of
chi-square test. reagents (Systematic error)
• The effects of multiple factors in a model Shift Values: one side or
system can be assessed either side of the mean
 Cause: Improper Quality assurance samples are checked on a daily 
calibration (Systematic basis to ensure proper performance of a laboratory proce
error) dure. 
Outliers Values: far from the main
set of values
Highly deviating values • Analysis of the results of quality assurance is based on 
Random or systematic acceptance criteria that define 
errors bias, trend in bias or imprecision that exceeds the perfor
Kurtosis Degree of flatness or mance attributes required of the system. 
sharpness
Precision Random error
Accuracy Systematic error • In the case of an inappropriate quality control outcome, 
Random error Causes: corrective action is taken to address the issue 
(Imprecision; -Mislabeling of the process, and all patient outcomes are repeated fro
Indeterminate) -Pipetting error m the time of the previous acceptable quality control outc
-Improper mixing of ome.
sample and reagents
-Voltage/Temperature
Quality assurance samples should not be used to check t
fluctuation
hat two processes, or two 
-Dirty optics
separate lots of reagents, yield the same results for patie
Parameters: SD and CV
nt samples, due to commutability limitations. 
Systematic error Causes:
(Inaccuracy/Determinate) -Improper calibration
-Deterioration of
• Proficiency testing offers external verification that a labo
reagents
ratory is using 
-Contaminated solution
the tool properly and in compliance with the requirements 
-Sample
of the supplier.
instability/unstable reagent
blanks
-Diminishing lamp II. LABORATORY QUALITY ASSESSMENT
power A. Definitions
-Incorrect sample and 1. Total quality management (TQM) is a managerial process
reagent volume that focuses on
Parameter: Mean improvement of the quality of all factors that affect laboratory
Multirule Shewhart Control rules + Control testing and
procedure chart performance. It consists of five elements: quality laboratory
Test method Westgard: at least 40 process, quality
samples control, quality assessment, quality improvement, and quality
Reference method Westgard: preferably planning.
100 samples
Analytical Run Control and patient 2. Quality assessment (QA): This is a systemic laboratory
specimens assayed, program,
evaluated, and report encompassing preanalytical, analytical, and postanalytical
together factors, that
Physiologic Limit Referred to as absurd monitors excessive variation in specimen acceptability, test
value methodologies,
POCT Performed by nonlab instruments, reagents, quality control, and personnel
personnel competencies. This
process is used to ensure accurate patient test results.
Quality Assurance Tripod:
3. Preanalytical error occurs during sample collection and
Program development
transport before
Assessment and
sample analysis and can include sample preparation and
monitoring
storage conditions.
Quality improvement
4. Analytical error occurs during the testing process and
Quality Patient Care Test request forms, clear includes problems
instruction for patient prep., related to reagents, instruments, controls, calibration,
specimen handling… performance of
Reference Range/ At least 120 individuals personnel, etc.
Interval Range/ Reference should be tested in each age 5. Postanalytical error occurs after the test is performed and
Values and sex category refers to clerical
errors, reporting of results, test interpretation, etc.
6. Accuracy is a measure of a laboratory test result's closeness to determine acceptability of results.
to the true value. a. Assayed control: Values are assigned by the manufacturer.
7. Precision is realized when repeated laboratory test results b. Unassayed control: Values are determined by each
yield the same individual laboratory
number; reproducibility. for their methods/instruments.
8. Reliability refers to the ability of laboratory testing to 17. Westgard multirules are statistical "rules" applied to
maintain accuracy and graphical summaries
precision over an extended period of time. of numerical quality control data to assess the acceptability of
9. Quality control (QC): A system used to monitor the such data.
analytical process to 18. Six Sigma is a data-driven, business approach to
detect and prevent errors that would impact on the accuracy performance improvement;
and precision of it is oriented toward process identification and process
laboratory test results; includes both statistical and improvement.
nonstatistical parameters. 19. Lean principles are an improvement trend to make work
a. Internal QC is performed by laboratory personnel using faster by providing
control materials ways to streamline through the removal of waste.
of known values and comparing the control values to 20. ISO 9000 Standards were established by the International
established, acceptable Organization for
ranges. The control material values are assessed using Levey- Standardization as a series of four standards for quality
Jennings management.
control charts and Westgard multirules to detect errors. B. Specimen Quality
b. External QC is performed by laboratory personnel when 1. Test result quality depends on the quality of the sample
analyzing specimens submitted.
sent to the laboratory by an external agency, and the results 2. Specimen quality depends on:
generated a. Patient preparation
are submitted to the agency for assessment. This type of b. Labeling procedures
assessment is c. Timing of specimen collection
known as proficiency testing. It is required by federal d. Special collection instructions
regulations for all e. Specimen handling and transport requirements
laboratories providing results for human diagnosis and/or f. Criteria for unacceptable specimens
treatment. C. Operating Instructions
10. Linearity check determines the lowest and highest values 1. Laboratory procedures should contain the following
that can be information: test
accurately measured by a particular method. This is an name, method principle, significance of test, patient
example of a preparation, test
nonstatistical QC parameter. specimen requirements, equipment and materials needed,
1 1 . Random errors affect precision, are unable to predict reagent preparation,
because they have no test procedure, calculations, quality control procedures,
known pattern, and may alternate between a positive or reference intervals,
negative direction. panic values, limitations of the procedure, and references,
12. Systematic errors are predictable and cause a constant including the
difference in results instrument user's manual. Each procedure must be reviewed,
that are consistently positive or negative or stay the same. Such signed, and
errors may be dated annually.
due to incorrect calibration, deteriorated reagents, instrument 2. Instrument user's manual and instrument maintenance
malfunction, etc. manual must
13. Delta check assesses the patient's most recent result for a be available, and all maintenance performed must be
particular test as documented.
compared to the patient's previous value; the difference D. Selecting Instruments
between the test 1. Selection criteria should include instrument cost, reagent
results (delta) is calculated and compared to established limits. cost, throughput,
14. Reference ranges are determined by each laboratory to fit technical support, personnel training, method linearity, range
their particular of methods
population. Intervals are generally constructed by adding and available, test methodologies, analytical sensitivity and
subtracting specificity, etc.
2 standard deviations from the mean. 2. Instruments are evaluated to determine instrument and
method accuracy,
15. Standard is material of known concentration (should be precision, systematic error, linearity, and calibration stability.
traceable to NIST)
that is used to calibrate an instrument or develop a standard E. Statistical Analysis/Chapter 9
curve. 1. Arithmetic mean (x) of a set of numbers is obtained by
16. Control is material of known value that is analyzed with adding all the
patient samples
numbers in the set and dividing the sum by the number of (high value) and the mean —2 standard deviations (low
values in that set. value) to include
2. Median is the middle value in a set of numbers that are 95% of the "healthy" population.
arranged according
to their magnitude.
3. Mode is the most frequently obtained value in a set of
numbers. Internal Quality Control
4. Standard deviation (s) reflects the variation of data values 1. Purpose: It is a comprehensive program involving statistical
around the analysis of
mean. control materials, which are analyzed with a batch of patient
5. Variance (s2) reflects dispersion around the mean and is the samples to
square of the determine acceptability of the run.
standard deviation. 2. Control material
6. Coefficient of variation (CV) reflects random variation of a. Commercially manufactured lyophilized or liquid
analytical materials that have the
methods in units that are independent of methodology, because same matrix as patient specimens and are used to evaluate the
it is a test process.
percentage comparison of the standard deviation divided by b. Control materials are handled exactly like patient
the mean. specimens: Analysis
7. Normal distribution is a symmetric distribution about the conditions (incubation time, analysis temperature, calculation,
mean, where etc.) and
95.45% of the values lie within ±2 s and approximately 5% preanalysis conditions if warranted (precipitation, protein-free
will normally filtrate, etc.).
fall outside. c. Control materials are selected so that values will be at
8. The sensitivity of a test is the percentage of individuals medically significant
with a specific levels. Generally, one control will have a value within the
disease that are correctly identified or predicted by the test as reference interval,
having the and a second control will have an abnormal (elevated) value.
disease. d. It is preferred that the same lot number of control material
9. The specificity of a test is the percentage of individuals be purchased
without the specific and used for at least a 1-year period.
disease that are correctly identified or predicted by the test as e. Lyophilized control material must be accurately
not having the reconstituted according
disease. to the manufacturer's directions to avoid vial-to-vial
10. Predictive value of a test utilizes the parameters of test variability. The
sensitivity and stability of the reconstituted material is important.
specificity as well as disease prevalence (i.e., incidence of a f. For qualitative controls, use materials that will provide
disease or both negative
condition). and positive results.
a. Positive predictive value is the percentage of people with 3. Data evaluation procedures for control materials
positive test a. Levey-Jennings control chart is constructed monthly for
results who have the disease. each control
b. Negative predictive value is the percentage of people with material using the mean ±3 standard deviations to construct a
negative test graph that
results who do not have the disease. allows visual detection of shifts and trends. The control value
F. Reference Intervals is plotted
1. Reference intervals (ranges) are calculated for each versus the established range, with the acceptable control
laboratory's menu of range
tests. Each laboratory serves a unique population, so the represented by ±2 standard deviations.
reference intervals 1) Control values that exceed the mean ±2 standard deviations
must be determined for that population. are
2. Use a minimum of 20 specimens from "healthy" people to generally considered unacceptable and alert personnel to
determine investigate
analyte values, calculate the mean and standard deviation, and the cause.
compare 2) Trend is a gradual change in the mean that is reflected as
to the reference interval suggested by the manufacturer. either a
3. Preferably, analyte values should be determined using a decrease or increase of consecutive control values (generally
minimum of 120 the
specimens from healthy people in each relevant sex and age number of consecutive observations signifying a trend is six or
category. more).
4. Calculate the mean and standard deviation. The change occurs only in one direction.
5. Reference intervals are calculated using the mean +2 3) Shift is a sudden change in the mean that is reflected as
standard deviations consecutive
control values above or below the mean. to the values established by the reference laboratories for the
4) A loss of precision is obvious on the chart when control purpose of
values become assessing the clinical laboratory's level of performance. This is
more dispersed. known as
b. Westgard multirule is a control procedure that utilizes proficiency testing.
control rules to 2. Proficiency testing: An agency sends proficiency samples
assess numerical quality control data; the control rules to a clinical
establish the limits laboratory to analyze, and the results generated are assessed by
for data rejection in a system with two controls. Other rules the agency
apply when for accuracy to determine the performance of the laboratory.
three controls are used. Assessment
1) I2s—1 control value exceeds the mean ±2 standard reports are sent to participating laboratories to assist with
deviations; warning performance
rule that triggers inspection of control values using the other analysis and test method and equipment selection. Federal
rejection CLIA '88 (Clinical
Laboratory Improvement Amendments '88) requires that all
rules that follow; only rule that is not used to reject a run; laboratories
results are performing human testing for diagnosis and/or treatment must
reportable use proficiency
2) I3s—1 control value exceeds the mean ±3 standard testing for all analytes it reports. Failure to comply can result
deviations; detects in sanctions,
random error including a complete closure of the laboratory.
3) 22s—2 consecutive control values exceed the same 2 3. Proficiency samples have a similar matrix to patient
standard specimens, are generally
deviation 0) limit (same mean +2 s or same mean -2 s); detects shipped in a lyophilized form with diluent, and are utilized in
systematic error proficiency
4) R4s—1 control value in a group exceeds the mean +2 s and testing programs.
a second
control value exceeds the mean -2 s, creating a 4 standard 4. Limitations of external quality control programs
deviation a. Some laboratories will treat proficiency samples
spread; detects random error differently than normal
5) 4ls—4 consecutive control values are recorded on one side patient specimens (i.e., special handling, running controls
of the mean before and after
and exceed either the same mean +1 s or the same mean -1 s; each proficiency sample, calibrating the assay before running
detects the proficiency
systematic error sample, special selection of personnel to perform the assay,
6) 10x—10 consecutive control values are recorded on one etc.).
side of the Such deviation from routine workload procedures will not
mean (either above or below the mean); detects systematic reflect the
error accuracy and precision of the laboratory.
4. Youden plot is a graphical technique for analyzing b. Proficiency samples do not reflect the preanalytical
interlaboratory data when component of
each laboratory has made two runs on the same analyte or one patient identification, collection, and handling procedures.
run on two There could
different analytes. The plot identifies within-laboratory and be problems in these areas that an external quality control
betweenlaboratory program is
variability. not designed to address.
H. External Quality Control 5. For a clinical laboratory to comply with CLIA '88, the
1. External quality control refers to a program where a laboratory must
clinical laboratory successfully participate in proficiency testing. In turn, the
contracts with an agency (e.g., College of American agencies that
Pathologists or American provide proficiency testing to clinical laboratories must be
Association of Bioanalysts) to receive and assay samples, the approved by
concentration the Centers for Medicare and Medicaid Services (CMS).
of which is unknown to the participating clinical laboratory.
The same samples
are sent by the agency to reference laboratories for analysis for
the purpose Chapter 11
of establishing target values and ranges of acceptability. The
results generated 1088 • CHAPTER16: COMPUTERS AND
by the participating clinical laboratory are sent to the agency LABORATORY INFORMATION SYSTEMS
for comparison I. DEFINITIONS
A. Data are raw facts that have no meaning until grouped computers in a community or city that shares information on
together or organized. patients. Hospitals,
B. Information is data organized and grouped to increase a reference laboratories, physician offices, pharmacies, health
user's knowledge. insurance companies,
C. A personal computer (PC), also referred to as a etc. can have access to patient information.
microcomputer or desktop O. Malware is malicious software that can damage computers
computer, is a stand-alone computer that contains a central and includes viruses,
processing unit (CPU), Trojans, spyware, etc.
monitor, hard drive, etc., and can be used for processing data. P. Informatics is the science of information, the practice of
D. An operating system is a computer program that controls processing information,
the basic operation and the development of information systems.
of a computer and allows other software to interact with the II. GENERAL COMPUTER INFORMATION
computer hardware A. Digital Data
(e.g., Windows Vista, UNIX, and Mac OS). 1. Computers process and store data with numbers.
E. A server is a computer with a large amount of memory and 2. Binary (base 2): Computers use a series of O's (zeroes) and
storage capacity that 1's (ones); 0 is off
stores data accessed by other computers, called clients or and 1 is on.
workstations. Programs 3. Bit: A 0 or 1
(applications) can also be stored on servers. 4. A Byte is a series of 8 bits. It takes one byte to represent one
F. A mainframe is a large-capacity computer designed to character. There
support many users at once are 2s, or 256, combinations of bytes. Storage capacity is
with little or no down time. The term can have different measured in
meanings, but today it often kilobytes (KB), 1024 bytes; megabytes (MB), 1024 kilobytes;
refers to computers compatible with the IBM System/360 or gigabytes
series of computers. (GB), 1024 megabytes.
G. Supercomputers are computers that, at the time of their B. Computer Hardware
production, are on the 1. The CPU contains millions of transistors and performs
forefront of processing speed. They contain hundreds of CPUs. mathematical and logical
H. A local area network (LAN) is a collection of hardware, operations. The speed of the CPU is measured in clock speed,
including printers and or gigahertz
PCs, or clients connected to at least one server through cables (GHz), which is the number of cycles per second. Cache
(hardwired) or via a memory is the location
wireless network. The PCs are able to send data and share files of data being processed by the CPU and is located on the CPU.
with others on the Cache memory is
network. the fastest memory on a computer, but it is also the most
I. An intranet is a network of computers and other hardware expensive. Modern
that is not accessible to computers have dual or quad processors.
anyone outside that organization or office. 2. Memory modules are the location where random access
J. A wide area network (WAN) is a computer network over a memory (RAM) is
large geographic stored. RAM contains data waiting to be processed or that has
area that crosses metropolitan or national boundaries. recently been
K. Computerized provider order entry (CPOE) is a method processed by the CPU. RAM requires continuous electricity to
of digital entry of be maintained;
instructions for the diagnosis and treatment of patients by a any data in RAM is lost when the computer loses power.
medical practitioner. Modern computers
L. An electronic health record (EHR) or electronic generally have 2-4 GB of RAM.
medical record (EMR) is 3. The motherboard is a circuit board connecting the other
a digital patient record that can include demographics, test components of the
results, medical history computer. The CPU, memory modules, and other circuit
and examination, images, etc. EHRs can be accessed via boards are plugged
computer over a network. into the motherboard. When the power is turned on, the
M. A hospital information system (HIS) is a powerful motherboard
computer system that distributes power to the integrated circuits and moves data
includes hardware and software responsible for storing patient, through the
business, and components. The electronic pathway for the movement of data
is referred
GENERAL COMPUTER INFORMATION • 1089 to as the databus.
employee data. An HIS is often linked to other digital
information systems (e.g., 1090 • CHAPTER16: COMPUTERS AND
laboratory information system). LABORATORY INFORMATION SYSTEMS
N. A community health information network (CHIN) is a 4. Input devices: A number of devices can input data into a
network of computer.
a. Keyboard
b. Pointing device (e.g., mouse and touch-sensitive pad) is stored sequentially, meaning the data can only be accessed in
used with a an ordered
graphical user interface (GUI). sequence. They are generally used to back up large amounts of
c. Bar code reader or scanner reads printed bar codes, a series data and
of parallel typically have a storage capacity of 4-20 GB. Sequential
lines that represent letters or numbers. Bar codes are used to storage is an
identify
patients and patient samples. GENERAL COMPUTER INFORMATION • 1091
5. Output devices effective use of space, but it takes longer to access the data
a. Monitor compared to
1) Resolution is measured in the number of pixels (picture accessing data on a hard drive.
elements) and d. USB flash drives contain a universal serial bus (USB)
the dot pitch. A pixel is the smallest piece of information in an connector and a
image. flash memory chip (circuit). Because of their compact size,
It is composed of three dots: red, green, and blue. A monitor large storage
with a capacity, and ease of use, these storage devices are widely
resolution of 1024 X 768 has 1024 columns and 768 rows of used.
pixels. e. Compact discs (CDs) and DVDs (digital versatile discs or
Dot pitch refers to the distance between dots of light of the digital
same color. video discs) are optical storage devices. Data are stored as
The smaller the dot pitch and the greater the number of pixels, either "pits"
the or "grounds" on the disks in thin closely spaced tracks. A laser
better is the image. is used to
2) Liquid crystal display (LCD) monitors have become the read the data on the disks. DVDs have tracks that are much
industry closer together
standard. They take up less space, are lighter, and use less and, therefore, they have greater storage capacity.
electricity 7. Cables are used to connect external components
than the older cathode ray tube (CRT) monitors. (peripherals) to the
b. Printers computer via the motherboard.
1) InkJet printers spray ink onto paper. a. Serial cables move one bit of data at a time. Serial cables
2) Laser jet printers use the precision of a laser to position are no longer
dots on a commonly used; they have been replaced by USB cables.
drum with magnetized toner. The particles are fixed to the b. Parallel cables move one byte of data at a time. Parallel
paper with cables had been
heat. LaserJet printers are faster but more expensive to used to connect printers and laboratory instruments to a
purchase computer. Parallel
compared to inkjet printers. Generally, however, the cost per connections have generally been replaced by USB
page is connections, which are
less for a laser jet printer. much faster and use thinner cables.
3) Plotters are vector graphic printing devices that print line c. USB cables are commonly used to connect peripherals. They
art by allow
moving a pen over the surface of paper. They are used for multiple devices to be connected through a single port; allow
large plug and
technical drawings (architectural) and computer-aided designs. play (hot swapping), where a device can be removed without
6. Storage devices restarting the
a. Hard drives use magnetized microscopic particles computer; and provide power to low-consumption devices.
embedded in a surface. d. IEEE 1394 interface (i.e., Firewire, Apple Inc.) is a high-
Data are added and retrieved using a read/write head. Hard speed serial
drives can be connection commonly used to connect digital cameras and
internal or external. Disk arrays are a series of linked, audio/visual
generally external, components to a computer.
hard drives with much larger storage capacity. Hard drives C. Electronic Communication
allow random 1. The Internet is a worldwide network of computers.
accessing of data, meaning the computer can directly read or 2. Transmission control protocol (TCP) is the protocol
write to any computers use to
location on the disk. exchange data on the Internet. It allows electronic mail (e-
b. Floppy disks also use magnetized particles, as do hard mail) and the
drives. Floppy content of Web sites to be sent electronically. TCP divides
disks are portable, but they have a much smaller storage messages and files
capacity. They into smaller pieces called segments.
have generally been replaced by other storage devices. 3. Internet protocol (IP) address is a unique address that
c. Tape drives, or streamers, read and write data to a magnetic electronic devices
tape. Data are
(e.g., computers and printers) use in order to communicate and software for receiving, processing, and storing laboratory
with each other on data and information.
a computer network. It can interface with laboratory instruments to transfer data into
4. Bandwidth refers to the rate data are transferred; it is patient records,
usually measured in evaluate quality control data, and store preventive maintenance
kilobits/second (Kbps). records. In addition,
a. A broadband connection is one that transfers a lot of an LIS can interface with an HIS, pathology information
digital data at once system, and other
or when multiple pieces of data are sent simultaneously. information systems.
Computers need a B. Components of an LIS
network interface card (NIC) to connect to a broadband 1. The LIS software user interface determines how the user
cable. Examples will interact with
of cable connections include: the system. It will have specific screens for entering data,
1) Ethernet cable (1 gigabit/second) sending reports,
2) Coaxial cable (10 megabits/second) reporting results, etc. The software will have features such as
security, access
1092 • CHAPTER 16: COMPUTERS AND control, file maintenance, etc.
LABORATORY INFORMATION SYSTEMS
3) Fiber optic cable (600 megabits/second) LABORATORY INFORMATION SYSTEMS • 1093
b. A dial-up connection over a telephone line is a 2. Request entry: Requests for laboratory tests to be
narrowband connection performed can be entered
(56 Kbps). A modem converts the computer's digital signal to through clients located in the nursing units or remote primary
analog. The care practitioner's
computer receiving the data must then use a modem to convert office. In the case of outpatients, requests can be entered when
the analog the patient arrives
signal back to digital. at the laboratory.
5. Wi-fi, or "wireless fidelity," allows wireless access to 3. Data (results) entry
computer networks via a. Electronic data interface (EDI) connections between an
radio waves. Although distances vary, with a standard antenna LIS and a clinical
distance is instrument allow automatic transfer of patient test results to the
limited to about 100 feet. LIS.
6. The World Wide Web, or the Web, is a body of b. Manual data entry: Laboratory scientist enters patient
information (documents) results at a client
interlinked and accessed via the Internet. Sir Tim Berners-Lee c. Release patient results: The results are added to the LIS,
is credited with but they are not
creating the Web in 1989. released to clients outside the laboratory until the results and
a. Hyperlinks: A navigational element in one document links quality
to another control are reviewed and verified. Alternatively,
section of the same document or to a different document autoverification can be
b. Hypertext transfer protocol (HTTP) is a communication used. In this case, the computer uses a set of instructions to
protocol for the determine if
transfer of information (hypertext) over a computer network. the results should be released. Because the results are not held
c. File transfer protocol (FTP) is a network protocol used for up for
uploading manual review, autoverification is quicker. To help with
documents to a Web server. verification,
d. Uniform resource locator (URL) is a string of characters reference ranges and panic values can be programmed into the
that provides LIS.
the address or location of a unique document available over the d. Point of care testing: Portable laboratory instruments, like
Internet. handheld
e. Hypertext markup language (HTML) and extensive analyzers, can connect to an LIS via a wireless connection.
markup language 4. Data storage
(XML) are standard languages used for Web pages. Web a. Redundant arrays of independent disks (RAID): LISs are
browsers read the regulated by
text (i.e., HTML code) and display the information. the Food and Drug Administration, and they are required to
7. Search engines use spiders or bots (short for robots) to have mirrored
retrieve information hard drives. Data are stored on two separate hard drives of the
found in Web pages and create a searchable database. LIS server.
8. Telemedicine is the use of technology to send healthcare- b. System backup: Each day the data are to be copied to a
related information tape, or other
(e.g., patient test results) for clinical diagnosis and treatment. portable storage device, and removed from the laboratory.
III. LABORATORY INFORMATION SYSTEMS 5. System security: Ongoing procedures to ensure the security
A. A laboratory information system (LIS) is a computer of patient data and
network of hardware
user profiles (usemames and passwords) to prevent medical, surgical, and diagnostic services and are designed to
unauthorized access must be communicate
in place. Users should have access only to the patient information about medical services and procedures among
information and LIS functions physicians and
needed to perform their job (minimum necessary use). other healthcare professionals. CPT codes are used for billing
Antiviral software purposes and
(e.g., McAfee and Norton) should be installed to protect the can be programmed into the LIS.
system from harmful 6. Quality control: An LIS can analyze quality control
malware, especially for networks with a Windows operating specimens and prepare
system. charts and reports (e.g., Westgard rules, Levey-Jennings
6. Barcoding can facilitate processing of clinical specimens. charts).
7. Interface: The LIS can be connected to clinical instruments 7. Quality assurance can provide reports on turnaround time,
and other documentation of
information systems through an EDI. An interface is typically critical result reporting, and corrected reports.
bidirectional, 8. Management reports: Cost per billable test calculations,
meaning information is sent to and from the instruments and test volume,
the information turnaround time, employee hours, workload data, etc.
systems. With a unidirectional interface, analyte results from 9. Encoding systems: Systemized Nomenclature of Medicine
an instrument —Clinical Terms
are sent to the LIS, but the LIS cannot send requests to the (SNOMED—CT) is a comprehensive database of
instrument. So that standardized terminology for
instruments and computers used in healthcare can healthcare. Once implemented, it will allow automatic data
communicate with each analysis over a wide
other, the Health Level 7 (HL7) communication standard was range of clinical information systems. Logical observation
adopted. HL7 is identifiers names and
an international committee formed in 1987 to formulate data codes (LOINC) is another database of universal standards for
standards, a set healthcare.
of rules that allow healthcare information to be shared and D. Selecting an LIS
processed in a 1. The process begins with a laboratory needs assessment,
uniform and consistent way. where data are
8. Manual procedures: If the computer system goes down, a collected on the information needs of the laboratory.
contingency plan 2. Needs are analyzed to determine feasibility of a system and
for manual procedures and forms needs to be in place. what is needed to
get the job done.
1094 • CHAPTER 15: COMPUTERS AND 3. Laboratory managers and administrators form a committee
LABORATORY INFORMATION SYSTEMS and prepare
9. System maintenance: LISs need to be shut down (taken a request for proposal (RFP). The RFP contains information
offline) periodically about the
for software upgrades and other maintenance. Occasionally, laboratory facility, lists specific requirements needed in an
the system will LIS, and poses
become nonresponsive (crash). questions about LISs. This information may include interface
10. Disaster recovery: Every laboratory needs a plan to capabilities to
restore the system after
system disruption by a storm, fire, or other hardware damaging LABORATORY INFORMATION SYSTEMS • 1095
situation. hospital information systems and laboratory instruments,
C. Information Provided by an LIS remote user access,
1. Patient demographics system requirements, custom features, hardware and software
2. Work lists maintenance
3. Data retrieval (inquiry) contracts, etc. The RFP is distributed to vendors.
a. Generate patient results: Flag critical values, print reports if 4. Vendors will respond to the RFP describing how their
requested, etc. systems will meet the
b. Perform delta checks: Results of an analyte assay are needs of the laboratory and the estimated cost of the systems.
compared to the 5. The RFP responses will be reviewed by the committee. To
most recent previously performed results on the same patient prevent information
c. Patient results can be retrieved electronically at a client or overload and confusion, only a few of the vendors, those that
via the Internet submitted an
with a Web browser. RFP response that match the needs assessment, should be
4. Reflex testing: If an initial test result is positive or outside selected to give
normal parameters, demonstrations.
the LIS can automatically order a second appropriate test. 6. Vendor demonstrations and visits to other laboratories
5. Current procedural terminology (CPT) codes: The CPT using the systems
codes describe help narrow the choices. Vendor demonstrations should be
scheduled within
a short time frame so that information is fresh in everyone's ordering, sample collection, testing, and
mind. reporting.
7. Selection is based on the system that can best meet the • Informatics plays a key role in assisting
laboratory's needs at physicians to manage
the lowest cost (i.e., the cost does not outweigh the benefit). laboratory orders (e.g., clinical pathways and
E. Installation decision support
1. The installation process is important and very time- systems) and results (e.g., clinical alerts,
consuming. It is critical to interpretive reports, and
identify any errors early in the process before the system is reflex tests). Together, these approaches
activated (goes live). maximize the usefulness of
2. Vendor representatives will install the server, clients, the laboratory to clinicians.
network connections, • Data exchange among diverse applications in
and software. health care depends on
3. Testing: A thorough test of individual components (unit coding protocols for identifying procedures and
test) and a test of the medical conditions,
system (integration test) are performed. as well as a communication language. At present,
4. Training laboratory personnel and other healthcare Current Procedural
providers on the LIS is an Terminology (CPT) is widely used to identify
expensive process. It is important to discuss this with the laboratory tests and
vendor before other medical procedures, but Logical
accepting a proposal. Management needs to know how many Observation Identifier Names
people the and Codes (LOINC) is a more powerful
vendor will train. It will become the responsibility of the nomenclature standard for
laboratory personnel laboratory tests. The International Classification of
receiving training to train others. Training will also be needed Diseases (ICD) is
for healthcare most commonly used for coding disease states,
providers outside the laboratory. whereas the
5. Communication: Before the LIS goes live, it is important Systematized Nomenclature of Medicine
to communicate to (SNOMED) is also widely
all members of the healthcare team about the planning and used in pathology and is being greatly enhanced
timeline of the in a new version
process. known as SNOMED-CT. Health Level Seven (HL7)
F. System Validation is the most
1. Validation of the laboratory information system is an prevalent communication standard in health care.
ongoing process of • The role of the pathologist will likely change as
proving the system performs its intended use initially and over the field of
time. computational pathology continues to evolve. It is
2. Validation consists of defining, collecting, maintaining, and focused on
reviewing incorporating multiple sources of data, analyzing
evidence that the system is performing consistently according the information,
to specification. and then presenting actionable knowledge to
It is tedious, difficult, and costly, but it must be done to assure maximize the benefits
that the system of medical decision making.
meets the needs of the laboratory. • The website for the Association for Pathology
Informatics
Certain concepts and terms specific to informatics
(www.pathologyinformatics.org) describes the
should be familiar
field of pathology
to pathologists working in an ever-modernizing
informatics as “[involving] collecting, examining,
laboratory
reporting, and
environment.
storing large complex sets of data derived from
• The laboratory information system (LIS) is
tests performed in
typically part of a hospital
clinical laboratories, anatomic pathology
or health care system network of clinical,
laboratories, or research
registration, patient
laboratories in order to improve patient care and
management, and financial systems that
enhance our
exchange information with
understanding of disease-related processes.” The
one another.
high-volume,
• The LIS supports workflow and information flow
detailed, and time-sensitive nature of the
in all steps of the
workflow in the clinical
laboratory testing process, including patient
laboratory has helped push the implementation of
registration, test
computing
technology to assist in information management.
Chapter 12 Level A laboratories, also known as sentinel
laboratories, may be the
Costs can be described in different ways, first to identify an unusual organism or cluster of
depending on how they isolates that may
relate to laboratory operations (direct/indirect), signal a bioterrorism event.
change with test • The responsibility of a Level A laboratory is to
volume (variable/fixed), pertain to staffing rule out suspected
(salary/nonsalary), or are biological agents rather than perform complete
associated with the useful life of supplies or identification or
equipment (operating/ highly complex analyses.
capital). Cost per reportable result is a key • Suspect samples must be handled safely and
indicator. legally (using chain of
• Reimbursement for laboratory services comes custody).
mostly from third-party • Specific protocols (and presumptive
payers such as Medicare (government) and identification criteria) must be
insurance companies applied for each biologic agent.
(nongovernment/private insurance), and • Category A (highest priority) biological agents
payments are almost always are easily
less than charges. disseminated, can cause high mortality, and can
• Inpatient laboratory testing charges are usually generate public
not reimbursed panic. They include bacterial (anthrax, plague,
directly; they are considered part of a per diem tularemia), viral
rate (i.e., general (smallpox, viral hemorrhagic fever) and toxin-
hospital daily room reimbursement rate) or a case mediated (botulism)
rate, such as agents.
diagnosis-related group rate (i.e., set rate for the • Level A laboratories play mostly a supportive
entire hospital stay, role in the management
regardless of actual length of stay). Thus inpatient of hospitalized patients who are victims of a
laboratory testing chemical terror attack
is usually considered a “cost center.” In contrast,
outpatient Category A Agents
laboratory charges are reimbursed directly; HIGHEST PRIORITY OF AGENTS THAT POSE A
therefore, outpatient NATIONAL
testing is usually considered a “revenue center.” SECURITY RISK
• For reimbursement purposes, Current Characteristics Agents
Procedural Terminology codes Easily disseminated and/or
describe tests, and International Classification of transmitted from person to
Diseases, Tenth person
Revision (ICD-10), codes describe the diagnosis. Can result in high mortality rates
Medical necessity with a major public health
requirements may limit reimbursement to those impact
tests associated with Might cause public panic
specific predefined diagnoses. Anthrax (Bacillus anthracis)
• Budgeting is the process of planning, Botulism (Clostridium botulinum)
forecasting, controlling, and Plague (Yersinia pestis)
monitoring the financial resources of an Smallpox (variola major)
organization. Tularemia (Francisella tularensis)
• A variety of financial tools are used to evaluate Viral hemorrhagic fevers—filoviruses
a capital project, such (i.e., Ebola, Marburg) and
as purchasing a chemistry analyzer. These arenaviruses (i.e., Lassa,
measure how long it takes Machupo)
to recoup an investment (payback) and how much
it generates in III. LABORATORY SAFETY
today’s dollars (net present value) and its rate of A. Regulatory Oversight
return (internal rate 1. Occupational Safety and Health Administration (OSHA)
of return). a. Federal agency charged with the enforcement of safety and
• Laboratory equipment can be acquired in health
different ways, including legislation
purchase, lease, and per-test payment. Each has b. Occupational Safety and Health Act of 1970 makes
pros and cons. employers
responsible for providing a safe and healthy workplace for
Chapter 13 their
employees. These inspections may be regularly scheduled or unannounced.
c. Hazardous Communication Programs, also known as the Inspections may also follow a complaint filed against a
Right to facility.
Know Standard: The purpose of this standard is to ensure that C. Personal Safety
chemical 1. Wash hands before leaving the laboratory and after taking
hazards in the workplace are identified and information off gloves.
concerning these 2. Do not mouth pipet.
hazards is communicated to employers and employees. 3. Tie back long hair and avoid loose sleeves/cuffs, rings,
2. Centers for Disease Control and Prevention (CDC): bracelets, etc.
Federal agency that 4. Do not apply cosmetics in the laboratory.
publishes numerous safety standards 5. Eating and drinking are forbidden in the laboratory.
3. The Joint Commission: Issues standards and grants 6. Housekeeping
accreditation to improve a. Maintain orderly work areas
the safety and quality of care provided to the public through b. Keep aisle-ways clear and free of tripping hazards
inspections of c. Keep floors dry to avoid slipping; attend to spills
healthcare facilities immediately
4. College of American Pathologists (CAP): Issues standards D. Personal Protective Equipment
and offers 1. OSHA requires that employers provide all necessary
accreditation through inspections personal protective
B. Safety Program equipment (PPE) to employees.
1. Accreditation organizations require clinical laboratories to 2. Eye protection: Goggles and face shield
have a formal 3. Protective clothing
safety program. The program needs to ensure that the a. The laboratory coat is designed to protect the clothing and
laboratory environment skin from
meets approved safety standards. chemicals that may be spilled or splashed. It should be worn
2. Safety officer or chair of the safety committee: buttoned
Responsibility up and with the sleeves extended to the wearer's wrist.
is to implement and maintain a safety program b. Foot protection is designed to prevent injury from
corrosive chemicals or
1002 • CHAPTER13: GENERAL LABORATORY heavy objects. If a corrosive chemical or heavy object were to
PRINCIPLES, QUALITY ASSESSMENT, AND SAFETY fall on the
3. Chemical hygiene officer (CHO): OSHA requires that
laboratories have a LABORATORY SAFETY • 1003
designated CHO whose responsibility is to provide technical floor, the most vulnerable portion of the body would be the
guidance in the feet. For this
development and implementation of the chemical hygiene reason, shoes that completely cover and protect the foot are
plan. worn in the
4. Material safety data sheet (MSDS) defines the toxicity of laboratory.
a chemical. The 4. Hand protection: Heat-resistant gloves for handling hot or
MSDS must be provided by the manufacturer and includes the cold objects (e.g.,
following dry ice) and latex or nitrile gloves to prevent exposure to
information: biological hazards
a. Physical data such as boiling point, vapor pressure, and must be available. Selection of protective gloves is based on
specific gravity chemical hazard
b. Threshold limit value (TLV): Exposure allowable for an and the tasks involved.
employee E. Safety Equipment
during one 8-hour day 1. Individual storage containers
c. Spill, disposal, and first aid procedures a. Selecting the best means of storage for chemical reagents
d. Personal protective equipment required to handle the will, to a great
chemical extent, depend on that reagent's compatibility with the
e. Additional toxicity must be listed; this would include container. A safety
identifying chemicals can is an approved container of no more than 5-gallon
as a carcinogen (substance or agent causing cancer), a capacity. It has a
mutagen (causes spring-closing lid and spout cover and is designed to safely
changes in DNA), or a teratogen (causes birth defects). relieve
5. Safety inspections pressure buildup within the container.
a. The laboratory should have a safety committee or inspection b. Sharps containers: Hard containers for the disposal of
team sharp objects such
periodically inspect the laboratory. as used phlebotomy needles, broken contaminated glass, and
b. Several federal, state, and private accreditation pipettes
organizations (e.g., CAP 2. Eye wash stations must be inspected and tested periodically
and The Joint Commission) conduct inspections of healthcare for proper
facilities. function.
3. Safety showers provide an effective means of treatment in downward at the front of the cabinet where the laboratorian is
the event that working.
chemicals are spilled or splashed onto the skin or clothing. The air can be exhausted into the room (Class 2A) or outside
They must be the building
inspected and tested periodically for proper function. (Class 2B).
4. Refrigerators c. Class 3 cabinets are gas-tight. The interior of the cabinet is
a. Standard refrigeration units are not appropriate for storing only
flammable accessible through glove ports.
materials. d. Chemical fume hoods and biological safety cabinets cannot
b. Laboratory refrigerators are not appropriate for storing be used
food for interchangeably. Fume hoods will not protect workers from
consumption. Each refrigerator and freezer must be labeled, infectious
"No food agents, and biological safety cabinets may not protect against
or beverages may be stored in this refrigerator." chemical
c. Each refrigerator and freezer must be monitored daily to vapors. In addition, chemicals can damage the HEPA filters in
ensure proper biological
functioning. safety cabinets.
5. Alarms are designed so that endangered personnel are F. Waste Collection and Disposal
alerted. All individuals 1. Discard all nonsharp biohazardous substances into
should become familiar with the exact location of the fire biohazard bags.
alarm stations 2. Dispose of used tubes in biohazard bags.
nearest to their laboratory. 3. Dispose of swab wrappings, band aid wrappings, used paper
6. Chemical spill kits towels, kit boxes,
a. Laboratories are equipped with clean-up kits for various and any other nonbiohazardous waste into regular trash bags.
types of spills. 4. Do not discard nonbiohazardous waste into red biohazard
Wear the appropriate PPE (i.e., gloves, goggles) when cleaning bags.
up spills. 5. Store chemicals in appropriate chemical can. Chemicals
b. Acid spills should not be poured
1) Apply neutralizer (or sodium bicarbonate) to perimeter of down sink drains.
spill. G. Mandated Plans
2) Mix thoroughly until fizzing and evolution of gas ceases. 1. Chemical hygiene plan
3) Transfer the mixture to a plastic bag, tie shut, fill out a a. OSHA requires laboratories to have a chemical hygiene
waste label, plan.
and place in a fume hood. b. List of responsibilities of employers and employees
c. Solvent spills c. Chemical inventory list
1) Apply activated charcoal to the perimeter of the spill. d. Copies of the MSDSs must be readily available.
2. Exposure control plan
1004 • CHAPTER 13: GENERAL LABORATORY a. OSHA requires that all laboratories have an exposure
PRINCIPLES, QUALITY ASSESSMENT, AND SAFETY control plan to
! minimize risk of exposure to bloodborne pathogens (BBPs).
2) Mix thoroughly until material is dry and no evidence of b. Regulates disposal of medical waste
solvent remains.
3) Transfer absorbed solvent to a plastic bag, tie shut, and LABORATORY SAFETY • 100S
place in fume 3. Ergonomic plan
hood. a. CAP requires laboratories to have an ergonomic plan to
7. Chemical fume hood minimize risk
a. The only safe place to work with some highly toxic and of work-related musculoskeletal disorders.
volatile chemicals b. Avoid awkward posture, repetitive motion, and repeated use
b. Partially enclosed ventilated work space for volatile of force.
chemicals c. Employer must provide training and appropriate equipment,
c. Chemical fume hoods are generally ducted and vent air and an
outside the building. assessment and documentation system.
d. Fume hoods are not to be used for the storage of hazardous 4. Transportation and shipping of clinical specimens
chemicals. a. Laboratories are responsible for preventing people from
8. Biological safety cabinets being exposed
a. Class 1 cabinets have an open front and are under negative to infectious agents during transport.
pressure. Air b. The Department of Transportation (DOT), International Air
is exhausted into the room after passing through high- Transport
efficiency particulate Association (IATA), and the International Civil Aviation
air (HEPA) filters. Organization
b. Class 2 cabinets provide added protection by forcing (ICAO) developed strict guidelines for the handling and
HEPA-filtered air shipping of
hazardous materials. Only special approved shipping with the potential to explode if bumped.
containers can be e. Sodium azide, a carcinogen, is sometimes used as a
used. Only individuals who have received training and have a preservative in
permit are laboratory reagents. When disposed of in the sewer, the
allowed to ship hazardous material. accumulation
H. Laboratory Hazards of copper and iron salts of azide may occur. These metallic
1. The United Nations (UN) established nine classes of salts are
hazardous materials. explosive, especially when subjected to mechanical shock.
a. Class 1—explosives f. Working with carcinogens requires special precautions such
b. Class 2—compressed gases as using
c. Class 3—flammable liquids a fume hood, wearing rubber gloves and a respirator, and
d. Class 4—flammable solids cleaning
e. Class 5—oxidizer materials contaminated glassware with a strong acid or organic solvent.
f. Class 6—toxic materials g. Chemical containers made of glass should be transported
g. Class 7—radioactive materials in rubber
h. Class 8—corrosive materials or plastic holders that will protect them from breakage.
i. Class 9—miscellaneous materials not classified elsewhere 4. Fire hazards
2. Warning labels a. Flammability is a measure of how easily a gas, liquid, or
a. The DOT requires all chemicals shipped in the U.S. have solid will ignite
labels based and how quickly the flame, once started, will spread.
on the UN hazardous material classification. Flammable and
b. DOT labels are diamond shaped with the classification inflammable both mean "to catch fire easily."
number in the b. Flammable liquids themselves are not flammable;
bottom corner. The hazard is also identified in words along the rather, the vapors from
horizontal the liquids are combustible. There are two physical
axis of the label. properties of a material
c. The DOT label is only on the shipping container. Once that indicate its flammability: flash point and volatility (boiling
received, the point).
laboratory must label each individual container in the shipping 1) The flash point of a material is the temperature at which a
container. liquid (or
d. Although OSHA mandates the use of labels or appropriate volatile solid) gives off vapor in quantities significant enough
warnings, to form
no single uniform labeling system exists for hazardous an ignitable mixture with air.
materials. 2) The volatility of a material is an indication of how easily
e. The National Fire Protection Association (NFPA) the liquid or
developed the 704-M solid will pass into the vapor stage. Volatility is measured by
Identification System, which most laboratories use. the boiling
1) The labels are diamond shaped, and each quadrant has a point of the material—the temperature at which the vapor
different pressure of
color: blue—health; red—flammability; yellow—reactivity; the material is equal to the atmospheric pressure. Volatile
and solvents
white—special information. The chemical is classified 0^ should be stored in small amounts in an explosion-proof
(least refrigerator.
3) The flash point of flammables is designated as less than
100G • CHAPTER 13: GENERAL LABORATORY 100°F, and that
PRINCIPLES, QUALITY ASSESSMENT, AND SAFETY of combustibles as greater than 100°F.
hazardous to most hazardous) in the areas of health, c. Xylene, ethanol, methanol, and acetone are flammable
flammability, and chemicals commonly
reactivity. used in clinical laboratories that must be stored in a
2) The chemical can be identified as a poison, water reactive, flammable liquid
etc. in the safety cabinet.
white quadrant. d. Some materials are pyrophoric, meaning that they can
3. Chemical hazards ignite
a. Approved spill kits must be nearby. spontaneously with no external source of ignition. Potassium
b. Concentrated acids must be diluted by adding them to water metal,
in the sink. for example, can react with the moisture in air.
c. Label all containers before adding the chemical.
d. Some chemicals can become more hazardous if stored for a LABORATORY SAFETY • 1007
prolonged e. Storage
time. Picric acid has the potential to form peroxides if stored 1) Flammable materials should never be stored near acids.
for a long 2) Storage areas should be cool enough to prevent ignition in
period of time and not used. The material can become shock the event
sensitive,
that vapors mix with air. Adequate ventilation should be
provided to
prevent vapor buildup.
3) Avoid storage of flammable materials in conventional
(non-explosionproof)
refrigerators. Sparks generated by internal lights or
thermostats
may ignite flammable material inside the refrigerator, causing
an
extremely dangerous explosion hazard.
4) Be aware of ignition sources in your laboratory area (heat
sources,
electrical equipment).
f. Handling
1) Use gloves and safety goggles when handling flammable
liquids
or vapors.
2) Dispensing of flammable or combustible liquids should only
be done
in a fume hood or in an approved storage room.
3) Do not use water to clean up flammable liquid spills.
g. Extinguishers
1) Extinguishers are classified according to a particular fire
type and are
given the same letter and symbol classification as that of the
fire.
a) Type A—combustibles: wood, cloth, paper, rubber, and
plastics
b) Type B—flammable liquids: oil, grease, and paint thinners
c) Type C—energized electrical equipment: electrophoresis
d) Type D—combustible metals: magnesium, titanium,
sodium,
lithium, potassium
2) Multipurpose extinguishers are highly recommended
because they
are effective against Type A, B, and C fires.
5. Biological hazards
a. The National Institutes of Health guidelines describe four
levels of
biosafety depending upon the biological agents isolated or
studied. The
levels are based on the virulence of the agents and the
availability of
effective treatments and vaccines.
1) Biosafety Level 1 laboratories handle agents that have no
known
potential for infecting healthy people.
2) Biosafety Level 2 laboratories are those laboratories that
work with
microorganisms associated with human diseases that are rarely
serious
and for which preventive or therapeutic interventions are often
available. Most clinical microbiology laboratories are Level 2.
3) Biosafety Level 3 is recommended for materials that may
contain
viruses not normally encountered in a clinical laboratory and
for
the cultivation of mycobacteria. Clinical laboratories offering
these
services must have a Level 3 facility. Working with
mycobacteria
requires the use of N95 HEPA filter respirators; surgical
masks
are not acceptable.
1008 • CHAPTER 13: GENERAL LABORATORY
PRINCIPLES, QUALITY ASSESSMENT, AND SAFETY
4) Biosafety Level 4 is required for work with dangerous and
exotic
agents that pose a high risk of aerosol-transmitted laboratory
infections
and life-threatening disease for which effective treatments are
limited.
b. Exposure risks
1) Accidental punctures with needles
2) Spraying (aerosols) or spilling infectious materials onto
desktop or floor
3) Cuts or scratches from contaminated object
4) Centrifuge accidents: Aerosols, broken tubes, etc.
c. Bloodborne pathogens are transmitted through contact with
infected
blood and body fluids and include human immunodeficiency
virus,
hepatitis B virus (HBV), and hepatitis C virus.
d. Use standard precautions and treat all blood or potentially
infectious
body fluids as if they are contaminated. Avoid contact
whenever possible,
and whenever it's not, wear personal protective equipment.
e. All surfaces, tools, equipment, and other objects that come
in contact
with blood or potentially infectious materials must be
decontaminated
and sterilized as soon as possible. Decontamination is
recommended with
5.25% chlorine bleach (sodium hypochlorite; NaOCl)
solution, a 1:10
dilution of household bleach. The diluted bleach solution
should be
made daily.
f. OSHA requires that employers offer employees HBV
vaccine if their
regular duties present a potential for exposure to the virus.
g. Before leaving the laboratory, laboratorians should wipe the
countertop
with a disinfectant, wash their hands in an antiseptic soap, and
remove
their laboratory coat.
h. Microbiology laboratories are engineered to maintain
negative air pressure
with respect to the administrative areas. This maintains airflow
into the
laboratory, minimizing the risk of airborne pathogens exiting
the
laboratory when a door is opened.
6. Compressed gases
a. Transportation of compressed gases is regulated by the
DOT.
b. NFPA labels should be attached to each cylinder.
c. Gas cylinders should be secured onto a hand truck for
transporting.
d. Gas cylinders must be stored in a vertical position chained
to a wall.
e. When in use, gas cylinders must be securely fastened to a
wall or
laboratory bench.
Category B Agents Rapidly growing 2 to 5 mm (overnight at 35° C),
Characteristics Agents nonhemolytic, nonpigmented, dry “ground-glass”
Moderately easy to surface colonies with irregular edges having
disseminate comma-shaped projections (Medusa head). The
Moderate morbidity and low colony has a sticky (tenacious) consistency when
mortality teased with a loop.
May require enhanced Centers Other criteria Nonmotile, catalase-positive,
for Disease Control and urease-negative,
Prevention diagnostic nitrate-positive, encapsulated bacillus that can be
capacity lysed by gamma phage (gamma phage typing is
Brucellosis (Brucella species) usually performed by Level B or C laboratory).
Epsilon toxin of Clostridium perfringens CO2, Carbon dioxide; CSF, cerebrospinal fluid.
Food safety threats (i.e., Salmonella sp., also susceptible and can transmit the pneumonic
Escherichia coli O157:H7, Shigella) plague to humans. Unlike
Glanders (Burkholderia malleri) anthrax, which cannot be transmitted person-to-
Melioidosis (Burkholderia person, pneumonic plague
pseudomallei) can be transmitted via large aerosol droplets from
Psittacosis (Chlamydia psittaci) a coughing patient.
Q fever (Coxiella burnetii) Yersinia pestis can be killed by
Ricin toxin from Ricinus communis polymorphonuclear cells, but they can
(castor beans) survive in monocytes, where they produce a
Staphylococcal enterotoxin B capsule to resist phagocytosis.
Typhus fever (Rickettsia prowazekii) These bacteria can then rapidly reach the lymph
Viral encephalitis (alphaviruses: system and bloodstream
Venezuelan equine encephalitis, and be disseminated to all organs, causing
eastern and western equine hemorrhage and necrosis.
encephalitis) Fewer than 100 organisms are necessary to cause
Water safety threats (i.e., Vibrio human infection.
cholerae, Cryptosporidium parvum) Studies have shown that the bacterium can
remain alive for up to 1 year
Category C Agents in soil and up to 270 days in live tissue. The
Characteristics Agents bacterium is killed after heat
Availability Hantavirus exposure (15 minutes at 72° C/160° F) and within
Ease of production and several hours of exposure
dissemination to sunlight (Tierno, 2002).
Nipah virus Clinical Features
Potential for high morbidity and Plague occurs in three clinical forms: bubonic,
mortality which is characterized by
Other emerging pathogens that swelling of the lymph nodes (buboes); pneumonic,
could be engineered for mass in which the lungs are
dissemination extensively involved; and septicemic, in which the
bloodstream is infected
Bacillus anthracis: Level A Laboratory Role with Yersinia pestis. The mortality rate of
Presumptively identify based on criteria below, untreated pneumonic plague is
and then submit culture 100%, and that of untreated bubonic plague is
to a Level B or C laboratory for final identification. about 50%.
Direct smears Samples such as blood, CSF, and Bubonic Plague. The incubation period of 2 to
skin (eschar) show 10 days is characterized
encapsulated gram-positive rods, single or in by malaise, high fever, and tender lymph nodes
chains. (buboes) that enlarge
Generally, spores not seen. and eventually necrose. Septicemia may occur,
Culture smears Large gram-positive bacilli (1-1.5 and bacteria can spread to
by 3-5 μm), which the central nervous system, the lungs (this
may be gram-variable after 72 hours; spores can produces the pneumonic disease,
be which can then be spread person-to-person), and
found in culture, especially under non-CO2 the rest of the body,
atmosphere but are nonswollen and are terminal eventually causing death.
or Pneumonic Plague. The 1- to 3-day incubation
subterminal. period is shorter than
Colonies on that of bubonic plague. Symptoms include high
sheep blood fever, cough, chest pain,
agar plates
and bloody sputum; pneumonia can lead to
respiratory and circulatory
collapse (Perry & Fetherson, 1997).
Practical Considerations
• Pneumonic plague patients transmit infection
through large particle
droplets (greater than 5 microns) generated by
coughing, talking,
or sneezing. A simple surgical-type mask can
protect workers or
family members. Droplet Precautions should be
maintained for 3
complete days of antibiotic therapy, after which a
person is no longer
contagious.
• Because “droplets” occur only within 3 to 5 feet
of a patient, central
air conditioning systems do not need to be fitted
with HEPA filters
(i.e., they do not need to provide bacteria- and
particle-free air).
• If an outbreak occurs, it is important to control
fleas, rats, and other
animals such as cats. Insecticides and repellents
are widely available.
• Hand washing is an essential prevention
strategy.
• Because Y. pestis does not have a spore form,
surfaces can be decontaminated
with a simple germicide, such as 10% bleach
solution. Skin
can be decontaminated by using any germicidal
soap product. Alcoholbased
hand sanitizers such as Purell can also be
effective.
• Clothing should be washed with a germicidal
detergent and hot water
(155° F), even though exposure is unlikely to
occur from reaerosolization
of bacteria from contaminated clothing.
• Stand clear (farther than 3 to 5 feet) of
individuals taking a coughing
fit or producing sputum with or without blood.
Brucellosis
History and Background
Brucellosis (Table 13-7) is a systemic zoonotic
disease caused by Brucella
melitensis, B. suis, B. abortus, and B. canis. These
bacteria ordinarily cause
disease in domestic animals, such as goats,
sheep, and camels (B. melitensis);
cattle (B. abortus); and pigs (B. suis). The primary
pathogen of dogs, B. canis
TABLE 13-6
Yersinia pestis: Level A Laboratory Role
Presumptively identify based on criteria below,
and then submit culture
to a Level B or C laboratory for final identification.
Direct smears More likely to see bipolar staining
(“safety pin”)
from clinical specimens (blood, sputum,
aspirates, etc.) than from cultures. Bipolarity is
better seen using Wayson or Wright-Giemsa
stain. Beware that bipolar staining is not always
observed and is not unique to Y. pestis.
Culture smears Plump gram-negative rods (1-2 by
0.5 μm),
single or in short chains.
Colonies on sheep
blood agar plates
Grow at 35° C (faster at room temperature) as
gray-white, nonhemolytic, translucent, pinpoint
colonies at 24 hours, but by 48 hours, colonies
are 1 to 2 mm in diameter, becoming
yellowish with age. Growth occurs with or
without carbon dioxide. Colonies can appear
as “fried egg” or with “hammered copper”
shiny surface. On MacConkey, grow as
pinpoint non–lactose-fermenting colonies after
24 hours; slightly larger at 48 hours.
Other criteria The bacterium is nonmotile at 35° C
to 37° C and
at room temperature (Y. pestis is the only
Yersinia that is nonmotile at room
temperature). It is oxidase and urease negative
and catalase positive; growth in broth is
flocculent and is described as “stalactite”;
clumps at side and bottom of tube.
TABLE 13-7
Brucella species: Level A Laboratory Role
Presumptively identify based on criteria below,
and then submit culture
to a Level B or C laboratory for final identification
and/or
confirmation, although most Level A laboratories
are able to
completely identify Brucella.
Direct smears Blood and/or bone marrow most
often submitted.
Brucella appears as faintly staining, small
gram-negative coccobacilli (0.5-0.7 by
0.6-1.5 μm), mostly seen as single cells
appearing like “fine sand.”
Culture smears Similar to above.
Colonies on sheep
blood (SBA)
and chocolate
(CA) agars
Usually not visible or are pinpoint at 24 hours; at
48 hours, colonies are tiny, nonpigmented, and
smooth with an entire edge, and are
nonhemolytic on SBA. Growth of some strains is
enhanced by carbon dioxide tension. Some
strains grow on MacConkey; Thayer-Martin can
be used as a selective medium. Blood cultures
are held for 21 days for suspect cases.
Other criteria These coccobacilli are catalase,
urease, and oxidase
positive (B. canis is variable). They are nonmotile
and do not require X and V factors. Brucellosis is
one of the most commonly reported
laboratoryacquired
infections. Automated systems are not
useful, nor are they recommended for
identification. Remember that sniffing culture
plates of Brucella can result in infection.