Simplifying Lab Test Interpretation

Simplifying Laboratory
Test Interpretation
Maria del Rosario, MD, MPH

Division of Infectious Disease Epidemiology
WVDHHR/BPH/OEPS
May 2011
WVDHHR/BPH/OEPS/DIDE 1
Objectives
• Review laboratory tests commonly
encountered in public health surveillance.
• Discuss laboratory test reports and practice

report interpretation using specific examples.
Disclaimer: This lecture is not intended to replace the advice and

recommendations of a healthcare provider.
Definition of Terms
• Normally sterile site: sites in the human body that are normally free from
organisms or foreign material, e.g. blood, joint, brain, etc.
• Unsterile site: sites in the human body that generally harbor
microorganisms, e.g. gut, oral cavity, nose, skin, etc
• Specimen: a sample of tissue (blood, urine, etc.) that may or may not
contain organisms
• Isolate: a population of organisms (bacteria) that has been separated from
a mixture
• Serotype: a group of closely related organisms with distinct characteristics.
• Assay: A test to detect or quantify a substance in a sample.
Laboratory Tests
 Detection Methods
o Microscopy
o Culture
o Antigen test*
 Identification Methods
o PCR*
o Viral load*
o PFGE
o Genotyping
 Serology
 Antimicrobial susceptibility
 Ancillary tests
*both detect and identify
Microscopy
Direct examination of a specimen (or may use stains) to
detect the presence of organisms.
Pros:
• Quick and easy
• Preliminary results
Gram negative
Cons: diplococci
• Not specific
Culture
The process of growing and propagating organisms in a media
that is conducive for their growth.
Pros:
• Confirm the organism
• Reproduce the organism and colony
use for additional testing
Cons:
• Delay in confirmation
• Require viable organism
• Difficult for fastidious
organisms
S. pneumoniae on blood agar plate
Antigen Test
Use of assay to detect the presence of antigen/s. Some assays are able to
differentiate antigens, some are not able to.
Technique Principle
Agglutination Known antiserum causes bacteria or other particulate antigens to
clump together or agglutinate
Complement Known antiserum mixed with the test antigen and complement is
fixation added. Sheep red blood cells and hemolysins are then added.
Positive test: no hemolysis, negative test: hemolysis
Enzyme-linked A rapid test where an antibody or antigen is linked to an enzyme
immunosorbant as a means of detecting a match between the antibody and
assay (ELISA) ; antigen.
Enzyme
immunoassay (EIA)
Fluorescent Fluorescent dye is attached to known antibodies. When the
antibody fluorescent antibody reacts with the antigen, the antigen will
fluoresce when viewed with a fluorescent microscope.
Result 1 Purpose of test
Test result interpretation
Polymerase Chain Reaction (PCR)
Method used to amplify a specific region of a DNA strand.
Pros:
• Simple process, eliminates tedious
work, results available within a day
• Does not require a viable organism
since only a strand of DNA is needed,
• Sensitive test
Cons:
• Sensitive – pick up environmental
contaminants
• Unable to distinguish between
certain species
Purpose of test
Result 2 Test result interpretation
Pulsed Field Gel Electrophoresis (PFGE)
A technique to separate large DNA molecules by applying an electric field that periodically
changes direction (electrophoresis)…to compare DNA banding patterns (fingerprints).
The outbreak strain of SalmonellaTyphimurium has been found in ill humans and in food samples during this
outbreak investigation.
Serology
• Serology: the study of blood serum, with
emphasis on testing of antibodies in the
serum
• Antigen: A substance which stimulates the

body to produce antibody; usually a
‘foreign’ substance
• Antibody: A protein molecule produced by

the body’s immune system in response to a
specific antigen. The antibody combines
with the antigen and disables it.
– Also called Immunoglobulins (e.g. IgG, IgM,
IgA, IgE)
– Referred to as anti-(name of antigen), e.g.
anti-HCV, anti-HAV
Antibodies
• IgM: type of antibody produced by the body, usually the first antibody to
appear in response to a foreign substance exposure, then eliminates the
organism in the early stages of immunity before there is sufficient IgG
• IgG: type of antibody that provides the majority of antibody-based

immunity against invading organisms. The only antibody that crosses the
placenta to provide immunity to the fetus
• Titer: the amount of antibodies present in the blood, usually as a result of

infection.
• Acute titer and Convalescent titer: At the acute stage of disease, serum is
tested (acute phase), followed by another blood draw and testing about 3
weeks (convalescent phase) later. IgG levels are compared and a 4-fold
increase between acute and convalescent samples usually indicate
infection.
Basic Anatomy of
Antibody Response to Infection
Human Parvovirus B-19: Disease and Immune Response
http://www.stanford.edu/group/virus/parvo/2005/B19.html
Antibody Testing
Pros:
• Screening tool
• Readily available
• Indicates response to antigen (even if antigen is not
detectable) – may indicate infection or immunity
Cons:
• Paired testing necessary for some diseases - may take a while
to get results, impact on patient management
• Unable to differentiate between immunity and disease
• Sensitivity and specificity:
 False-negative result: compromised immune system
 False-positive result: liver disease, low disease prevalence
Result 3
IFA
Type of test
Purpose of test
Ehrlichia chaffeensis Infection
Laboratory criteria for diagnosis
Supportive:
Serological evidence of elevated IgG or IgM antibody reactive with E. chaffeensis
antigen by IFA, ELISA, dot-ELISA, or assays in other formats (CDC uses an IFA
IgG cutoff of ≥1:64 and does not use IgM test results independently as
diagnostic support criteria.), OR …
Confirmed:
Serological evidence of a fourfold change in immunoglobulin G (IgG)-specific
antibody titer to E. chaffeensis antigen by IFA between paired serum
samples (one taken in first week of illness and a second 2-4 weeks later), OR
Detection of E. chaffeensis DNA …OR
Demonstration of ehrlichial antigen…, OR
Isolation of E. chaffeensis from a clinical specimen…
Hepatitis A Antibody Tests
Hepatitis A antibody Total

•Anti-HAV Total
•Antibody to Hepatitis A Virus
•HAV Ab Total
- measures both IgM and IgG
Hepatitis A antibody IgM

•Anti-HAV, IgM
•Antibody to Hepatitis A Virus, IgM
•HAVAb, IgM
Type of test
Result 4 Purpose of test
HCV RNA HCV RNA HCV RNA HCV
RNA
Hepatitis C Testing - 1
SEROLOGIC TESTS
• Enzyme Immunoassay (EIA) for Anti-HCV

 Positive: past or current infection
 Verification of Anti-HCV (+) screening test
1. Reflex supplemental testing*: follow-up with more specific serologic
test, e.g. HCV RIBA or NAT
2. Signal-to-cut-off ratio (s/co): predict supplemental test-positive
results ≥95% of the time, s/co dependent on test type
• HCV RIBA* (Recombinant Immunoblot Assay)

 Detects antibodies to individual HCV antigens and confers increased
specificity compared to EIA-2
 Some RIBA-positive patients are HCV RNA-negative
VIRAL LOAD TESTS
 Measure HCV RNA (genetic material)
 Detects actively replicating virus
 2 types:
A. Qualitative test - detects presence of HCV RNA virus (result: positive/negative)

• Nucleic Acid Test (NAT)* for HCV RNA using RT-PCR
 Detects HCV RNA in the blood
 Very sensitive
B. Quantitative test – measures the amount of virus in 1 ml of blood, use to assess response
to treatment
• Branched-chain DNA (bDNA)
 Easy and cheap, especially for large number of samples
 Only measures viral loads greater than 50 IU/ml
• Transcription-mediated Amplification (TMA)

 New, easy
 Amplifies and detects viral genetic materia;l in the blood
 Can measure viral loads as few as 5-10 IU/ml
GENOTYPING
• HCV Genotype
 6 genotypes, >50 subtypes
 clinical importance: counseling and treatment
 epidemiology
LIVER FUNCTION TEST

• ALT
• SGPT
Type of test
Result 5a Purpose of test
Hepatitis C, past or present
Clinical Case Definition

• No symptoms are required…
Laboratory criteria for diagnosis
• 1 or more of following 4 criteria: Anti–HCV positive (repeatedly reactive) EIA
verified by at least 1 additional more specific assay, OR
• HCV RIBA positive, OR
• NAT positive for HCV RNA (including genotype), OR
• Anti-HCV screening-test-positive with a signal to cut-off ratio predictive of a true
positive as determined for the particular assay and posted by CDC.
Case classification
• Confirmed: laboratory confirmed and does not meet the case definition for acute
hepatitis C.
• Probable: anti-HCV positive (repeat reactive) by EIA and has ALT or SGPT values
above the upper limit of normal, but the anti-HCV EIA result has not been verified
by an additional more specific assay or the signal to cut-off ratio is unknown.
Result 5b
Type of test
Purpose of test
 Interpretation of
o Test 1
o Test 2
Test 1
Test 2
Antimicrobial Susceptibility
MIC (minimum inhibitory
concentration)
• lowest concentration of
antimicrobials that will inhibit
the growth of organisms. MICs
are important to confirm
resistance of organisms to an
antimicrobial agent. MIC
Methods:
• Disk diffusion test
• E test
• Broth dilution test
Sample 6
Type of test
Purpose of test
Sample 7
Type of test
Purpose of test
Ancillary Tests
• CBC and WBC
• CSF cells
• Liver function tests – ALT, AST, bilirubin

Tips when reviewing a laboratory
report
• Is the organism (or disease) reportable?
• When was the specimen obtained in relation
to onset of illness?
• Was the source from a normally sterile site?
• Were antibiotics used prior to specimen
collection?
Summary
• Basic understanding of a laboratory test is key to
maximizing its use.
• Laboratory tests have ‘strengths’ and

‘weaknesses’.
• Timing is everything!
(between disease onset and specimen collection)
Thank you
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Simplifying Laboratory

Maria del Rosario, MD, MPH

• Discuss laboratory test reports and practice

Disclaimer: This lecture is not intended to replace the advice and

• Antigen: A substance which stimulates the

• Antibody: A protein molecule produced by

• IgG: type of antibody that provides the majority of antibody-based

• Titer: the amount of antibodies present in the blood, usually as a result of

Hepatitis A antibody Total

- measures both IgM and IgG

Hepatitis A antibody IgM

• Enzyme Immunoassay (EIA) for Anti-HCV

• HCV RIBA* (Recombinant Immunoblot Assay)

A. Qualitative test - detects presence of HCV RNA virus (result: positive/negative)

• Transcription-mediated Amplification (TMA)

LIVER FUNCTION TEST

Clinical Case Definition

• Liver function tests – ALT, AST, bilirubin

• Laboratory tests have ‘strengths’ and

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