Transcript LECTURE ON SEROLOGICAL DIAGNOSIS OF INFECTIOUS …

LECTURE ON SEROLOGICAL
DIAGNOSIS OF INFECTIOUS
DISEASES AND TUMOR MARKERS
ROBERTO D. PADUA JR., MD, DPSP
DEPARTMENT OF PATHOLOGY AND LABORATORY DIAGNOSIS
FATIMA COLLEGE OF MEDICINE
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• SEROLOGY
• The scientific study of blood sera and their effects
• Subdivision of immunology concerned with in-vitro
Ag-Ab reaction
• Concerned with the laboratory study of the
activities of the components of serum that
contribute to immunity
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• IMMUNOLOGY
• The study of the molecules, cells, organs and
systems responsible for the recognition and
disposal of foreign (non-self) material
• The study of how the body components respond
and interact
• The desirable and undesirable consequences of
immune interactions
• The ways in which the immune system can be
manipulated to protect or treat disease
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• IMMUNITY
• The ability of an organism to resist infection by
means of the presence of circulating antibodies
and white blood cells
• Distinctive characteristics of the immune system
 Specificity
 Memory
 Mobility
 Replicability
 cooperativity
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• METHODS OF DETECTION OF ANTIBODIES
1. Immuno-precipitation Assays
= detect antibodies in solution
= qualitative indication of the presence of
antibodies
= end-point is visual flocculation of the
antigen and antibody in suspension
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• METHODS OF DETECTION OF ANTIBODIES
2. Complement Fixation
= based on the activation or fixation of
complement following binding of
complement factors to Ag-Ab immune
complexes
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• METHODS OF DETECTION OF ANTIBODIES
3. Neutralization
= the ineffectivity of an organism or the
activity of toxin is neutralized by
specific antibody
= rarely used for diagnostic purposes
= mainly used to detect antibody formation
after vaccination
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• METHODS OF DETECTION OF ANTIBODIES
4. Particle Agglutination
= relatively simple and fast
= capable of detecting lower concentration of
antibodies
= designed to detect antibodies to viruses,
subsequent to interaction or vaccination
= utilize Ag coated latex particles, coal particles,
bentonite particles or erythrocytes
= direct and indirect methods
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• METHODS OF DETECTION OF ANTIBODIES
5. Immunofluorescence
= requires use of microscope equipped to
provide ultraviolet illumination or an
instrument capable of irradiating the
assay with UV light and detecting the
resultant fluorescence with a
fluorometer
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• METHODS OF DETECTION OF ANTIBODIES
6. Enzyme Immunoassay
= the most sensitive
= usually indirect assay that depends on the use of
an antihuman IgG or IgM antibody conjugate
= the antibody conjugate (if present) is made to
attach to enzyme which catalyzes conversion
of the substrate to a colored product which
will then be read with the use of a
spectrophotometer
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• METHODS OF DETECTION OF ANTIBODIES
7. Radioimmunoassay
= high sensitivity
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• Microbial antigen detection provides direct
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evidence of infection, and is preferred for
diagnosis of infection over antibody detection
(indirect evidence of infection)
However, not all infectious agents have
available antigen assays or culture techniques
making the detection of specific antibodies
diagnostically useful
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• Infectious Disease Indicators, Nonspecific
• Acute phase reactants
• Limulus lysate assay
– Detects trace amounts of endotoxin from all gram (-)
bacteria
– Presence in CSF = gram (-) bacterial meningitis
– Rapid clearance from blood makes serum test unreliable
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• Molecular Biology
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Nucleic acid amplification
DNA sequencing and typing
Direct molecular probe (in situ hybridization)
Nucleic acid quantitation
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• Molecular Biology
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Uses:
• Cases requiring increased sensitivity and specificity of
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identification
Cases requiring faster report turnaround time
Confirmation of culture
Identification of organisms that are non-viable or cannot
be cultured
Identification of fastidious, slow growing organisms
Identification of organisms that are dangerous to culture
Identification of organisms in small numbers or in small
volume specimens
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• Molecular Biology
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Uses:
• Density of amplifiable DNA correlates with microbial
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density
Monitoring of disease progression or initiation or
modification of therapy
Drug susceptibility testing
Differentiation of antigenically similar organisms
Molecular epidemiology and infection control
Disease diagnosis by characterization of genetic materials
without direct identification of infectious agent
Determination of virulence of antimicrobial resistance
genes
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• SYPHILIS
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The most commonly acquired spirochete disease in the
U.S.
A complex sexually transmitted disease that has a highly
variable clinical course
Over 50,000 cases reported in 1990 in the U.S.
Causative agent is Treponema pallidum
No natural reservoir in the environment, requires living
host
Spiral shaped and motile due to peri-plasmic flagella
Variable length
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• SYPHILIS
• Three other pathogens in the group Treponema
which are morphologically and anti-genetically
similar to T. pallidum, differences are in
characteristics of lesions, amount of systemic
involvement and course of the disease
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T. pertenue (Yaws)
T. endemicum (non-venereal syphilis)
T. carateum (pinta)
T. cuniculi (rabbit syphilis)
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• SYPHILIS
• Mode of Transmission
– Organism is very fragile, destroyed rapidly by heat, cold
and drying
– Sexual transmission most common, occurs when
abraded skin or mucous membranes come in contact
with open lesion
– Can be transmitted to fetus
– Rare transmission from needle stick and blood
transfusion
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• SYPHILIS - - Stages of the Disease
1. Primary stage
= primary lesion is chancre
= the lesion heals spontaneously after 1-5 weeks
= swab of chancre smeared on slide, examined
under dark-field microscope, spirochetes will be
present
= 30% become serologically positive one week
after appearance of chancre, 90% positive after
three weeks
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• SYPHILIS - - Stages of the Disease
2. Secondary Stage
= occurs 6-8 weeks after initial chancre, becomes
systemic, patient highly infectious
= characterized by localized or diffuse
mucocutaneous lesions, often with generalized
lymphadenopathy
= primary chancre may still be present
= secondary lesions subside in about 2-6 weeks
= serology tests nearly 100% positive
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• SYPHILIS - - Stages of the Disease
3. Latent Stage
= stage of infection in which organisms persists in
the body of the infected person without causing
symptoms or signs
= this stage may last for years
= one-third of untreated latent stage individuals
develop signs of tertiary syphilis
= after 4 years it is rarely communicable sexually
but can be passed from mother to fetus
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• SYPHILIS - - Stages of the Disease
4. Tertiary Stage
= occurs anywhere from months to years
after secondary stage, typically between 10
to 30 years
= gummatous syphilis
= cardiovascular syphilis
= neurosyphilis
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• SYPHILIS
• Congenital Syphilis
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Transmitted from mother to fetus
Fetus affected during the second or third trimester
40% result in syphilitic stillbirth
Live-born infants show no signs during first few
weeks
= 60-90% develop clear or hemorrhagic rhinitis
= skin eruptions (rash) especially around mouth,
palms of hands and soles of feet
= general lymphadenopathy, hepatosplenomegaly,
jaundice, anemia, painful limbs & bone abnormality
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• SYPHILIS - - DIAGNOSIS
• Evaluation based on 3 factors
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Clinical findings
Demonstration of spirochetes in clinical specimen
Present of antibodies in blood or CSF
= more than one test should be performed
= no serological test can distinguish between
other treponemal infections
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• SYPHILIS - - DIAGNOSIS
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Laboratory Testing
A. Direct examination of clinical specimen by dark-field
microscopy or fluorescent antibody testing of sample
B. Non-specific or non-treponemal serological test to
detect reagin, utilized as screening test only, not
diagnostic
= Reagin is an antibody formed against cardiolipin
= Found in sera of patients with syphilis as well as
other diseases
= Non-treponemal tests become positive 1-4 weeks
after appearance of primary chancre, in secondary
stage may have false positive due to prozone, in
tertiary 25% are negative, after successful
treatment will become non-reactive after 1 to 2
years
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• SYPHILIS - - DIAGNOSIS
• Laboratory Testing
C. Specific Treponemal antibody tests are used as a
confirmatory test for a positive reagin test
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = SYPHILIS
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NON-TREPONEMAL SEROLOGICAL TESTS – REAGIN
TEST
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Venereal Disease Research Laboratory=VDRL
= Flocculation test, antigen consists of very fine particles that
precipitate out in the presence of reagin
= Utilizes antigen consists of cardiolipin, cholesterol and
lecithin
= serum must be heated to 56 C for 30 minnutes to remove
anti-complimentary activity which may cause false
positive
= reported as Non-reactive, weakly reactive and reactive
= used primarily to screen CSF
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = SYPHILIS
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NON-TREPONEMAL SEROLOGICAL TESTS – REAGIN
TEST
2. Rapid Plasma Reagin – RPR
= general screening test
= can not be performed on CSF
= the VDRL cardiolipin antigen is modified with choline
chloride to make it more stable and is attached to
charcoal particles to allow macroscopic reading, the
antigen comes prepared and is very stable
= serum or plasma may be used for testing, serum is not
heated
= results are read macroscopically
= appears to be more sensitive than the VDRL
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = SYPHILIS
• NON-TREPONEMAL SEROLOGICAL TESTS –
REAGIN TEST
3. Other tests which use modified VDRL Ag
A. USR – unheated serum reagin test
= modified VDRL Ag, uses choline
chloride/EDTA
= microscopic flocculation test
B. RST – reagin screen test
= modified VDRL Ag with Sudan Black
= Sudan Black makes flocculation reaction
macroscopically visible
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = SYPHILIS
• SPECIFIC TREPONEMAL TESTS
1. Treponema pallidum Immobilization Test –
TPI
= live T. pallidum become immobilized by
antibody in serum of infected persons
= cumbersome and expensive, no longer
used in U.S.
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = SYPHILIS
• SPECIFIC TREPONEMAL TESTS
2. Treponema pallidum Hemagglutination –
TPHA
= adapted to microtechniques (MHA-TP)
= tanned sheep RBC’s are coated with T.
pallidum antigen from Nichol’s strain
= positive result is agglutination of RBC’s
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = SYPHILIS
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SPECIFIC TREPONEMAL TESTS
3. Fluorescent treponemal antibody absorption test (FTA-ABS)
= one of the most used confirmatory test
= diluted, heat inactivated serum added to Reiter’s strain of T.
pallidum to remove cross reactivity due to other
Treponemes
= slides are coated with Nichol’s strain of T. pallidum and add
absorbed patient serum
= slides are washed and incubated with Ab bound to a
fluorescent tag
= after washing again the slides are examined for
fluorescence
= requires experienced personnel to read
= highly sensitive and specific, but time consuming to perform
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = SYPHILIS
• SPECIFIC TREPONEMAL TESTS
4. ELISA
= tubes coated with T. pallidum antigen
= antibody in serum attaches to antigen
= following washing, add an anti-antibody
tagged with enzyme alkaline
phosphatase
= detectable color changes occur
Sensitivity and Specificity of Serologic Tests for
Untreated Syphilis at Different Stages
Serologic Test for Syphilis in Various Conditions
Algorithm for Positive Serologic Test for Syphilis
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = SYPHILIS
• PROBLEM AREAS
1. Biologic False Positives (BFP)
A. Collagen diseases such as arthritis, LE,
etc., sometimes result in increased
amount of reagin
B. Certain infections : IM, malaria, leprosy
C. Other treponemal infections
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = SYPHILIS
• PROBLEM AREAS
2. False negatives
A. Very early in disease or latent, inactive
stage
B. Immunosuppressed patients
C. Consumption of alcohol prior to testing
(temporary)
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = SYPHILIS
• PROBLEM AREAS
3. Congenital syphilis
A. Non-treponemal tests on cord blood or
baby serum detect IgG antibody,
maybe of maternal origin
B. Detection of IgM lacks sensitivity
C. Western blot has demonstrated high
sensitivity and specificity
D. Recommended that all mothers be tested
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = SYPHILIS
• PROBLEM AREAS
4. Cerebrospinal Fluid tests
A. Used to determine if Treponemes have
invaded the CNS
B. VDRL utilized to confirm neurosyphilis
C. Lacks sensitivity
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = SYPHILIS
• CORRELATION OF TREATMENT WITH
TEST RESULTS
A. Treatment at the primary stage, serology
tests become non-reactive after 6 months
B. Treatment at secondary stage, tests usually
non-reactive after 12-18 months
C. If treatment is not initiated until 10 or more
years, the reagin tests probably positive for
life
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
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LYME’S DISEASE
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Disease first recognized in 1977 in Lyme, Connecticut
Causative organism is Borrelia burgdorferi
Can be cultured but it is very difficult
Organism has been isolated from blood, CSF, skin lesions
and joint fluid
= Can be transmitted perinatally, causing intrauterine death
= Vector of transmission is the Ixodes tick
= Must remain attached a minimum of 24-48 hours for
transmission to occur
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = LYME’S DISEASE
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STAGES OF THE DISEASE
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Localized rash – erythema chronicum migrans
Dissemination to multiple organ system
= occurs by way of the bloodstream
= may occur weeks to months after infection
= migratory pain may occur in the joints, tendons and bones
= neurologic  Bell’s palsy, peripheral neuropathy, aseptic
meningitis
= cardiac include carditis and arrythmia
3. Chronic disseminated
= characterized by chronic arthritis
= affects the large joints, especially the knee
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = LYME’S DISEASE
• Diagnostic criteria
• Isolation of organism from clinical specimen or
• Diagnostic titers of IgG and IgM in serum or CSF
or
• Significant change in serum titers of IgG or IgM
in paired acute and convalescent sera
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = LYME’S DISEASE
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LABORATORY DIAGNOSIS
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Diagnosed clinically, confirmed serologically
Antibodies to antigens of B. burgdorferi can be detected by latex
agglutination, IFA, ELISA, and Western Blot
Serological tests are often falsely negative during early weeks.
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Specific IgM Abs usually appear 2- 4 weeks after erythema
migrans, peak after 3-6 weeks of illness, decline to normal after
4-6 months
IgG titers appears more slowly (4-8 weeks after the rash), peak
after 4-6 months, may remain high for months or years
Western Blot is most sensitive
IFA and ELISA are more commonly performed due to ease of
procedure, but are subject to false positives due to either
spirochete diseases and some autoimmune diseases
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = STREPTOCOCCAL INFECTION
• STREPTOCOCCAL SEROLOGY
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Streptococci are gram (+), beta-hemolytic, spherical,
ovoid, or lancet-shaped organisms which are catalase
negative and seen in pairs or chains
Divided into groups or serotypes based on cell wall
components  Streptococcus pyogenes belongs to
Lancefield group A and it is believed the M protein is the
chief virulent factor of this group
Numerous exo-antigens are produced and excreted as the
cell metabolizes (Streptolysin O, DNase, Hyaluronidase,
Nicotinamide, Adenine dinucleotidase (NADase),
Streptokinase)
Culture and rapid screening tests detect early infection
Sequelae include Rheumatic Fever and Acute GN
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = STREPTOCOCCAL INFECTION
• GROUP A STREPTOCOCCAL INFECTION
• Two major sites of infection : upper respiratory
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tract and skin
Upper respiratory tract = sore throat, tonsillar
exudate
Skin = pyoderma or impetigo
Suppurative complications = erysipelas, scarlet
fever, septic arthritis, meningitis
Non-suppurative complications = RF or Poststreptococcal GN
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = STREPTOCOCCAL INFECTION
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GROUP A STREPTOCOCCAL INFECTION
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Rheumatic Fever
= only certain serotypes of S. pyogenes is involved
= develops as sequelae in 2-3% untreated upper respiratory
infections
= symptoms occur about 18 days after sore throat
= Group A streptococcus share antigenic determinants with
host tissue, especially heart and even joints
= inflammation of mitral valve most serious
= 30-60% of patients may suffer permanent disability
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = STREPTOCOCCAL INFECTION
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GROUP A STREPTOCOCCAL INFECTION
B. Post-Streptococcal Glomerulonephritis
= follows Streptococcal infection of skin or pharynx
= occurs about 10 days following initial infection
= characterized by damage to glomeruli of the kidneys
= renal function impaired due to reduction in glomerular
filtration rate, results in edema and HPN
= renal failure not typical
= one theory is damage caused by antigen-antibody
complexes depositing in kidneys
= complement is activated resulting in low levels
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = STREPTOCOCCAL INFECTION
• LABORATORY TESTING
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Most reliable test is culture and identification of the
organism from infected site
Rapid streptococcal screening tests from the throat
exudates have high specificity but low sensitivity, 60-85%
Detection of Streptococcal antibodies most useful in
Streptococcal sequelae
The most useful antibodies are : ASO, anti-DNase B, antiNADase, anti-Hyaluronidase
Serological evidence of disease is based on elevated or
rising titer of Streptococcal antibodies
Four-fold (2 tube dilution) rise in titer is considered
clinically significant
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = STREPTOCOCCAL INFECTION
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LABORATORY TESTING
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Anti-Streptolysin O Titer (ASO Titer)
= two of the toxins produced are Streptolysin S, which is oxygen
stable, non-antigenic and Streptolysin O (SLO), which is
oxygen labile and antigenic
= SLO is a hemolysin which is toxic to many tissues, including heart
and kidneys
= evokes an antibody response (anti-SLO) which neutrolizes the
hemolytic action of SLO
= the test is specific for ASO, it does not test for antibodies to any
other Streptococcal exotoxins
= normal values will vary, <125 Todd units for adults, 5-125 Todd
units for children, recent Strep infections 250 Todd units for
adults, 333 Todd units for children
= a single titer is of little significance unless extremely elevated,
titers performed over a period of time will give the most information
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = STREPTOCOCCAL INFECTION
• LABORATORY TESTING
2. Anti-DNase B Testing
= may appear earlier than ASO
= increased sensitivity for detection of
glomerulonephritis preceded by streptococcal
skin infection
= macro- and micro-titer, ELISA, and neutralization
techniques are available
= Neutralization technique has advantage of
stability of reagents
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = STREPTOCOCCAL INFECTION
• LABORATORY TESTING
3. Anti-Hyaluronidase Testing
= test patient serum for antibodies which inhibit
action of Hyaluronidase
= after performance of the test, a clot will form into
the tubes where enzyme activity of
Hyaluronidase has been neutralized by
patient antibody
= Hyaluronidase produced by patients with throat
or skin infections, ASO produced in response
to throat infections only
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = STREPTOCOCCAL INFECTION
• LABORATORY TESTING
4. Streptozyme Testing
= hemagglutination procedure to detect antibodies
to numerous Streptococcal antigens
= sheep RBC’s are coated with Streptolysin,
Streptokinase, Hyaluronidase, DNase, and
NADase
= patient serum diluted 1 : 100, mixed with sheep
RBC’s and observed for agglutination
= rapid and simple to perform, more false positive
and negative results occur
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
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SEROLOGY OF VIRAL INFECTIONS
A.
Hepatitis
= general term meaning inflammation of the liver, usually
accompanied with fever, nausea, vomiting and
jaundice
= can be caused by radiation, chemicals, disease processes
such as autoimmune disease, viruses and cancer
= 5 distinct viruses – A, B, C, D and E
= all of these are RNA viruses except hepatitis B which is a
DNA virus
= initial infection may be clinically silent
= chronic carrier state may develop and may result to liver
failure due to cirrhosis, hepatocellular carcinoma, or
fulminant hepatitis
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = VIRAL HEPATITIS
• Hepatitis A virus (HAV)
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Transmitted by fecal oral route
Occurs worldwide
Most hepatitis epidemics are due to HAV
Progress of infection:
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Incubation of 2-7 weeks, may be asymptomatic or may
include jaundice
Clinical illness develop abruptly and include fever,
anorexia, vomiting, fatigue and malaise
Increase in serum transaminases
RUQ pain, dark urine and pale stool
Recovery 2-4 weeks, no carrier state
Mortality 0-1%
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = VIRAL HEPATITIS
• Hepatitis A virus (HAV)
• Antibody and antigen markers
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First and most clinically useful is IgM antibody to
HAV
IgM indicates acute infection, appears 4-5 weeks
after exposure
IgM disappears in 3-6 months, replaced by IgG
anti-HAV
IgG peaks during convalescence and may remain
detectable for life
Time course of Hepatitis A virus (HAV)
infection
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = VIRAL HEPATITIS
• Hepatitis B virus (HBV)
• Old term “serum hepatitis”, incubation period of 4-26 weeks
• Route of infection is usually parenteral, direct inoculation
• Incidence of infection is 140,000-320,000 cases per year
resulting in 5-6,000 deaths per year
• Duration of acute infection ranges from 4-8 weeks with
symptoms similar to HAV
• 10% progress to chronic
• One-third of chronic at risk of developing chronic active
hepatitis, cirrhosis and/or hepatocellular carcinoma
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = VIRAL HEPATITIS
• Hepatitis B virus (HBV) = Lab Diagnosis
• Involve the detection of three marker system
• Hepatitis B surface antigen (HBsAg) is the first to
appear, appears 2-4 weeks during late incubation,
marker of choice for recent infection
• Anti-Hepatitis B surface antigen (anti-HBs) is the
last antibody to appear, may persist for life
• Between disappearance of HBsAg and appearance
of anti-HBs is known as the core window
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = VIRAL HEPATITIS
• Hepatitis B virus (HBV) = Lab Diagnosis
• IgM antibody to Hepatitis B core antigen (anti-
HBc) may be the only detectable marker during
the core window, differentiates recent infection
from chronic carrier state
• Third marker is Hepatitis Be antigen (HBeAg),
appearance of HBeAg and anti-HBe, closely
coincide with HBsAg
Hepatitis B viral genome
Spread of Hepatitis B virus (HBV) in the
body
Symptoms of typical acute viral hepatitis B infection
correlated with the four clinical periods of this disease
Clinical outcomes of Acute Hepatitis B infection
The serologic events associated with the
typical course of acute HBV infection
Interpretation of Serologic Markers of Hepatitis B
Virus Infection
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = VIRAL HEPATITIS
• Hepatitis D virus (HDV)
• Requires infection with Hepatitis B
• Route of transmission the same as HBV
• Can occur as coinfection or superinfection
Consequences of delta virus infection
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = VIRAL HEPATITIS
• Hepatitis D virus (HDV) = Serological
markers
• HDAg found early, disappears rapidly, not very
useful
• IgM anti-D and total anti-HD (IgM and IgG)
detected during acute phase
• Presence of IgM anti-D and HBsAg together with
IgM anti-HBc indicates co-infection
• Absence of IgM anti-HBc indicates superinfection
• Presence of anti-HD indicates chronic infection
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = VIRAL HEPATITIS
• Hepatitis C virus (HCV)
• Clinically and epidemiologically similar to HBV
• 60-70% of HCV patients will develop chronic
hepatitis, 10-20% cirrhosis and 15% hepatocellular
carcinoma
• HCV and HBV may be present as co-infections
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = VIRAL HEPATITIS
• Hepatitis C virus (HCV) = Serological
Markers
• Serological profile not fully developed
• Present of HCV antibodies only indicates present or
past infection
• Can have false negative in some patients
Outcomes of Hepatitis C infection
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = VIRAL HEPATITIS
• Hepatitis E virus (HEV)
• Similar to HAV in transmission and clinical course
• Found primarily in developing countries, Africa and Asia
• Results in acute hepatitis, no risk of chronic hepatitis
• Pregnant women with HEV may develop fulminant liver
failure and death
• No distinctive markers, diagnosis based on symptoms for
exposed individuals in endemic countries
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = VIRAL HEPATITIS
• Hepatitis G virus
• Independently discovered 1995-1996 by 2
separate research groups
• RNA virus
• Transmissible by blood-borne route
• Found in patients with acute or chronic liver dse.
• Exact clinical significance needs to be further
defined
• ELISA and Western Blot methods have been
developed
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HERPES VIRUS
B. HERPES VIRUS GROUP
= includes EBV, CMV, Herpes simplex
virus type I and II, Varicella-zoster
virus
= DNA viruses that remain within nucleus
while completing life cycle
= most infections are subclinical and
result in latent stage
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HERPES VIRUS GROUP
• Epstein-Barr Virus (EBV)
• Spread through oral transmission of infective saliva
and is the cause of infectious mononucleosis
• Other diseases – Burkitt’s lymphoma,
nasopharyngeal carcinoma, B-cell lymphoma
• Virus may become reactivated and is the
suggested cause of chronic fatigue syndrome
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HERPES VIRUS GROUP
• Epstein-Barr Virus (EBV)
– Characteristics of infection
• 4-7 week incubation, acute self limiting
• Enlarged LN in the neck, sore throat, fever, rash
• Malaise, lethargy, extreme tiredness
• Liver and spleen involvement and enlargement
• Hematology : high WBC, over 20% atypical
reactive lymphocytes
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HERPES VIRUS GROUP
• Epstein-Barr Virus (EBV)
– Serological testing = may involve screening tests to
detect heterophile antibodies
• Heterophile antigens are a group of similar antigens found in
unrelated animals
• Heterophile antibodies produced against heterophile antigens
of one species will cross react with others
• Forssman antigen is an example of a heterophile antigen and
is found on the RBC’s of many species
• Forssman antibodies formed against Forssman antigens will
agglutinate sheep RBC’s
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HERPES VIRUS GROUP
• Epstein-Barr Virus (EBV)
– Infectious Mononucleosis slide tests
• Horse RBC’s possess antigens which react with the
antibody associated with IM
• Patient serum mixed with horse RBC’s,
agglutination is positive
• Not diagnostic, must look at total clinical picture
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HERPES VIRUS GROUP
• Epstein-Barr Virus (EBV)
– EBV specific antibodies may be measured
• Must know pattern of appearance of EBV antigens
• Most valuable is IgM antibody to viral capsid antigen (VCA),
indicates a current infection (best marker), lasts about 12
weeks
• Can also detect anti-early antigen (EA), recent infection and
anti-EB nuclear antigen (EBNA), older infection
• ELISA and immunofluorescence techniques most commonly
used
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HERPES VIRUS GROUP
• Cytomegalovirus
• Transmission occurs from person to person
• Symptoms resemble IM but has negative test for
EBV
• In babies may cause life-threatening illness
resulting in CNS involvement, hearing loss, and
mental retardation
• Seen in patients with deficient immune system,
AIDS, transplantation
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HERPES VIRUS GROUP
• Cytomegalovirus
– Immunologic response
• For best diagnostic results, lab tests for CMV antibody should
be performed by using paired serum samples
• One blood sample should be taken upon suspicion of CMV,
and another one taken within 2 weeks. A virus culture can be
performed at any time the pt. is symptomatic
• IgM antibodies produced against early and intermediate-early
(IE) CMV antigens, last for 3 to 4 months
• IgG appear shortly after and peak at 2 to 3 months
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HERPES VIRUS GROUP
• Cytomegalovirus
– Laboratory Diagnosis
• Range from culture and cytologic techniques to
DNA probes, PCR and serologic techniques
• Detection of antibodies indicator of recent infection
• Viral culture lack sensitivity and are time
consuming and expensive
• Microscopic examination of biopsy specimens,
urine sediment or peripheral blood may reveal the
typical cytomegalic cell with “owl’s eye” inclusion
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HERPES VIRUS GROUP
• Cytomegalovirus
– Laboratory Diagnosis
• Detection of CMV Ag in cells more appropriately detected by
•
•
•
•
•
immunofluorescent techniques using monoclonal antibodies
ELISA is the most commonly available serologic test for
measuring antibody to CMV
The result can be used to determine if acute infection, prior
infection, or passively acquired maternal antibody in an
infant is present
Other tests include various fluorescence assays, indirect
hemagglutination, and latex agglutination
Screening tests using coated latex particles compare
favorably to more complex tests for antibody detection
False positives can occur = RA and Ebstein-Barr antibodies
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HERPES VIRUS GROUP
• Herpes Simplex Virus (HSV)
– Laboratory testing
• Recovery of the virus in cell culture is considered the “gold
standard” for detection of this virus from sources other than
CSF, culture helpful in differentiating types of HSV
• Direct examination using immunofluorescence or
immunoperoxidase staining of cells from lesion
• DNA probes, ELISA, latex agglutination, RIA and indirect
immunofluorescence
• Serology is not very useful because there is a high
prevalence of antibody in the normal population
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HERPES VIRUS GROUP
• Varicella-Zoster Virus
– Laboratory testing important to distinguish
VZV from other infections, selection of
antiviral drugs, or determining immune status
of individuals
• PCR is now the routine testing method for VZV
• Direct fluorescent antibody staining and viral
culture techniques may be used for the detection
of VZV in most specimen types
• IgG and IgM antibody tests by ELISA may be used
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = GERMAN MEASLES
• Rubella Virus
– Laboratory testing
• Performed primarily for diagnosis of acquired infections and
to determine immune status of pregnant patients
• Some tests detect IgG antibodies, other IgM
• Methods include : hemagglutination inhibition, passive
hemagglutination, neutralization, hemolysis in gel,
complement fixation, fluorescent immunoassay, RIA, ELISA
and latex agglutination
• Method depends on volume of testing, turn around time,
complexity, expense and whether a qualitative or quantitative
test is needed
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = MEASLES
• Rubeola
• Serology testing provides best means of confirming a
measles diagnosis
• Methods to detect Rubeola antibodies include :
hemagglutination inhibition, endpoint neutralization,
complement fixation, IFA and ELISA
• In addition to signs and symptoms, diagnosis confirmed by
presence of Rubeola specific IgM antibodies or four-fold rise
in IgG antibody titer in paired samples taken after rash to 10
to 30 days later
• IgM test highly depended on time of sample collection with
3-11 days after rash being optimal
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = MUMPS
• Mumps
• Methods to detect mump antibodies include : complement
•
•
•
•
fixation, hemagglutination inhibition, hemolysis-in-gel,
neutralization assays, IFA and ELISA
Current or recent infections indicated by presence of specific
IgM antibody in single sample which can be detected within
5 days of illness
Fourfold rise in specific IgG antibody in 2 samples collected
during acute and convalescent phases
Fluorescent antibody staining for mumps antigens developed
but not widely used
Cross-reactivity between antibodies to mumps and
parainfluenza viruses has been reported in test for IgG
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Human Immunodeficiency Virus (HIV)
• Etiologic agent of AIDS
• Discovered independently by Luc Montagnier of France and
Robert Gallo of the US in 1983-1984
• Former names of the virus include :
Human T cell Lymphotrophic virus (HTLV-III)
 Lymphadenopathy associated virus (LAV)
 AIDS associated retrovirus (ARV)

• HIV-2 discovered in 1986, antigenically distinct virus endemic
in West Africa
• One million people infected in US, 30 Million worldwide are
infected
• Leading cause of death of men aged 25-44 and 4th leading
cause of death of women in this age group in the US
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Structural genes
• Gag is p55 from which three core proteins (p15,
p17 and p24) are formed
• Env gene codes for envelope proteins gp160,
gp120 and gp41
• Pol codes for p66 and p51 subunits of reverse
transcriptase and p31 an endonuclease
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Immunologic Manifestations
• Early stage slight depression of CD4 count, few symptoms,
temporary
• Window of up to 6 weeks before antibody is detected, by 6
months 95% positive
• During window p24 antigen present, acute viremia and
antigenemia
• Antibodies produced to all major antigens
First antibodies detected produced against gag proteins
p24 and p55
 Followed by antibody to p51, p120 and gp41
 As disease progresses, antibody levels decreases

SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Immunologic Manifestations
• Immune abnormalities associated with increased
viral replication
 Decrease
in CD4 cells
 B cells have decreased response to antigen
 CD8 cells initially increase and may remain elevated
 As HIV infection progresses, CD4 T cells drop
resulting in immunosuppression and susceptibility of
patient to opportunistic infections
 Death comes due to immuno-incompetence
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Laboratory diagnosis of HIV infection
1. Methods utilized to detect
• Antibody
• Antigen
• Viral nucleic acid
• Virus in culture
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Laboratory diagnosis of HIV infection
2. ELISA Testing
= first serological test developed to detect HIV
infection
= antibodies detected include those directed against
p24, gp120, gp160 and gp41, detected first in
infection and appear in most individuals
= used for screening only, false positives do occur
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Laboratory diagnosis of HIV infection
4. Western Blot Testing
= most popular confirmatory test
= antibodies to p24 and p55 appear earliest
but decrease or become undetectable
= antibodies to gp31, gp41, gp120, and
gp160 appear later but are present
throughout all stages of the disease
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Laboratory diagnosis of HIV infection
4. Western Blot Testing = interpretation of result
 no bands, negative
 in order to be interpreted as positive a minimun
of 3 bands directed against the following antigens
must be present : p24, p31, gp41 or gp120/160
 CDC criteria require 2 bands of the following :
p24, gp41 or gp120/160
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Laboratory diagnosis of HIV infection
4. Western Blot Testing = interpretation of result
 indeterminate results are those samples that produce bands
but not enough to be positive, may be due to the following:
1. prior blood transfusions, even with non-HIV-1 infected blood
2. prior or current infection with syphilis
3. prior or current infection with malaria
4. autoimmune diseases
5. infection with other human retroviruses
6. second or subsequent pregnancies in women
*** run an alternate HIV confirmatory assay
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Laboratory diagnosis of HIV infection
5. Indirect immunofluorescence assay
= can be used to detect both virus and
antibody to it
= antibody detected by testing patient serum
against antigen applied to a slide, incubated,
washed and a fluorescent antibody added
= virus is detected by fixing patient cells to slide,
incubating with antibody
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Laboratory diagnosis of HIV infection
6. Detection of p24 HIV antigen
= p24 antigen only present for short time, disappears when
antibody to p24 appears
= anti-HIV-1 bound to membrane, incubated with patient serum,
second anti-HIV-1 antibody attached to enzyme label is added
(sandwich technique), color change occurs
= optical density measured, standard curve prepared to
quantitate results
= positive confirmed by neutralizing reaction, preincubate
patient sample with anti-HIV, retest, if p24 present immune
complexes form preventing binding to HIV antibody on
membrane added
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Laboratory diagnosis of HIV infection
6. Detection of p24 HIV antigen
= test not recommended for routine screening as
appearance and rate of rise are unpredictable
= sensitivity lower than ELISA
= most useful for the following
a. early infection suspected in seronegative patient
b. newborns
c. CSF
d. monitoring disease progress
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Laboratory diagnosis of HIV infection
7. Polymerase Chain Reaction (PCR)
= looks for HIV DNA in the WBC’s of a person
= amplifies tiny quantities of the HIV DNA present, each cycle of
PCR results in doubling of the DNA sequences present
= the DNA is detected by using radioactive or biotiny lated
probes
= once DNA is amplified it is placed on nitrocellulose paper and
allowed to react with a radio-labeled probe, a single stranded
DNA fragment unique to HIV, which will hybridize with the
patient’s HIV DNA if present
= radioactivity is determined
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Laboratory diagnosis of HIV infection
8. Virus isolation
= definitively diagnose HIV
= best sample is peripheral blood, but can use CSF,
saliva, cervical secretions, semen, tears or material
from organ biopsy
= cell growth in culture is stimulated, amplifies
number of cells releasing virus
= cultures incubated one month, infection confirmed
by detecting reverse transcriptase or p24 antigen in
supernatant
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Laboratory diagnosis of HIV infection
9. Viral Load Tests
= viral load or viral burden is the quantity of HIV-RNA that
is in the blood
= measures the amount of HIV-RNA in one milliliter of blood
 take 2 measurements 2-3 weeks apart to
determine baseline
 repeat every 3-6 months in conjunction with CD4
counts to monitor viral load and T-cell count
 repeat 4-6 weeks after starting or changing
antiretroviral therapy to determine effect on viral load
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Laboratory diagnosis of HIV infection
10. Testing of neonates
= difficult due to presence of maternal IgG antibodies
= use tests to detect IgM or IgA antibodies, IgM lacks
sensitivity, IgA more promising
= measurement of p24 antigen
= PCR testing maybe helpful but still not detecting
antigen soon enough : 38 days to 6 months to be
positive
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = DENGUE
• Dengue fever
• Transmitted by mosquitoes
• There are 4 known distinct serotypes ( dengue
virus 1, 2, 3 and 4)
• In children , infection is often sub-clinical or causes
a self-limited febrile disease
• Secondarily infected with a different serotype,
dengue hemorrhagic fever or dengue shock
syndrome
Algorithm for Serologic Testing for AIDS
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = DENGUE
• Dengue fever
• Dengue IgG/IgM Rapid Test is a solid phase
immunochromatographic assay for the rapid
qualitative and differential detection of IgG and
IgM antibodies to dengue virus in human serum,
plasma or whole blood. This test can also detect all
4 Dengue serotypes by using a mixture of
recombinant Dengue envelop proteins
Rapid Test for Dengue
Rapid Test for Dengue
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES =DENGUE
• Dengue fever
– Interpretation of the test
• IgG and IgM positive = indicative of a late primary or early
•
•
•
•
secondary dengue infection
IgM positive = indicative of primary Dengue infection
IgG positive = indicative of secondary or past dengue
infection
Negative = retest in 3-5 days if Dengue infection is
suspected
Invalid = insufficient specimen volume or incorrect
procedural technique. Repeat the test using a new test
device
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = Typhoid Fever
• Typhoid Fever
• Caused by Salmonella typhi
• Rapid detection is now available in the market
Typhidot = a qualitative detection test against a specific
antigen of Salmonella typhi. It can detect both IgG and
IgM separately and simultaneously. Thus, indicating the
status of acute infection, convalescence or previous
exposure
 Salmonella typhi IgG/IgM Rapid test = an
immunochromatographic assay for rapid, qualitative and
differential detection of IgG and IgM antibodies to
Salmonella typhi in human serum, plasma or whole blood

Typhidot
Typhoid Fever Rapid test
Typhoid Fever Rapid Test
H. Pylori Rapid test
Malaria Ab Rapid test
Rapid test for TB
Rapid test for Chlamydia
Rotavirus/Adenovirus Rapid test
Rapid test for Rubella
Rapid test for RSV
Rapid test for Tetanus
Rapid test for Legionella
Rapid test
TUMOR MARKERS
TUMOR MARKERS
• What are they?
• Are substances usually proteins, that are produced by the
body in response to cancer growth or by the cancer tissue
itself and certain benign (noncancerous) conditions
• Detected in higher than normal amounts in the blood, urine,
or body tissues
• Some tumor markers are specific for one type of cancer,
while others are seen in several cancer types
• Measurements can be useful – when used along with x-rays,
or other tests in the detection and diagnosis of some types of
cancer
TUMOR MARKERS
• Measurements of tumor marker levels alone are
not sufficient to diagnose cancer for the
following reasons:
– Tumor marker levels can be elevated in people with
benign conditions
– Tumor marker levels are not elevated in every person
with cancer – especially in early stages of the disease
– Many tumor markers are not specific to a particular
type of cancer
TUMOR MARKERS
• Characteristics required of the “ideal” tumor marker
– The following are desirable
• 100% accuracy in differentiating between healthy
•
•
•
•
•
individuals and tumor patients
Ability to detect all tumor patients, if possible at a very
early stage
Organ specificity, so that information is provided on the
localization of the tumor
Correlation between the concentration of the marker freely
circulating in serum and the individual tumor stages
Ability to indicate all changes in tumor patients receiving
treatment
Prognostic value of the tumor marker concentration
TUMOR MARKERS
• Clinical Uses of Tumor Markers
• Early detection of the tumor
• Differentiating benign from malignant conditions
• Evaluating the extent of the disease
• Monitoring the response of the tumor to therapy
• Predicting the recurrence of the tumor
TUMOR MARKERS
• CARCINO-EMBRYONIC ANTIGEN (CEA)
• A complex glycoprotein with a MW of
approximately 180,000 daltons
• First discovered in patients with adenocarcinoma
of the colon in 1965
• Metabolized primarily by the liver with a circulating
half-life ranging from 1 to 8 days
• Hepatic diseases, including extrahepatic biliary
obstruction, intrahepatic cholestasis and
hepatocellular disease, may impede clearance
rates and increase serum concentrations
TUMOR MARKERS
• CARCINO-EMBRYONIC ANTIGEN (CEA)
• Normally, it is present in the fetal intestine,
pancreas and liver during the first 2 trimesters of
gestation
• Normal colonic mucosa and pleural and lactating
mammary tissue bind to anti-CEA antiserum;
however, the quantity of CEA or CEA-like molecules
expressed in these tissues is much less than that
observed in malignant tumors
• Normal range is from 0 to 2.5 to 3.0 ng/ml as
determined by radioimmunoassay
TUMOR MARKERS
• CARCINO-EMBRYONIC ANTIGEN (CEA)
– Benign conditions that
• Cigarette smoking
• Emphysema
• Gastric ulcer
• Pancreatitis
• Diverticulitis
• BPH
cause elevated CEA
Bronchitis
Gastritis
Hepatic disease
Polyps of colon & rectum
Crohn’s disease
Renal disease
TUMOR MARKERS
• CARCINO-EMBRYONIC ANTIGEN (CEA)
– Malignant conditions causing elevation of CEA in
addition to adenocarcinoma of colon & rectum --- Ca
of the pancreas, lung, breast, stomach, thyroid gland
and female reproductive tract
– Of these non-colonic CA, levels of CEA are most
commonly elevated in CA of the pancreas (65-90%)
and lung (52-77%)
– The magnitude of elevation of levels of CEA correlates
with stage of disease to a lesser extent
TUMOR MARKERS
• Alpha-FETOPROTEIN (AFP)
• An oncofetal protein that was first discovered in
1963 in the serum of mice with hepatoma
• Normal fetal protein synthesized by the liver, yolk
sac, and GIT that shares sequence homology with
albumin
• A major component of fetal plasma, reaching a
peak concentration of 3mg/ml at 12 weeks of
gestation -- following birth, it clears rapidly from
the circulation, having a half-life of 3.5 days
• Concentration in adult serum <20ng/ml
TUMOR MARKERS
• Alpha-FETOPROTEIN (AFP)
– Benign conditions causing elevation of AFP
• 2nd and 3rd trimesters of pregnancy
• Cirrhosis
• Acute and chronic hepatitis
• Hepatic necrosis
TUMOR MARKERS
• Alpha-FETOPROTEIN (AFP)
– Malignant conditions causing elevation of AFP aside
from hepatoma
• Teratocarcinoma of the testis and embryonal Ca (70%)
• Carcinoma of the pancreas (23%)
• Carcinoma of the stomach (18%)
• Carcinoma of the lung (7%)
• Carcinoma of the colon (5%)
*** In patients with hepatoma, the incidence of elevation of
levels of AFP correlates with tumor burden
TUMOR MARKERS
• HUMAN CHORIONIC GONADOTROPIN
(HCG)
• A glycoprotein hormone with a MW of 45,000
daltons
• Composed of 2 polypeptide chain – alpha and beta
 Alpha-chain
is common to several glycoprotein
hormones secreted by the anterior pituitary
 Beta- chain is unique and confers structural and
functional identity to these hormones. Homology
exists with human luteinizing hormone and may
cause immunologic cross-reactivity. Basis of det’n.
TUMOR MARKERS
• HUMAN CHORIONIC GONADOTROPIN (HCG)
• Circulating half-life is 12 to 20 hours
• Normally secreted by placental tissue with highest circulating
•
•
•
•
levels occurring at 60 days of gestation
Significant elevation occurs during pregnancy and in patients
with trophoblastic neoplasms or nonseminomatous germ cell
tumors
It maybe secreted in small amounts by the testis, pituitary
gland and GIT
Maybe elevated in some benign conditions – peptic ulcer
disease, inflammatory intestinal disease and cirrhosis
In patients with trophoblastic disease, levels of HCG correlate
with tumor burden, prognosis of patient and response to
therapy
TUMOR MARKERS
• CALCITONIN
• A peptide hormone composed of 32 amino acids with a MW
•
•
•
•
of 3,149 daltons
A hypocalcemic factor secreted by C cells of the thyroid
gland
Serum half-life is 12 minutes and normal levels are <0.1
nanogram/ml using radioimmunoassay
Marked elevations are observed in medullary carcinoma of
the thyroid
Primary clinical application is to detect familial medullary
carcinoma of the thyroid which is transmitted as an
autosomal dominant pattern

Secretion normally fluctuates in these patients,
provocative tests (pentagastrin stimulation or calcium
infusion) greatly increased the sensitivity of this test to
detect MCT
TUMOR MARKERS
• CALCITONIN
– Other neoplasms less frequently associated
with increased levels
• Small cell carcinoma of the lung
• Carcinoma of the breast
• Carcinoid
• Hepatoma
• Renal cell carcinoma
• Zollinger-Ellison syndrome
TUMOR MARKERS
• CALCITONIN
– Benign conditions associated with increased
level
• Pancreatitis
• Hyperparathyroidism (primary and secondary)
• Paget’s disease of bone
• Pulmonary disease
TUMOR MARKERS
• CATECHOLAMINE METABOLITES
• Most commonly assayed catecholamine metabolites are
vanillylmandelic acid (VMA) and homovanillic acid (HVA),
which are metabolites of norepinephrine and dopamine,
respectively
• Urinary levels of this metabolites can be accurately measured
from a single urine specimen using gas chromatographic
techniques – requires avoidance of tea, coffee, fruit and
vanilla from the diet 72 hours before urinary sampling
• Most useful in diagnosing and monitoring patients with
NEUROBLASTOMA
TUMOR MARKERS
• CATECHOLAMINE METABOLITES
• Neuroblastoma is a malignant lesion of the neural
crest tissue, which most commonly occurs in
children
• Elevated urinary levels of VMA and HVA are
observed in 75 to 95% of patients
• Improved survival time was reported in patients
with a ratio of urinary VMA to HVA of ≥1.5
TUMOR MARKERS
• PROSTATIC ACID PHOSPHATASE
• First proposed as a marker of advanced carcinoma of the
prostate in 1938
• Acid phosphatases are group of enzymes that are also
present in lower concentrations in the bone, kidney, liver,
spleen, and intestine
• PAP is a glycoprotein with a MW of 100,000 daltons, which
consists of two identical subunits
• Levels can be elevated in some benign conditions—
osteoporosis, hypoparathyroidism, hyperthyroidism, prostatic
surgical treatment, catheterization of the urinary tract and
benign prostatic hypertrophy
TUMOR MARKERS
• PROSTATIC ACID PHOSPHATASE
• Other malignant conditions with elevated PAP – multiple
myeloma, osteogenic sarcoma and bony metastases
• Can be measured by biochemical or immunologic methods;
radioimmunoassay is much more sensitive than chemical
determination
• In one study, a direct correlation was observed between
reduced levels of serum acid phosphatase and a 50%
reduction in the mass of the tumor after therapy, thus, PAP
has definite limitations as a tumor marker for carcinoma for
prostate
TUMOR MARKERS
• ADRENOCORTICOTROPHIC HORMONE (ACTH)
• Most frequently observed ectopic hormone produced by
neoplasms
• First reported in 1928 with small cell carcinoma of the lung
• Associated with other malignant diseases – adenocarcinoma
and squamous cell carcinoma of the lung, carcinoid,
pancreatic islet cell tumor, carcinoma of the breast,
carcinoma of the colon, pheochromocytoma, thymoma,
medullary thyroid carcinoma and carcinoma of the ovaries
• Benign conditions – COPD, obesity, HPN, DM
TUMOR MARKERS
• ADRENOCORTICOTROPHIC HORMONE (ACTH)
• Ectopic secretion of ACTH can be differentiated from ACTH
that originates in the pituitary gland by the dexamethasone
suppression test; failure to suppress plasma cortisol levels
with high dose dexamethasone suggests ectopic secretion of
ACTH
• It has no value in screening for carcinoma and pretreatment
levels demonstrate no correlation to patient survival time or
stage of disease
• It lacks the sensitivity and specificity to be clinically useful for
screening, staging, or predicting response to therapy
TUMOR MARKERS
• ANTIDIURETIC HORMONE (ADH)
• Small cell carcinoma is most commonly associated with
•
•
•
•
ectopic secretion of ADH
Secretion of ADH may be detected biochemically or may
present clinically as SIADH
Other malignant diseases with ectopic secretion – carcinoma
of pancreas, bronchial carcinoid tumors, carcinoma of the
adrenal cortex, thymomas, carcinoma of the bladderand
prostate
Benign conditions – pulmonary disease, disorders of the CNS,
anesthetics, and ingestion of drugs
Not a useful marker for screening of carcinoma, staging or
monitoring response to therapy
TUMOR MARKERS
• CA 125
• An antigen present on 80% of nonmucinous
ovarian carcinomas
• Defined by a monoclonal antibody (OC125) that
was generated by immunizing laboratory mice with
a cell line established from human ovarian
carcinoma
• Elevated in other cancers – endometrial,
pancreatic, lung, breast, and colon
• Elevated in benign conditions – menstruation,
pregnancy, endometriosis
TUMOR MARKERS
• CA 19-9
• A monoclonal antibody generated against a colon
carcinoma cell line to detect a
monosialoganglioside found in patients with
gastrointestinal adenocarcinoma
• Elevated in gastric cancer (21-42%), colon cancer
(20-40%), pancreatic cancer (71-93%)
TUMOR MARKERS
• PROSTATE-SPECIFIC ANTIGEN (PSA)
• Found in normal prostatic epithelium and secretions but not
•
•
•
•
•
in other tissues
It is a glycoprotein whose function may be to lyse the
seminal clot
Highly sensitive for the presence of prostatic cancer
Elevation correlated with stage and tumor volume
Predictive of recurrence and response to treatment
Has prognostic value in patients with very high values prior
to surgery are likely to relapse
TUMOR MARKERS
• PROSTATE-SPECIFIC ANTIGEN (PSA)
• Present in low concentrations in the blood of adult
males
• It is produced by both normal and abnormal
prostate cells
• Benign elevations – prostatitis and BPH
COMMON TUMOR MARKERS CURRENTLY IN USE
Tumor Markers
Cancers
What else?
Sample
AFP (Alphafetoprotein)
Liver, germ cell
cancers of
ovaries or testes
Also elevated
during pregnancy
blood
CA 15-3
Breast and
others including
lung and ovaries
Also elevated in
benign breast
conditions;
blood
CA 19-9
Pancreatic,
sometimes
colorectal and
bile ducts
Also elevated in
pancreatitis and
inflammatory
bowel disease
blood
CA 125
ovarian
Also elevated with blood
endometriosis,
some other
diseases and
benign conditions;
not recommended
as a general
screen
COMMON TUMOR MARKERS CURRENTLY IN USE
Tumor markers
Cancers
What else?
Sample
CEA (CarcinoColorectal, lung,
embryonic antigen breast, thyroid,
pancreatic, liver,
cervix, and
bladder
Elevated in other
blood
conditions such as
hepatitis, COPD,
colitis, pancreatitis
and in cigarette
smokers
Estrogen
Receptors
breast
Increased in
hormone
dependent cancer
tissue
hCG (human
chorionic
gonadotropin)
Testicular and
trophoblastic
Elevated in
pregnancy,
testicular failure
Blood, urine
Her-2/neu
breast
Oncogene that is
present in
multiple copies in
20-30% of
invasive breast
cancer
tissue
COMMON TUMOR MARKERS CURRENTLY IN USE
Tumor
markers
Cancer
What else?
Sample
Monoclonal
Immunoglobulins
Multiple Myeloma
and
Waldenstrom’s
macroglobulinemi
a
Overproduction of
an Ig or Ab,
usually detected
by protein
electrophoresis
Blood, tissue
Progesterone
Receptors
breast
Increased in
hormone
dependent cancer
tissue
PSA, total and
free
prostate
Elevated in BPH,
prostatitis and
with age
blood
LESS COMMON TUMOR MARKERS
Tumor
Markers
Cancers
What else?
Sample
B2M (Beta-2 Multiple
microglobulin myeloma,
lymphomas
Crohn’s
disease,
hepatitis
Blood
BTA (Bladder
tumor
antigen
CA 72-4
(Cancer
antigen 72-4
Bladder
Gaining
acceptance
Urine
Ovarian
No evidence
that is better
than CA 125
Blood
LESS COMMON TUMOR MARKERS
Tumor
Markers
Cancers
What else?
Sample
Calcitonin
Thyroid
Medullary
carcinoma
Also elevated in
pernicious
anemia and
thyroidits
Blood
NSE (Neuronspecific enolase
Neuroblastoma,
small lung
cancer
May be better
Blood
than CEA for ff.
this kind of lung
cancer
NMP22
Bladder
Not widely used
Prostate-specific Prostate
membrane
antigen (PSMA)
Urine
Not widely used, Blood
levels increase
normally with
age
LESS COMMON TUMOR MARKERS
Tumor
Markers
Cancers
What else?
Sample
Prostatic acid
phosphatase
(PAP
Metastatic
prostate cancer,
myeloma, lung
cancer
Not widely used Blood
anymore,
elevated in
prostatitis and
other conditions
S-100
Metastatic
melanoma
Not widely used
TA-90
Metastatic
melanoma
Not widely used, Blood
being studied
Thyroglobulin
Thyroid
Used after
thyroid is
removed to
evaluate
treatment
Blood
Blood