INTERPRETATION OF HEPATITIS B

PANEL (HBV 5-IN-1)TEST RESULTS

DERBAN, ISAAC (BMS)

OUTLINE
 INTRODUCTION
 Brief description
 Hepatitis B virus
 Hepatitis B Profile Test (5 – in – 1)
 HBV MARKERS & SIGNIFICANCE
 TEST PRINCIPLE
 SAMPLE COLLECTION AND PREPARATION
 TEST PROCEDURE
 READING THE TEST RESULTS
 INTERPRETATION OF TEST RESULTS
 DISCUSSION
 INTRODUCTION OF HBV 5-IN-1 INTERPRETER SOFTWARE
 SUMMARY
 INTRODUCTION (HBV)
 Hepatitis B Virus

 Hepatotropic DNA virus

 The core of the virus contains

 DNA Polymerase
 Core Antigen and Envelope antigen

 The core is enclosed in a coat that

contains lipid, protein and
carbohydrate and expressed as the
surface antigen.
INTRODUCTION (HBV)
 Three distinct antigen-antibody systems are associated with HBV
infection:

 Hepatitis B surface antigen (HBsAg) and its antibody anti-HBs

(HBsAb)

 Hepatitis B core antigen (HBcAg) and its antibody anti-HBc (HBcAb)

 Hepatitis B e antigen (HBeAg) and its antibody anti-HBe (HBeAb)

 Clinically, HBsAg, HBsAb, HBeAg, HBeAb and HBcAb are the

important markers in the diagnosis of HBV infection.
INTRODUCTION (HBV PROFILE)
 HBV Profile (5-in-1) is a 5 test (within
one device) immunochromatographic
based one step in vitro test

 It is designed for the qualitative

detection of
 HBsAg
 HBsAb
 HBeAg
 HBeAb
 HBcAb

 It is intended to be used as screening

test and as an aid in the diagnosis of
infection with HBV
 HBV MARKERS AND SIGNIFICANCE
 Hepatitis B surface  Antibody to hepatitis B
antigen (HBsAg) surface antigen (HBsAb)
 First serologic marker to  Is a marker of immunity
appear  Its presence indicates an
 Is a marker of infectivity i.e. immune response to
Confirm HBV infection 1. HBV infection
 Its presence indicates either 2. Vaccination
acute or chronic HBV
infection
 HBV MARKERS AND SIGNIFICANCE
 Hepatitis B "e" antigen  Antibody to hepatitis B
(HBeAg) "e" antigen (HBeAb)
 Is a marker of a high degree of  may be present in an
HBV infectivity infected or immune person
 It correlates with high level of  In persons with chronic
HBV replication i.e. Reflects HBV infection, its presence
the presence of circulating suggests
intact virions, DNA 1. Low viral titer
polymerase and HBV DNA 2. Low degree of infectivity.
 It is primarily used to help
determine the clinical
management of patients with
chronic HBV infection
 HBV MARKERS AND SIGNIFICANCE
 Antibody to hepatitis B core antigen (HBcAb)
 Is a marker of
 Acute (IgM)
 Chronic (IgG)
 Resolved HBV infection
 It is not a marker of vaccine-induced immunity
 It may be used in pre-vaccination testing to determine previous
exposure to HBV infection
 Types:
1. IgM antibody subclass of anti-HBc
2. IgG antibody subclass of anti-HBc (Total HBcAb)
HBV MARKERS AND SIGNIFICANCE

 IgM antibody subclass of anti-HBc.

 Positivity indicates recent infection with HBV
 Its presence indicates acute infection.
 NB: Detected using the 7 –in -1 test kit.
HBV MARKERS AND SIGNIFICANCE

 IgG antibody subclass of anti-HBc (Total HBcAb)

 is a marker of past or current infection with HBV.
 Total HBcAb and HBsAg are both positive (in the absence
of IgM anti-HBc), this indicates chronic HBV infection.
 TEST PRINCIPLE
 Detection of HBsAg, HBsAb  Strip/Lateral Flow
and HBeAg Immunochromatographic
 Sandwich immunoassay device is used for the
technique. detection HBsAg, HBsAb,
 Detection of HBeAb and HBeAg, HBeAb and HBcAb
HBcAb
 Competitive immunoassay
 Detection of HBsAg
 HBsAg test is a double antibody sandwich
immunoassay.
 Colloidal gold conjugated anti-HBsAg antibody
complexes are dry-immobilized in the test device.
 When the sample is added,
 it migrates by capillary diffusion through the strip
rehydrating the gold conjugate complexes.
 If present, HBsAg will react with the gold conjugate
complexes forming particles.
 These particles will continue to migrate along the
strip until the Test Zone (T)
 where they are captured by anti-HBsAg antibodies
immobilized there
 a visible red line appears.
 If there is no HBsAg in sample, no red line will appear
in the Test Zone (T).
 The gold conjugate complexes will continue to
migrate alone
 until they are captured in the Control Zone (C)
by immobilized goat anti-mouse IgG antibody
aggregating a red line, which indicates the validity
of the test.
 Detection of HBsAb
 HBsAb test is a double antigen sandwich
immunoassay.
 Colloidal gold conjugated HBsAg complexes are dry-
immobilized in the test device.
 When the sample is added
 it migrates by capillary diffusion through the strip
rehydrating the gold conjugate complexes.
 If present, HBsAb will react with the gold conjugate
complexes forming particles.
 These particles will continue to migrate along the
strip until the Test Zone (T)
 where they are captured by HBsAg immobilized
there
 a visible red line appears.
 If there is no HBsAb in sample, no red line will
appear in the Test Zone (T).
 The gold conjugate complexes will continue to
migrate alone
 until they are captured in the Control Zone (C) by
immobilized goat anti-HBsAg antibody aggregating a
red line, which indicates the validity of the test.
 Detection of HBeAg
 HBeAg test is a double antibody sandwich
immunoassay.
 Colloidal gold conjugated anti-HBeAg antibody
complexes are dry-immobilized in the test device.
 When the sample is added
 it migrates by capillary diffusion through the strip
rehydrating the gold conjugate complexes.
 If present, HBeAg will react with the gold conjugate
complexes forming particles.
 These particles will continue to migrate along the
strip until the Test Zone (T)
 where they are captured by anti-HBeAg antibodies
immobilized there
 a visible red line appears.
 If there is no HBeAg in sample, no red line will
appear in the Test Zone (T).
 The gold conjugate complexes will continue to
migrate alone
 until they are captured in the Control Zone (C)
by immobilized goat anti-mouse IgG antibody
aggregating a red line, which indicates the validity
of the test.
 Detection of HBeAb
 HBeAb test is a competitive immunoassay.
 Colloidal gold conjugated anti-HBeAg antibody
complexes are dry-immobilized in the test device.
 When the specimen is added,
 it migrates with the gold conjugate complexes by
capillary diffusion through the strip.
 If present, HBeAb will compete with gold conjugate
complexes for the limited amount HBeAg
immobilized in the Test Zone (T).
 It will prevent the gold conjugate complexes from
reacting with HBeAg
 no red line appears in the Test Zone (T).
 If there is no HBeAb in the specimens
 gold conjugate complexes will react with HBeAg
 a visible red line appears.
 To serve as a procedural control,
 a red line will always appears in the Control Zone
(C)
 which indicates the validity of the test.
 Detection of HBcAb
 HBeAb test is a competitive immunoassay.
 Colloidal gold conjugated anti-HBeAg antibody
complexes are dry-immobilized in the test device.
 When the specimen is added,
 it migrates with the gold conjugate complexes by
capillary diffusion through the strip.
 If present, HBeAb will compete with gold conjugate
complexes for the limited amount HBeAg
immobilized in the Test Zone (T).
 It will prevent the gold conjugate complexes from
reacting with HBeAg
 no red line appears in the Test Zone (T).
 If there is no HBeAb in the specimens
 gold conjugate complexes will react with HBeAg
 a visible red line appears.
 To serve as a procedural control,
 a red line will always appears in the Control Zone
(C)
 which indicates the validity of the test.
SAMPLE COLLECTION AND PREPARATION
 Whole Blood:
 Collect whole blood specimens following regular
clinical laboratory procedures.
 Heparinized capillary tubes must be used for collecting
whole blood samples. Do not use hemolyzed blood
samples.
 Whole blood specimens should be used immediately
after collection.
SAMPLE COLLECTION AND PREPARATION
 Serum or Plasma
 Collect serum or plasma specimens following regular
clinical laboratory procedures.
 Only those specimens that are clean, clear and with good
fluidity can be used for the assay.
 Those specimens that are apparently hemolyzed, extremely
thickened or with very high fat level are NOT suitable for
the assay.
 Storage: A specimen should be refrigerated if not used the
same day of collection. Specimens should be frozen if not
used within 3 days of collection. Avoid freezing and thawing
the specimens more than 2-3 times before use.
 TEST PROCEDURE
 Remove the test device from its
protective foil wrapper by tearing at
the notch. Holding the strip in a
vertical position
 Add 60ul (2 drops) of whole blood,
serum or plasma to each sample well
and simultaneously start timing.
 Read the results after 15 minutes. Test
results should not be interpreted after
20minutes.
 To avoid confusion, discard the test
device after interpreting the result.
 NB: The actual technical
specifications are subject to the
insert provided with the
product.
 READING THE TEST RESULTS
 PositiveTest Results
 HBsAg strip : One magenta band appears on
the control region, a magenta band also
appears on the test region (lower portion of
the read area).
 HBsAb strip : One magenta band appears on
the control region, a magenta band also
appears on the test region (lower portion of
the read area).
 HBeAg strip: One magenta band appears on
the control region, a magenta band also
appears on the test region (lower portion of
the read area)
 HBeAb strip: One magenta band appears on
the control region, No visible band on the test
region (lower portion of the read area)..
 HBcAb strip : One magenta band appears on
the control region, No visible band on the test
region (lower portion of the read area).
 READING THE TEST RESULTS
 Negative Test Results
 HBsAg strip : One magenta band appears on
the control region, No visible band on the test
region (lower portion of the read area).
 HBsAb strip : One magenta band appears on
the control region, No visible band on the test
region (lower portion of the read area)
 HBeAg strip: One magenta band appears on
the control region, No visible band on the test
region (lower portion of the read area).
 HBeAb strip: One magenta band appears on
the control region, a magenta band also
appears on the test region (lower portion of
the read area).
 HBcAb strip : One magenta band appears on
the control region, a magenta band also
appears on the test region (lower portion of
the read area).
 INTERPRETATION OF TEST RESULTS

 Interpretation of test  Brief Description of the

results is done using software.
 The correct reading of the
test results.
 HBV markers and their
significance.
 INTERPRETATION OF TEST RESULTS
(Assessment – Discussion)
HBV 5 – in – 1Test Results  What is the No. Of Bands/Result
 HBsAg – Negative
 HBsAb – Negative
 HBeAg – Negative
 HBeAb – Negative
 HBcAb – Negative

INTERPRETATION
 Patient has not been exposed
to HBV; thus, susceptible
 INTERPRETATION OF TEST RESULTS
(Assessment – Discussion)
HBV 5 – in – 1Test Results  What is the No. Of Bands/Result
 HBsAg – Negative
 HBsAb – Negative
 HBeAg – Negative
 HBeAb – Negative
 HBcAb – Positive

INTERPRETATION
 Resolved infection (most
common)
 Resolving acute infection
 INTERPRETATION OF TEST RESULTS
(Assessment – Discussion)
HBV 5 – in – 1Test Results  What is the No. Of Bands/Result
 HBsAg – Positive
 HBsAb – Negative
 HBeAg – Negative
 HBeAb – Negative
 HBcAb – Negative

INTERPRETATION
 Exposure to Hepatitis B virus
 INTERPRETATION OF TEST RESULTS
(Assessment – Discussion)
HBV 5 – in – 1Test Results  What is the No. Of Bands/Result
 HBsAg – Negative
 HBsAb – Positive
 HBeAg – Negative
 HBeAb – Negative
 HBcAb – Negative

INTERPRETATION
 Past exposure to HBV with
recovery
 Immune due to Hepatitis B
vaccination
 INTERPRETATION OF TEST RESULTS
(Assessment – Discussion)
HBV 5 – in – 1Test Results  What is the No. Of Bands/Result
 HBsAg – Positive
 HBsAb – Negative
 HBeAg – Negative
 HBeAb – Negative
 HBcAb – Positive

INTERPRETATION
 Chronic Hepatitis B infection
 Patient is a carrier (Carrier
State)
 INTERPRETATION OF TEST RESULTS
(Assessment – Discussion)
HBV 5 – in – 1Test Results  What is the No. Of Bands/Result
 HBsAg – Negative
 HBsAb – Positive
 HBeAg – Negative
 HBeAb – Negative
 HBcAb – Positive

INTERPRETATION
 Immune due to natural
infection
 Exposure to HBV with
recovery or immunity
 INTERPRETATION OF TEST RESULTS
(Assessment – Discussion)
HBV 5 – in – 1Test Results  What is the No. Of Bands/Result
 HBsAg – Positive
 HBsAb – Negative
 HBeAg – Negative
 HBeAb – Negative
 HBcAb – Positive (IgM)

INTERPRETATION
 Acute Hepatitis B infection
 INTERPRETATION OF TEST RESULTS
(Assessment – Discussion)
HBV 5 – in – 1Test Results  What is the No. Of Bands/Result
 HBsAg – Positive
 HBsAb – Negative
 HBeAg – Positive
 HBeAb – Negative
 HBcAb – Negative

INTERPRETATION
 No interpretation. Repeat
test, read results at the right
time and also check the
expiry date of the kits
 INTERPRETATION OF TEST RESULTS
(Assessment – Discussion)
HBV 5 – in – 1Test Results  What is the No. Of Bands/Result
 HBsAg – Positive
 HBsAb – Negative
 HBeAg – Negative
 HBeAb – Positive
 HBcAb – Positive

INTERPRETATION
 Chronic Hepatitis B infection
with non-replicating virus
 INTERPRETATION OF TEST RESULTS
(Assessment – Discussion)
HBV 5 – in – 1Test Results  What is the No. Of Bands/Result
 HBsAg – Positive
 HBsAb – Negative
 HBeAg – Positive
 HBeAb – Negative
 HBcAb – Positive

INTERPRETATION
 Chronic Hepatitis B infection
with active replicating virus
 INTERPRETATION OF TEST RESULTS
(Assessment – Discussion)
HBV 5 – in – 1Test Results  What is the No. Of Bands/Result
 HBsAg – Positive
 HBsAb – Positive
 HBeAg – Positive
 HBeAb – Positive
 HBcAb – Positive

INTERPRETATION
 No interpretation. Repeat
test, read results at the right
time and also check the
expiry date of the kits
 INTERPRETATION OF TEST RESULTS
(Assessment – Discussion)
HBV 5 – in – 1Test Results  What is the No. Of Bands/Result
 HBsAg – Positive
 HBsAb – Positive
 HBeAg – Positive
 HBeAb – Negative
 HBcAb – Negative

INTERPRETATION
 No interpretation. Repeat
test, read results at the right
time and also check the
expiry date of the kits
 INTERPRETATION OF TEST RESULTS
(Assessment – Discussion)
HBV 5 – in – 1Test Results  What is the No. Of Bands/Result
 HBsAg – Positive
 HBsAb – Positive
 HBeAg – Negative
 HBeAb – Positive
 HBcAb – Positive

INTERPRETATION
 No interpretation. Repeat
test, read results at the right
time and also check the
expiry date of the kits
 INTERPRETATION OF TEST RESULTS
(Assessment – Discussion)
HBV 5 – in – 1Test Results  What is the No. Of Bands/Result
 HBsAg – Negative
 HBsAb – Negative
 HBeAg – Positive
 HBeAb – Negative
 HBcAb – Negative

INTERPRETATION
 No interpretation. Repeat
test, read results at the right
time and also check the
expiry date of the kits
 INTERPRETATION OF TEST RESULTS
(Assessment – Discussion)
HBV 5 – in – 1Test Results  What is the No. Of Bands/Result
 HBsAg – Positive
 HBsAb – Negative
 HBeAg – Positive
 HBeAb – Positive
 HBcAb – Negative

INTERPRETATION
 No interpretation. Repeat
test, read results at the right
time and also check the
expiry date of the kits
 SUMMARY
 HBV Profile (5-in-1) is a 5
test (within one device)
immunochromatographic
based one step in vitro test
 the qualitative detection of
 HBsAg
 Hepatitis B Virus  HBsAb
 HBeAg
 Three distinct antigen-
 HBeAb
antibody systems are
 HBcAb
associated with HBV
infection.
 Clinically, HBsAg,  It is intended to be used as
HBsAb, HBeAg, HBeAb screening test and as an aid in
and HBcAb are the the diagnosis of infection
important markers in the with HBV
diagnosis of HBV infection.
SUMMARY

 Hepatitis B surface Antigen  Antibody to HBeAg

(HBsAg) (HBeAb)
 Is a marker of infectivity  Is a marker of low degree
 Antibody to HBsAg of HBV infectivity (In
(HBsAb) chronic patient)
 Is a marker of immunity  Antibody to Hepatitis B
 Hepatitis B envelope
core antigen (HBcAb)
Antigen (HBeAg)  Is a marker of
 Is a marker of high degree  Acute (IgM)
of HBV infectivity  Chronic (IgG)
 Resolved HBV infection
SUMMARY: READING OF TEST RESULTS

 POSITIVE RESULTS  NEGATIVE RESULTS

SUMMARY:INTERPRETATION OF RESULTS
HBsAg HBsAb HBeAg HBeAb HBcAb (Total) Remark/Interpretation

Negative Negative Negative Negative Negative Susceptible

Indicate the number of bands to be seen on the panel
1 1 1 2 2
Negative Negative Negative Negative Positive Resolving acute infection or Resolved infection
Indicate the number of bands to be seen on the panel (most common)
1 1 1 2 1

Positive Negative Negative Negative Negative Exposure to Hepatitis B virus

Indicate the number of bands to be seen on the panel
2 1 1 2 2
Negative Positive Negative Negative Negative Past exposure to Hepatitis B virus with recovery
Indicate the number of bands to be seen on the panel or Immune due to Hepatitis B vaccination
1 2 1 2 2

Positive Negative Negative Negative Positive Chronic Hepatitis B infection or Carrier state
Indicate the number of bands to be seen on the panel
2 1 1 2 1
Negative Positive Negative Negative Positive Immune due to natural infection/Exposure to
Indicate the number of bands to be seen on the panel Hepatitis B virus with recovery or immunity
1 2 1 2 1

Positive Negative Negative Negative Positive (IgM) Acute Hepatitis B infection

Indicate the number of bands to be seen on the panel
2 1 1 2 1
Positive Negative Negative Positive Positive Chronic Hepatitis B infection with non-replicating
Indicate the number of bands to be seen on the panel virus
2 1 1 1 1
Positive Negative Positive Negative Positive Chronic Hepatitis B infection with active
Indicate the number of bands to be seen on the panel replicating virus
2 1 2 2 1
 REFERENCES
 Giannakos, Alexander, "DEVELOPMENT OF A PAPER BASED LATERAL FLOW
DEVICE FOR THE DETECTION OF IαIPVIA COMPETITIVE ELISA" (2014). Open Access
Master'sTheses. Paper 397.

 M. Sajid et al., Designs, formats and applications of lateral flow assay: A literature review,
Journal of Saudi Chemical Society (2014),http://dx.doi.org/10.1016/j.jscs.2014.09.001

 Interpretation of Hepatitis B serologic test results , www. cdc.gov/hepatitis

 Lumiquick, Rapid HBV Panel test- manual

 Gerbers, R. Development of Enhanced Lateral Flow Test Devices for Point Of

CareDiagnstics. s.l. : University of Rhode Island Department of Mechanical,Industrial and
Systems Engineering, 2013.

 Föllscher, W. Development of a Platform for Lateral Flow Test Devices with the
Capability of Using Multiple Fluids. s.l. : University of Rhode Island ,Department of
Mechanical, Industrial and Systems Engineering, 2013.
 THANKS FOR YOUR
ATTENTION AND PARTICIPATION