Serology Testing 101:

The Basics

W. Barry Lee, MD, FACS

Georgia Eye Bank, Atlanta, GA
EBAA 2009 Annual Meeting, Seattle
 I have no financial interest in this material
 Contents for Review
 Plasma dilution
 Tests required by FDA
 Future testing requirements by FDA
 Non-required and conflicting test
results
 Tissue Recalls
 Plasma Dilution
 What is plasma dilution?
 Why do we care?
 How do we calculate it?
BASIC PHYSIOLOGY
 What is Plasma
 Liquid portion of blood
 Approximately 55% of blood volume
 Contents
 Water
 Proteins (albumin, antibodies,clotting factors)
 Electrolytes
 Hormones, metabolic products
 Viral particles / antigens
 Physiological Response to
Shock or Hemorrhage
 Loss of blood volume
 Efflux of RBCs and plasma proteins
 Increased heart rate and vasoconstriction
in attempt to restore fluid to blood vessels
 Slow redistribution of plasma proteins over
24-48 hours
PLASMA DILUTION
 What is Plasma Dilution?
 Loss of plasma proteins leads to dilution of
bloodstream
 Addition of IV fluids/blood products further
dilutes what remains
(IgG, IgM,viral particles, etc.)
 Concentration of Ig & viral antigens may drop
below limits for detection tests
 FALSE NEGATIVE TEST RESULT
 Causes of plasma dilution
 Hemorrhage / blood loss
 IV fluid administration
 Blood
 Whole blood

 RBC’s

 Colloids (large molecules)

 Albumin, Fresh frozen plasma, Platelets

 Crystalloids (small molecules)

 Salt solutions (Normal saline, Lactated Ringers)

 Glucose solutions (D5W)

 Total parenteral nutrition (TPN)

G1.220 Infectious Disease Testing
of Medical Standards
 Plasma Dilution Donor Evaluation
 Did donor blood loss occur ?
 Did the donor receive “Infusion/transfusion of
crystalloids and/or colloids and blood” ?
 EBAA Medical Standards
 “FDA regulations shall be used to record
infusion/transfusion volumes given to each
donor within 48 hours prior to obtaining the
blood sample for infectious disease testing”
 Every donor with blood loss
 Every donor age 12 and under receiving
infusion/transfusion preceding sampling.
Samples not suitable for testing
 Colloids and/or crystalloids were administered
& their total volume exceeds the donor’s
plasma volume
 Whole blood/RBCs or a combination of blood,
colloids, and crystalloids were administered &
volume exceeds the donor’s blood volume
 Calculate the blood/colloids/crystalloids
against donor plasma volume. Reject the
sample for testing if either of these
calculations exceeds the appropriate volume
 FDA-Required Tests in 2005

Donor Eligibility
 HIV I/II antibodies (cadaveric test kit)
 Hepatitis B surface antigen (cadaveric test kit)
 Hepatitis C antibodies (cadaveric test kit not required)
 Syphilis (RPR)
 positive screening can be overridden by negative confirmatory
treponema-specific test

17
 Current FDA-Required Tests
 Donor Eligibility
 HIV I/II antibodies (cadaveric test kit)
 HIV I/HCV NAT*
 Hepatitis B surface antigen (cadaveric test kit)
 HBV core antibody
 Hepatitis C antibodies
 Syphilis (RPR)
 positive screening can be overridden by negative
confirmatory treponema-specific test
 Pending FDA Requirements
 Final Guidance on Donor Eligibility (pending)
 FDA to announce implementation dates for:

 HCV antibodies*
 HBV NAT*
 WNV NAT*
 Trypanosoma cruzi antibodies
 *test kit approved for cadaveric blood
 Nucleic Acid Testing
Shortens the seronegative window

Antibody/Ag Nucleic Acid

Testing Testing

HIV 16-22 days 7-11 days

Hepatitis B 59 days 39-49 days

Hepatitis C 70-82 days 7-11 days

 Nucleic Acid Testing
Probability of missing an infected tissue donor

EIA EIA + NAT

HIV 1/700x106 1/1000x106

Hepatitis B 1/82x106 1/98x106

Hepatitis C 1/7x106 1/54x106

Clinically significant? Cost/benefit?

 Effectiveness

 Since institution of serologic testing for

HIV, hepatitis in 1980s:
 >600,000 corneal transplants in USA
 HIV transmission: zero cases
 Hepatitis B transmission: zero cases
 Hepatitis C transmission: zero cases
 HIV
 Human Immunodeficiency Virus 1
 etiologic agent for AIDS
 present in tears, cornea
 Human Immunodeficiency Virus 2
 immune deficiency, neurologic disease
 less virulent than HIV 1
Serology Time Course
 HIV
 Negative HIV 1 & 2 screening is required.
 Available tests include:
 IgG, IgM antibody to HIV 1 and 2 (EIA, RIA, IF)
 envelope, core, polymerase proteins: gp41, gp120,
gp160, gag p24, gag p17, p66, p32 (Western blot)
 viral nucleic acid (NAT by PCR or TMR)
 reverse transcriptase (co-cultivation)
 Risk Estimate: HIV

 Transmission: parenteral and sexual contact

 Risk after exposure to HIV (+) blood:
 Percutaneous exposure (needle stick): 0.3%
 Mucous membrane exposure : 0.09%
 Potential for transmission via transplant
 Avascular cornea = lower risk
 no reported cases, despite (+) donors

 probably very low

 No reported cases of HIV seroconversion associated

with corneal transplantation
 Hepatitis B Virus
 Acute hepatitis, no sequelae (80-85%)
 More serious problems (15-20%)
 fulminant fatal hepatitis
 chronic infectious hepatitis
 cirrhosis
 hepatocellular carcinoma
 Transmissible via corneal transplant
Hepatitis B Virus:
Structure
 Chronic Hepatitis B: Serology

HBsAg

Total anti-HBc
Titer

IgM anti-HBc

4 8 12 16 20 24 28 32 36 52 Years

Weeks after Exposure

 Hepatitis B Virus

 Negative HBsAg screening is required.

 Other tests (EIA, RIA) include:
 anti-HBc (core antibody)
 HBV NAT (nucleic acid testing)
 anti-HBs (surface antibody)
 HBeAg/anti-HBe (e antigen/ e antibody)
 Screening vs. confirmatory tests
 Hepatitis B Virus

Interpretation of Core Antibody Results

 Core-positive donors may not be used

 Risk Estimate: Hepatitis B
 Transmission: parenteral and non-parenteral
 Risk after exposure to HBsAg (+) blood
(needle-stick):
HBeAg Disease Seroconversion
Positive 22-31% 37-62%
Negative 1-6% 23-37%
 Avascular cornea = lower risk
 Transmission of hepatitis B via corneal
transplant has been reported
 Hepatitis C Virus
 Acute hepatitis, no sequelae (25%)
 Chronic infection (75%)
 cirrhosis (20-50%)
 hepatocellular carcinoma
 mortality (5-10%)
 Potential for transmission via transplant
 no reported cases (6 transplanted corneas)
 very low, but HCV RNA present in cornea
 Hepatitis C Virus
 Negative HCV antibody screening required
 Other tests available include:
 confirmatory immunoblot assays
 viral RNA (NAT by PCR or TMR)
 EIA for core antigen
 Screening vs. confirmatory tests
 Hepatitis C Virus
 Limitations of HCV antibody test (EIA):
 poor indicator of past vs. current infection
 high false positive rate in U.S.
 prolonged window prior to seroconversion
 negative in 10% of those infected with HCV
 HCV RNA has been detected in corneas
from seropositive donors
 HCV NAT may be better
 Risk Estimate: Hepatitis C

 Transmission: less clear – sexual, parenteral

 Risk after exposure to HCV (+) blood:
 Percutaneous exposure (needle stick): 1.8%
 Mucous membrane exposure : “rare”
 No reported cases of hepatitis C infection
related to corneal transplantation
 Risk is probably nil if confirmatory testing of
initially positive specimen is negative
 Syphilis
 Treponema pallidum
 Different stages of disease
 Multi-organ clinical findings

 Potential for transmission via transplant

 no reported cases
 no transmission in animal studies
 extremely unlikely
 Syphilis
 Negative screening is required:
 RPR, STS, VDRL
 Confirmatory tests:
 TPI: preferred, lower false (+) rate
 FTA-ABS, MHA-TP, PK-TP
 May remain positive for years after resolution

 Tissue transplantable if confirmatory test is

negative
 Risk Estimate: Syphilis
 Transmission: sexual, parenteral
 Transmission risk after corneal storage in
Optisol GS is probably nil
 No reported cases of syphilis transmission
attributed to corneal transplantation
 Human T-Lymphotropic Virus
 HTLV-1
 adult T-cell leukemia
 HAM/TSP
 HTLV-2
 no clear association with human disease
 Potential for transmission via transplant
 no reported cases
 probably very low
 Human T-Lymphotropic Virus

 Screening for HTLV antibodies via EIA,

IFA: not required
 Confirmatory testing: Western blot, RIA
on purified viral protein
 Tissue from donors with HTLV-1 or
HTLV-2 infection shall not be offered for
surgical purposes.
 G1.280: Non-required
Laboratory Results

 “Non-required positive serology tests

must be reported to the eye bank's
medical director, who must review and act
on them, or the eye bank must have a
standard policy regarding the action to be
taken in response to that test.”
 G1.280: Non-required
Laboratory Results

 All conflicting serology results must be

reported to:
 the eye bank's medical director, who must
review and act on them
 the EBAA, within 45 days
Non-Required Tests: What to Do
 If test indicates donor may be infectious,
do not offer tissue for surgical use
 Use discretion in other cases, erring on
the side of caution
 If offered for surgery, discuss
implications with operating surgeon
 G1.290: Tissue Recall
 Positive test results or information about
behavioral risks or medical history, received
after release of tissue, that indicate a risk for
transmission of a relevant communicable
disease must be reported to:
 the eye bank's medical director
 the consignee (i.e. the transplanting surgeon or
distributing eye bank), within 45 days
 the EBAA, within 45 days
 the FDA, within 45 days
 Recall

 Consignee notification of positive results

for a relevant communicable disease,
after tissue release

 Does not require removal of tissue that

has already been transplanted

 FDA notification necessary

 Recalls: What To Do

 Perform confirmatory tests

 Specific confirmatory tests:
 HIV 1/2 Ab: Western blot (HIV 1,2), nucleic
acid
 HBsAg: confirmatory neutralization assay
 Hepatitis B core antibody: anti-HBs
 Hepatitis C antibody: immunoblot
 Recommendations to Surgeon
 Encourage discussion of results with
recipient
 Recommend recipient testing, immediately
and in 6-12 weeks, 6 months
 Infectious disease consultation if recipient
tests positive
 No data on risk/benefit of tissue removal or
post-exposure prophylaxis (PEP)
 Serology Testing Summary
 An important tool for determining donor eligibility
 Highly effective at reducing the risk of systemic
disease transmission
 FDA requirements for testing remain in flux as
new infectious disease risks develop
 Careful risk/benefit and cost/benefit evaluation
must continue with new test recommendations