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Jeanne V. Linden, M.D., M.P.H.
Director
Telephone: (518) 485-5341
Fax: (518) 485-5342
btraxess@health.state.ny.us

Blood and Tissue Resources Program

Guide For The Interpretation and Management of Tissue Donor Screening Test Results

Blood and Tissue Resources Program
Wadsworth Center
New York State Department of Health

April 1997
Adapted from materials prepared by the New York Blood Center, New York, New York

TABLE OF CONTENTS

TABLES and APPENDICES

GENERAL ASPECTS OF TISSUE DONOR SCREENING

TESTS UTILIZED IN SCREENING FOR INFECTIOUS DISEASES

Table 1 lists the assays routinely performed on tissue donors, except for indicators of infection for some sexually transmitted diseases for which testing is performed only on reproductive tissue donors. Results of screening tests determine the suitability of tissue for transplantation into a recipient, and the acceptability of the donor, if living, for future donations.

Serologic Screening tests detect either specific antigens or specific antibodies to infectious agents -- hepatitis B surface antigen (HBsAg), or antibodies to the core of hepatitis B (anti- HBc) and to hepatitis C (anti-HCV), human immunodeficiency virus types 1 and 2 (HIV-1/HIV-2), human T-cell ly,mphotropic virus types I and II (HTLV-I and HTLV-II) and Treponema pallidum.

False reactive tests results are common in donor screening, because of the high sensitivity of the tests. Table 1 lists the prevalence of reactive test results among healthy blood donors and in the estimated false positive rate for each assay. The section below on Sensitivity, Specificity and Safety discusses reasons for false positive results.

All donor samples are subjected to initial screening tests. If the initial screen is negative, the donated tissue is considered suitable for transplantation. Initially reactive samples for any test are repeated in duplicate. If one or more of the duplicate tests is reactive, the sample is considered repeatedly reactive. Repeatedly reactive results usually lead to rejection of the donor. When possible, these samples are subjected to confirmatory tests for assessment of the test results' significance for the health of the donor. Supplemental/confirmatory assays are not available for all screening tests. See Appendix A for guidance on interpretation of supplemental/confirmatory test results.

Donor rejection is the usual consequence of a repeatedly reactive screening test. This is an extremely conservative approach to preserve the safety of the tissue supply.

Reentry procedures may be performed on living donors deferred because of repeatedly reactive screening results for HIV and negative confirmatory test results. Tests are performed on two separate samples collected six months apart. When results of the tests called for by the re-entry protocol are negative, the donor may be accepted into the donor base. Reentry protocols for HBsAg and HCV have also been developed by the Food and Drug Administration (FDA).

SENSITIVITY, SPECIFICITY AND THE SAFETY OF THE TISSUE SUPPLY

The major objective of the screening process is to protect the recipient. Thus, the safety of the tissue supply depends on the sensitivity of screening assays. Sensitivity is the ability of a test to identify true positives. Specificity is the ability of a test to avoid false positives. An assay able to detect all true positives would have 100% sensitivity. These two variables are interrelated: a very sensitive test is often less specific, and vice versa. Approaches to increase assay sensitivity frequently result in loss of assay specificity, with consequent increase in the number of samples with non-specific (false positive) test results.

Tissue banks ensure the safety of transplant recipients by enhancing the sensitivity of screening tests. Consequently, in order to detect most of the donors who are truly positive for an infectious disease marker, many false positive test results are generated.

Reagent manufacturers, the FDA, and many scientists have attempted to deal with these issues by developing confirmatory tests for distinguishing true positive from false positive results. Unfortunately, there is no confirmatory test for HBcAb. Positivity in this assay causes the deferral of about 1% of blood donors.

VARIABILITY OF TEST RESULTS

Not infrequently, results of tests on living donors repeated in a clinical laboratory do not agree with those obtained during donor screening. Quite often, clinical laboratories utilize assay kits from manufacturers different from those used at tissue banks and blood centers. Most discrepant test results observed with assay kits of different manufacturers occur with false positive samples. Retesting a sample using a test supplied by a different manufacturer is one approach to ensure dentification of false reactive samples. Confirmatory tests, when available, also assist in clarification of the discrepancy. However, donors with discordant results should not be accepted in any event.

On rare occasions, true positive samples in one assay kit produce negative results in another assay kit, and subsequent investigation confirms the fact that there was no technical error in the performance of the assay. This may occur because of different assay sensitivity, or assay configuration or sometimes for unknown reasons. Discrepant results are usually communicated to the manufacturer and are subject to intense review.

Appendix B provides a detailed list of variability among assay kits.

SAMPLE IDENTITY

Sample identity errors are extremely rare. However, the potential for sample mix-up is a concern sometimes expressed by donors who are notified of positive screen test results. Major clinical laboratories have sophisticated specimen handling systems that ensure sample identity. At large blood centers, blood donor samples are barcoded at the time of collection, tests are performed on the primary barcoded tube, barcodes are read, samples are loaded automatically on assay trays, and results are read on automated instruments then are transmitted electronically to the computer system. Thus, there is no manual transcription of labels, sample numbers or test results. Confirmatory tests are more complex and cannot be automated, but extensive manual checks and test repetition ensure sample identity.

HEPATITIS B SURFACE ANTIGEN (HBsAg)

Screening method

Enzyme immunosorbent assay (EIA or ELISA). The assay is extremely sensitive and detects the presence of viral antigen in a donor specimen.

Use in donor screening

HBsAg identifies carriers of hepatitis B virus who may transmit the agent to recipients. The prevalence of HBsAg is high among intravenous drug users, homosexual males and in populations from areas in which hepatitis B is endemic, such as Asia, Africa, the Caribbean and Latin America.

Testing algorithm

All samples reactive on EIA are to be retested in duplicate. If two of the three results are reactive, the sample is considered repeatedly reactive for HBsAg. Donors who test repeatedly reactive on HBsAg screening are considered unsuitable, regardless of the outcome of confirmatory testing.

Confirmatory assay

Samples repeatedly reactive on HBsAg screening are subjected to confirmation using a "neutralization" assay. In this assay, antibodies specific to HBsAg are added to samples of donor serum or plasma. The screening test result is considered confirmed and the sample considered positive for HBsAg if specific inhibition of the initial reaction is observed. Samples that are not confirmed are considered negative for HBsAg, and the initial result is classified as false positive. Only 20 to 40% of samples reactive on EIA screening for HBsAg are confirmed as positive in the neutralization assay.

HBV vaccine

Both recombinant and plasma-derived hepatitis B virus (HBV) vaccines induce formation of antibodies to the surface antigen of HBV (HBsAb). Thus, vaccinated individuals who had tested negative for HBsAg remain negative after vaccination and may qualify as donors. HBV vaccines do not transmit HBV.

Immunoglobulins

Treatment of individuals positive for HBsAg with immunoglobulin or with hepatitis B immunoglobulin (HBIg) may cause transient disappearance of HBsAg from the circulation or suppress the presence of HBsAg in recently infected individuals.

MEDICAL EVALUATION OF LIVING DONORS WITH REPEATEDLY REACTIVE RESULTS FOR HBsAg

EIA screen repeatedly reactive, confirmation positive, HBcAb positive or negative

These results may indicate active HBV infection or carrier status. A number of individuals infected with HBV become carriers, i.e., continue to have detectable viral particles present in the circulation despite resolution of the infectious process. The presence of HBcAb in some HBsAg-positive individuals further confirms this fact. In the absence of clinical history or symptoms, there is no immediate significance associated with confirmed HBsAg reactivity. However, approximately 10% of individuals infected with HBV may develop chronic liver disease and require medical evaluation and, when appropriate, therapy.

It should be noted that other tests for HBV may remain negative for several weeks after infection. Thus, in the absence of clinical symptoms, a waiting period of 8 to 12 weeks is recommended before repeating these tests in order to allow sufficient time for evidence of seroconversion to develop.

HBsAg has important implications for contacts of the positive individual. Modes of transmission of HBV include blood (needle sticks, exposure through broken skin), sexual and vertical (mother to child) transmission. Evaluation and vaccination of negative contacts are recommended. Worldwide programs of HBV vaccination and protection of newborns with hepatitis B immunoglobulin are expected to decrease substantially the prevalence of HBV infection.

EIA screen repeatedly reactive, confirmation negative, HBcAb negative, ALT normal

These results reflect non-specificity of assay systems. If HBcAb is negative, alanine aminotransferase (ALT) activity is normal, and there are no clinical symptoms, no retesting or follow up of living donors is recommended.

EIA screen repeatedly reactive, confirmation negative, with positive HBcAb or elevated ALT

If ALT is elevated or HBcAb is positive, medical evaluation of living donors is indicated.

Significance of discordant test results

Discrepancy among test results obtained with different HBsAg assay kits is not infrequent and is related to different characteristics of test kits. Usually these inconsistencies occur with samples which are negative on HBsAg confirmation. In the absence of other hepatitis markers or clinical symptoms, HBsAg EIA screen results negative on confirmation have no significance for the health of the donor. Inconsistent reactivity of confirmed positive samples is observed on rare occasions, because of variations in the concentration of circulating HBsAg or differences in sensitivity of test kits.

HEPATITIS B CORE ANTIBODY (HBcAb)

Screening method

Enzyme immunosorbent assay (EIA or ELISA). Different commercial kits are not standardized for antibody levels.

Use in donor screening

The prevalence of core antibodies to HBV is high among intravenous drug users, homosexual males and populations from areas in which hepatitis B is endemic, such as Asia, Africa, the Caribbean and Latin America.

Testing algorithm

All samples reactive by EIA are to be retested in duplicate. If two of the three results are reactive, the sample is considered repeatedly reactive for HBcAb. Donors who test repeatedly reactive on HBcAb screening may be considered unsuitable. Tissue from a donor testing positive for HBcAb may be made available for transplantation, provided the tissue container is appropriately so labelled and the approval of the medical director is documented. A thorough evaluation must be performed concluding that the donor is not infectious for hepatitis B. Such evaluation should include testing for the presence of anti-HBs.

Confirmatory assay

Not available.

Interpretation of HBcAb reactivity

IgG antibodies to hepatitis B core develop in the course of infection by the virus and remain detectable for many years. IgM antibodies may be detected for a short period of time after infection and denote recent infection. Donor screening tests detect both IgM and IgG antibodies. Levels of antibody do not necessarily correlate with clinical course of disease. About 70% of individuals positive for HBcAb also have antibodies to the surface antigen of HBV, which protect the individual against future re-infection with HBV. However, studies have shown that even individuals with HBsAb occasionally may carry viral DNA and may thus be infectious.

Immunoglobulins

Individuals who recently received preventive treatment with immunoglobulins or with hepatitis B immunoglobulin (HBIg) may become transiently positive for HBcAb because of passive transfer of antibodies. The test becomes negative after clearance of the passive antibodies, a process which may take several months. Immunoglobulins do not transmit HBV or other viral infections.

HBV vaccine

Both recombinant and plasma-derived HBV vaccines induce formation of HBsAb only. Thus, individuals who tested negative for HBcAb remain negative after vaccination and are acceptable as donors.

MEDICAL EVALUATION OF LIVING DONORS WITH POSITIVE HBcAb RESULTS

Isolated HBcAb positivity, no other infectious disease markers

In the absence of clinical history or symptoms, no significance is associated with HBcAb reactivity. Repetition of the screening test or performance of other tests for hepatitis is not recommended.

HBcAb positivity with positive HBsAg or HCV test, or elevated ALT

May be associated with acute or chronic liver disease due to viral infection and requires medical evaluation.

Tests for HBV and HCV may remain negative for several weeks after infection. Thus, in the absence of clinical symptoms, a waiting period of 8 to 12 weeks after exposure is recommended before ordering these tests, in order to allow for seroconversion.

Significance of discordant test results

Discordance among test results obtained with different HBcAb assay kits is not infrequent because of the different characteristics of the tests. Usually, these inconsistencies occur with false positive samples and samples with values close to the assay cut-off. In the absence of other hepatitis markers or clinical symptoms, these results have no significance for the health of the donor.

ANTIBODIES TO HEPATITIS C VIRUS (HCV)

Testing method

Enzyme immunosorbent assay, EIA or ELISA. The EIA detects antibodies to recombinant antigens of HCV. The first generation assay was licensed in May, 1990. In March, 1992 a second generation multiantigen EIA became available for donor screening. Sensitivity of the first generation assay was estimated at about 80%. Sensitivity of the second generation assay is estimated at above 90%.

Use in donor screening

HCV is thought to be responsible for the majority of the cases of non-A, non-B hepatitis. Discovery of the virus and subsequent introduction of HCV screening have substantially reduced transmission of hepatitis C (non-A, non- B) by transplantation.

The prevalence of HCV in the U.S. population varies between 1 and 3%. The prevalence of EIA repeatedly reactive individuals among blood donors is 0.6%. About 60% of these individuals are positive on confirmatory tests. The mode of transmission of HCV has not been identified in more than half the cases. HCV prevalence is high among intravenous drug users. Vertical (mother- to-child) and sexual transmission have been reported.

Testing algorithm

All samples initially reactive on EIA are to be retested in duplicate. If two of three results are reactive, the sample is considered repeatedly reactive for HCV. Donors who test repeatedly reactive on HCV screening are considered unsuitable regardless of the outcome of confirmatory testing.

Confirmatory assay

In June, 1993, the FDA licensed a confirmatory assay for HCV antibodies, the RIBA HCV 2.0 strip immunoassay (SIA). The format of the assay is a strip immunoblot containing four antigens in bands; three proteins from non-structural regions of the HCV genome and one protein from the viral nucleocapsid or core. Studies of blood donors indicate that 40 to 60% of those testing repeatedly reactive on screening tests are confirmed as positive, with 15% testing indeterminate.

HBV vaccine

There is no antigenic relationship between HBV and HCV. Thus, HBV vaccine does not affect HCV testing results.

Influenza vaccine

Several of the individuals who received an influenza vaccine in 1991 became simultaneously reactive in certain donor screening tests, particularly those for HIV-1, HTLV-I and HCV (first generation test kits). All these individuals had negative or inconclusive results in confirmatory tests. The finding was traced to IgM antibodies induced by the immunization. These antibodies produced non-specific results by adhering to the plastic support used in the assay. The non-specific reaction lasted from eight to 12 weeks and disappeared. The phenomenon was observed with tests from different manufacturers and with individuals receiving other vaccines. Preliminary studies indicate that these samples are not reactive in the newer assay kits for antibodies to HIV-1/2 and to HCV (second generation). Nevertheless these donors should be rejected.

Immunoglobulins

There is no documented evidence of protection from immunoglobulins in individuals exposed to HCV. Immunoglobulin preparations do not transmit HCV or other viruses. In 1991, in response to FDA recommendations, manufacturers of plasma derivatives introduced HCV screening of plasma donated for further manufacture. Thus, some batches of immunoglobulins which predate the requirement for screening may contain antibodies to HCV. Therefore, donors who received immunoglobulins may become transiently positive on screening tests for HCV because of passively acquired antibodies.

MEDICAL EVALUATION OF LIVING DONORS WITH REPEATEDLY REACTIVE RESULTS FOR HCV

About 50% of individuals infected with HCV develop chronic liver disease, and some may progress to cirrhosis. This is the major focus of follow up of individuals with positive HCV screening tests. The development of chronic liver disease may be controlled by therapy with interferon.

HCV EIA screen repeatedly reactive, ALT normal

Approximately 40% of cases with these results represent true infection with HCV. HCV screening tests and the ALT test should be repeated in 8 to 12 weeks. If possible, the sample should be subjected to supplemental assays for HCV.

HCV EIA screen repeatedly reactive, ALT elevated

These results may indicate active HCV infection. The patient should be medically evaluated and, if appropriate, treated.

Implications of HCV infection for contacts of positive individuals are unclear at the present time. Modes of transmission of HCV include blood (needle sticks and exposure through broken skin), sexual and vertical (mother-to-child) transmission. Evaluation of contacts may be recommended.

It should be noted that tests for HCV may remain negative for several weeks after infection. Thus, in the absence of clinical symptoms, a waiting period of 8 to 12 weeks is recommended before repeating these tests, in order to allow sufficient time for evidence of seroconversion to develop.

Significance of discordant test results

Discordance among test results obtained with different HCV assay kits is not infrequent and is related to different characteristics of test kits. Usually these inconsistencies occur with false positive samples. In general, in the absence of other hepatitis markers or clinical symptoms, HCV EIA screen results not repeated from one test kit to another have no significance for the health of the donor.

ANTIBODIES TO HUMAN IMMUNO-DEFICIENCY VIRUS TYPES 1 AND 2 (HIV-1/ HIV-2)

Screening method

Enzyme immunosorbent assay, EIA or ELISA. The assay is extremely sensitive and detects the presence of antibodies to HIV-1 and HIV-2 in donor specimens. It should be noted that many public health laboratories and clinical laboratories licensed by New York State do not use donor screening tests; instead, they use assays for HIV-1 alone, because of the limited number of cases of HIV-2 identified in the U.S to date.

Use in donor screening

Screening for antibodies to HIV-1 became available in 1985. The sensitivity and specificity of available screening tests has improved substantially over the years. A combined screening test for HIV-1 and HIV-2 was introduced in February, 1992. This test is more sensitive for HIV-1 than the HIV-1 test.

Testing algorithm

All samples reactive on EIA are to be retested in duplicate. If two of the three results are reactive, the sample is considered repeatedly reactive for HIV- 1/HIV-2 and is subjected to additional more specific tests for confirmation. Donors who test repeatedly reactive on HIV-1/HIV-2 screening are considered unsuitable regardless of the outcome of confirmatory testing.

Confirmatory/supplemental assays

Samples repeatedly reactive on HIV- 1/HIV-2 screening are subjected to confirmation using an FDA-licensed Western blot or immunofluorescence assay for HIV-1, an FDA- licensed specific EIA assay for HIV-2, and, if needed, an investigational Western blot assay for HIV-2.

Influenza vaccine

Several of the individuals who received an influenza vaccine in 1991 became simultaneously reactive in certain donor screening tests, particularly HIV-1, HTLV- I and HCV 1.0 assays. All these individuals had negative or inconclusive results in confirmation assays. The finding was traced to IgM antibodies induced by the immunization. These antibodies produced non-specific results by adhering to the plastic support used in the assay. The non-specific reaction lasted from 8 to 12 weeks and disappeared. This was observed with tests from different manufacturers and with individuals receiving other vaccines. Preliminary studies indicate that samples from these immunized individuals are not reactive in the newer assay kits for antibodies to HIV-1/HIV-2 and HCV (second generation).

Immunoglobulins

Commercial preparations of human immunoglobulins do not transmit viral infections, including HIV. Immunoglobulins do not interfere with HIV screening test results because all donors of plasma used in the manufacture of immunoglobulins are screened for HIV.

INTERPRETATION AND MANAGEMENT OF HIV-1/ HIV-2 TEST RESULTS AND MEDICAL EVALUATION OF LIVING DONORS WITH REPEATEDLY REACTIVE RESULTS FOR HIV-1/HIV-2

EIA screen negative

These donors are considered negative for antibodies to HIV. Confirmatory assays for HIV are not performed on samples negative on HIV EIA testing. According to the manufacturers and to the FDA, confirmatory tests have been designed to be performed only when EIA screening is reactive. Unnecessary performance of these tests often generates indeterminate test results that distress donors and are not useful in donor management. Between 10 and 15% of normal donors who are negative on EIA screen for HIV present bands on the Western blot assay for HIV-1. This phenomenon is due to cross reacting antibodies unrelated to HIV-1. An extremely small number of individuals with negative EIA screening test results for HIV may have been infected but are still in the "window" period of seroconversion, that is, the time period when antibodies cannot be detected by current screening tests.

EIA screen repeatedly reactive, confirmatory test negative

Management of donors with this sequence of test results depends on assessment of risk for HIV exposure. Among individuals at risk are homosexual males, intravenous drug users and their sexual partners, heterosexual men and women who have had unprotected sex with partners at HIV risk or with multiple partners, persons with hemophilia, and sexual partners of individuals in the above categories.

a. Individuals without risk behavior
All studies performed on blood donors to date have clearly shown that, in the absence of clinical history of HIV-risk behavior, the above results are non-specific and have no significance for the health of the donor.

b. Individuals with risk behavior
Repeatedly reactive EIA screening results may reflect early stages of seroconversion because the EIA screening test is more sensitive than supplemental/confirmatory tests. In living donors, screening tests should be repeated in 4 to 8 weeks.

EIA screen repeatedly reactive, confirmatory tests indeterminate or inconclusive

Management of these donors also depends on an assessment of risk of exposure to HIV, as described above.

a. Individuals without risk behavior

Approximately 30% of donors with repeatedly reactive results on EIA screening tests have indeterminate or inconclusive results on supplemental/confirmatory tests. All studies performed among blood donors to date have clearly shown that, in the absence of clinical history of risk behavior, indeterminate or inconclusive confirmatory test results are due to cross-reactive antibodies unrelated to HIV and have no significance for the health of the donor.

b. Individuals with risk behavior

Individuals with risk behavior and inconclusive or indeterminate supplemental/confirmatory test results may be in the process of seroconversion. In living donors, screening tests should be repeated in 4 to 8 weeks.

EIA screen repeatedly reactive, confirmatory tests positive

These individuals are considered positive for HIV infection. Living donors should be referred to their own physician or to specialized medical care. HIV-1/HIV-2 positivity has important implications for contacts of the positive individual. HIV may be transmitted by exposure to blood (needle sticks), by sexual contact, and from a pregnant mother to her child. Evaluation of contacts is important and should be conducted. The overwhelming majority of HIV-positive individuals are infected with HIV-1. A very small number of cases of HIV-2 infection have been identified in the U.S. There are no major clinical differences between infection with HIV-1 and HIV-2.

Significance of HIV discordant test results

Discordance among EIA screening test results obtained with different HIV-1/HIV-2 assay kits are not infrequent and are related to the different characteristics of various commercial test kits. In the vast majority of cases, positive samples produce consistent results, regardless of test manufacturer or kit lot. Inconsistencies sometimes occur among samples negative or indeterminate on HIV-1/HIV-2 confirmation. In the absence of HIV risk behavior, these results have no significance for the health of a living donor. It should be noted that donor tests used by blood centers are substantially different from those in use by commercial laboratories and local departments of health and may produce different results particularly, with negative, indeterminate or inconclusive samples.

Discrepancies among Western blot results from tests of different manufacturers may also be observed, especially in samples with indeterminate results. Band patterns may vary among different lots of assay kits from the same manufacturer and among assay kits from different manufacturers.

ANTIBODIES TO HUMAN T-CELL LYMPHOTROPIC VIRUS TYPES I AND II (HTLV-I AND HTLV-II)

Screening method

Enzyme immunosorbent assay, EIA or ELISA. The assay is very sensitive for detection of antibodies to HTLV-I. Because of relatively high cross-reactivity between HTLV-I and HTLV-II (60 to 80%), the assay also detects antibodies to HTLV-II in donor specimens. It should be noted that assay kits are calibrated against panels of well-documented specimens from HTLV-I infected individuals and are not calibrated for HTLV-II detection. More sensitive versions of the test are being actively developed.

Use in donor screening

Screening for antibodies to HTLV-I/II became available in late 1988 and has been applied to all blood donors in the U.S. since that time. These viruses may be transmitted by transfusion of cellular blood products. Approximately 25% of recipients of seropositive blood units seroconvert.

Approximately 55% of HTLV-positive blood donors in the New York metropolitan area are typed as HTLV-I and 45% as HTLV-II. Most (95%) individuals infected with HTLV-I or HTLV-II never develop clinical disease. About 5% of those infected with HTLV-I are at risk of developing disease over their lifetime. HTLV-II positivity association with disease has been rare. A few cases of HTLV-II positivity associated with neurological disease have been reported.

HTLV-I is considered the etiologic agent of adult T-cell leukemia (ATL), a particularly aggressive form of leukemia characterized by hypercalcemia, high levels of circulating IL-2 and "flower cells" in peripheral blood. HTLV-I is also considered the etiologic agent of tropical spastic paraparesis (TSP), also called HTLV-I-associated myelopathy (HAM). TSP is a progressive neurological disease affecting limbs and sphincters.

HTLV-I is an endemic virus with high prevalence in many areas of the world, including southern islands of Japan, the Caribbean, Central and South America, Central Africa, Iran, India, southeastern U.S. and certain areas of New York City. The prevalence in certain areas of Kyushu, Japan, reaches 7 to 10%. Studies in Jamaica and Trinidad indicate a prevalence of 5%. HTLV-I is transmitted by breast milk during breast-feeding, by sexual contact and by blood exposure.

HTLV-II is prevalent among several Native American populations in the southwestern U.S., in Florida, and in Central and South America. HTLV-II is also endemic among intravenous drug users in the U.S.

Testing algorithm

All samples reactive on EIA are to be retested in duplicate. If two of the three results are reactive, the sample is considered repeatedly reactive for HTLV-I/II and is subjected to supplemental testing. Except for eye donors and donors of tissue subjected to viral inactivation, donors who test reactive on HTLV-I/II screening are generally considered unsuitable, regardless of the outcome of confirmatory testing.

Confirmatory assays

Samples repeatedly reactive on HTLV-I/II screen are subjected to supplemental tests using investigational assays. There are no FDA-licensed confirmatory testing kits at the present time. Samples are subjected to an investigational Western blot assay containing the recombinant protein p21e. The purpose of these assays is to ascertain the presence of antibodies to HTLV and to differentiate between the two viruses where possible.

Influenza vaccine

Several of the individuals who received an influenza vaccine in 1991 became simultaneously reactive in certain donor screening tests, particularly HIV-1, HTLV-I and HCV 1.0 assays. All these individuals had negative or inconclusive assay results in confirmatory tests. This was traced to IgM antibodies induced by the immunization. These antibodies produced non-specific results by adhering to the plastic support used in the assay. The non-specific reaction lasted from 8-12 weeks and disappeared. The phenomenon was observed with tests from different manufacturers and in individuals receiving other vaccines. Preliminary studies indicate that these samples are not reactive in the newer assay kits for antibodies to HIV-1/HIV-2 and to HCV (second generation).

HTLV-I/II assays currently in use are the same as those in use in 1991. Thus, some non-specific results related to influenza vaccine may occur.

Immunoglobulins

Plasma products and commercial preparations of human immunoglobulins have not been reported to transmit HTLV-I/II. Donors of plasma used in the manufacture of immunoglobulins are not screened for HTLV-I/II. Consequently, there is a theoretical possibility that patients who receive immunoglobulins may become reactive in the screening assay because of passive transfer of antibodies. This finding has not yet been documented, probably because of the very low prevalence of antibodies to HTLV-I/II among U.S. blood donors.

INTERPRETATION AND MANAGEMENT OF HTLV-I/II TEST RESULTS AND MEDICAL EVALUATION OF LIVING DONORS WITH REPEATEDLY REACTIVE RESULTS FOR HTLV-I/II

Donors frequently confuse HTLV results with HIV results and need reassurance that, while HTLV-I and HTLV-II are retroviruses, they are not the etiologic agents for HIV disease. Infection with HTLV-I or HTLV-II bears no relationship or resemblance to infection with HIV or AIDS.

EIA screen negative

These donors are considered negative for antibodies to HTLV-I and probably HTLV-II. However, several cases of HTLV-II infection with negative EIA screening for HTLV-I have been reported.

EIA screen repeatedly reactive, confirmatory tests negative

These individuals are considered negative for HTLV-I and HTLV-II. EIA screening results are false positive because of a non-specific reaction. No further action is recommended. Retesting of living donors is not recommended because seroconversion is extremely rare.

EIA screen repeatedly reactive, confirmatory tests indeterminate or inconclusive

Management of these results also depends on risk exposure assessment for HTLV-I or HTLV-II.

a. Individuals who are not at risk

These are individuals who were not born in endemic areas, are not intravenous drug users, and have had no sexual partners from endemic areas or who were intravenous drug users. Approximately 20% of donors with repeatedly reactive results on EIA screening have indeterminate or inconclusive results on supplemental/confirmatory tests. All studies performed to date on blood donors have clearly shown that, in the absence of clinical history, indeterminate or inconclusive confirmatory test results are due to cross-reactive antibodies unrelated to HTLV-I or HTLV-II, and have no significance for the health of the donor.

The screening test may be repeated in 4 to 8 weeks, and should be repeated after six months. Individuals who do not become positive on confirmatory tests (i.e., remain negative or have inconclusive results) after six months are considered clinically negative for HTLV.

b. Individuals at risk

Individuals at risk include those born in endemic areas or who use intravenous drugs, and their sexual partners. These individuals may be infected with HTLV-I or HTLV-II. Individuals with at-risk behavior and inconclusive or indeterminate confirmatory test results may be in the process of seroconversion. Screening tests should be repeated in 4 to 8 weeks and again six months after donation. If seroconversion does not occur within six months, an asymptomatic individual with reactive EIA screening and indeterminate or inconclusive confirmatory tests may be considered clinically negative for HTLV. Intravenous drug users require regular medical assessment.

EIA screen repeatedly reactive, confirmatory test positive

These individuals are considered positive for HTLV-I or HTLV-II infection, and should be referred to their own physicians or to specialized medical care. HTLV-I/II infection has implications for contacts of positive individuals. Modes of transmission of HTLV include breast feeding, blood (needle sticks and exposure through broken skin) and sexual contact. Evaluation of contacts is recommended. About 40% of positive individuals are infected with HTLV-I. They should be monitored at regular intervals for potential development of HTLV-I-associated disease. Since HTLV-II-associated disease appears to be rare, in the absence of clinical symptoms, monitoring may not be necessary.

Significance of discordant HTLV-I/II test results

Discordance among EIA screening test results obtained with different HTLV-I assay kits is not infrequent and is related to different characteristics of different test kits. Usually these inconsistencies occur among samples which are negative on HTLV-I/II confirmation. However, some of the discrepant samples have been shown to contain antibodies to HTLV-II which were detected by one assay kit and not by another.

Discrepancies in confirmatory test results are frequent, because of the lack of FDA-licensed tests and because of the wide variety of confirmatory algorithms utilized by different laboratories. Band patterns vary among lots of assay kits and among assay kits from different manufacturers.

SYPHILIS SEROLOGY

Use in donor screening

Treponema pallidum is rarely detected in the circulation and does not survive long under tissue storage conditions. Some believe that positive syphilis serology correlates with risk behavior, and therefore, the syphilis test is a surrogate for other sexually transmitted diseases. Commonly used manual screening tests include reagin-based tests, such as RPR (rapid plasma reagin) and VDRL (Venereal Disease Research Laboratory). Automated screening tests include the Olympus PK-TP (a microhemagglutination assay that does not use a non-specific control consisting of unsensitized cells).

Testing Algorithm

Samples reactive on a syphilis screen are to be retested in duplicate. If two of three results are reactive, the sample is considered repeatedly reactive for syphilis and is referred for confirmatory testing.

Confirmatory assay

Samples repeatedly reactive on syphilis screening are subjected to confirmatory testing by FTA-ABS (fluorescent treponemal antibody absorption), an indirect immunofluorescence assay, or MHA-TP (microhemagglutination- Treponema pallidum).

Interpretation

If confirmatory testing is positive, the donor should be rejected. If confirmatory testing is negative, the tissue may be used. In the absence of confirmatory testing, donors should be rejected; however, the medical director may consider an exception to this guideline for donors of avascular eye tissue.

MANAGEMENT OF LIVING DONORS WITH REACTIVE SCREENING RESULTS

Specific tests for syphilis, such as the PK-TP, MHA-TP and FTA-ABS, detect antibodies to treponemal antigens and indicate past exposure to Treponema pallidum. These tests remain positive for long periods of time regardless of specific treatment. About 1% of positive results on FTA-ABS, MHA-TP or PK-TP are false positive in a low-prevalence population. Reagin-based tests are reactive only during acute stages of syphilis infection and often become negative after specific antibiotic treatment.

Screening with PK-TP

Since, unlike MHA-TP, the PK-TP test does not utilize unsensitized cells for controls and it is not reactive during acute stages of syphilis infection, supplemental testing should include both FTA-ABS (or MHA-TP) and a reagin test (RPR, VDRL)

PK-TP screen repeatedly reactive, FTA-ABS (or MHA-TP) positive, reagin test (RPR, VDRL) negative

A positive FTA-ABS result with a negative RPR result indicates past infection and does not require medical management. Local health departments generally do not follow up on patients with negative reagin tests (RPR).

PK-TP screen repeatedly reactive, FTA-ABS (or MHA-TP) positive, reagin test (RPR, VDRL) positive

These results indicate that the individual has active infection. Evaluation and treatment are indicated. Positive syphilis serology has important implications for sexual contacts of the individual. Public health laws in many states require reporting of active syphilis infection to local health departments, which may, when appropriate, perform contact tracing. Evaluation and treatment of infected contacts are recommended.

PK-TP screen repeatedly reactive, FTA-ABS (or MHA-TP) negative, reagin test (RPR, VDRL) negative

Often these results reflect non-specificity of assay systems. For instance, some individuals carry heterophile antibodies which react with the chicken red blood cells used as reagents in the assay kit. Since PK-TP does not make use of non-specific controls (unlike MHA-TP), it is impossible to differentiate true positives from non-specific reactions. In the absence of clinical history, an individual with negative FTA-ABS results for syphilis is considered negative for syphilis infection. No follow up or test repetition is recommended.

PK-TP screen repeatedly reactive, FTA-ABS (or MHA-TP) negative, reagin test (RPR, VDRL) positive

Reagin tests such as the RPR produce non-specific results under many circumstances, including recent vaccinations, viral pneumonia, intravenous drug use, lupus erythematosus, mononucleosis, malaria and leprosy. Such results require medical investigation.

Screening with RPR (VDRL)

RPR (VDRL) screen repeatedly reactive, FTA-ABS (or MHA-TP) confirmation positive

These results indicate that the individual has active infection. For living donors, evaluation and treatment are indicated. Positive syphilis serology has important implications for sexual contacts of the individual. Public health laws in many states require reporting of active syphilis infection to local health departments, which may, when appropriate, perform contact tracing. Evaluation and treatment of infected contacts are recommended.

RPR (VDRL) screen repeatedly reactive, FTA-ABS (or MHA-TP) negative

Reagin tests such as RPR produce non- specific results under many circumstances, including recent vaccinations, viral pneumonia, intravenous drug use, lupus erythematosus, mononucleosis, malaria and leprosy. Such results require medical investigation.

Significance of discordant test results

Discordance among test results obtained with different treponemal assay kits is related to assay construction. These inconsistencies usually occur with samples negative on FTA-ABS confirmation. Inconsistent reactivity of confirmed positive samples is observed only on rare occasions and may be due to differences in sensitivity of assay kits.