The following is a list of factors that may interfere with the performance and results of donor screening assays. These factors explain many of the discrepancies found between test results obtained during donor screening and test results obtained in clinical laboratories.

PATIENT-RELATED

Seroconversion
In some instances, changes in test results reflect infection and conversion of a patient from negative to reactive for the assay. Most of viral infections for which donors are screened may have rather benign acute symptoms and remain unnoticed (HBV, HCV, HIV-1, HIV-2, HTLV-I and HTLV-II)

SPECIMEN-RELATED

High levels of immunoglobulins
Such as in rheumatoid factor and autoimmune disease.

Specimen quality and storage conditions
Hemolyzed or lipemic specimens may produce false results; samples stored under inappropriate conditions (old samples, and samples exposed to high heat, or frozen and thawed several times) may also produce false results. Post-mortem specimens may be associated with an increase in false positive results.

"Sticky sera"
Certain specimens produce false reactive results in many different assays; some of these specimens are from patients from areas highly endemic for parasitic diseases, such as Africa and Latin America).

Recipients of influenza vaccine
Several individuals who received influenza vaccine in 1991 became simultaneously reactive in certain donor screening tests, particularly HCV 1.0, HIV-1 and HTLV-I assays. These results were traced to IgM antibodies from immunization that adhered to the plastic support used in the assay. All these patients had negative or inconclusive results in supplemental/confirmatory tests. The non-specific reaction lasted from 8 to 10 weeks. The same phenomenon was observed in individuals receiving other vaccines.

Animal virus to which humans may be exposed
Some indeterminate results in Western blot for HIV have been attributed to cross-reactivity of antibodies directed against feline and bovine leukemia virus.

ASSAY KIT-RELATED

Unexpected cross-reactions with test components
Animal reagents used in buffers and conjugates (gelatin, bovine and human albumin, rabbit, goat or horse sera)

Cells for growing the virus used as antigen
A well known example is reactivity of sera from multiparous women found in early HIV tests. The antigen had been contaminated with DR4 from the H9 cells used to grow HIV.

E. coli or yeast used to produce recombinant antigens
Among the assays that use recombinant antigens are HBcAb, anti-HIV-1/HIV-2, and anti-HCV.

Fusion proteins used for cloning recombinant proteins
Some donors produce antibodies to SOD (superoxide dismutase), which is the fusion protein used for cloning HCV recombinant antigen.

Continuous variables, discrete decisions
Assay kits produce a continuous range of color that is transformed into a numerical value. A cut- off is then mathematically calculated. Values equal to or above the cut-off are considered positive, and below the cut-off, non-reactive. Many sources of variation cause changes in cut-off and sample values. It is extremely rare to obtain the same value twice on the same sample. The coefficient of variation is about 10%.

Establishment of assay cut-off
The assay cut-off is established by the kit manufacturer and approved by the FDA based on extensive clinical trials and statistical analysis of the data. All assay cut-offs have been placed in a "safe" region of the assay curve, in order to preserve sensitivity. Unfortunately, the establishment of "safe" cut-offs compromises assay specificity. Many non-specific results observed in donor samples are caused by this "safe" cut-off.

Samples with values close to the assay cut-off
Because of the coefficient of variation, samples with values close to the cut-off may produce positive or negative results when repeated. However, because the cut-off has been placed in a "safe" region of the assay curve, the vast majority of these samples are truly negative.

Testing protocols
Assay protocols approved by the FDA have been designed with the knowledge that certain samples may give variable test results. Consequently, such protocols require duplicate repeat testing of all initially reactive samples. If the two repeat tests are negative, the sample is considered negative. If one or more of the repeat tests are positive, the sample is considered repeatedly reactive.

Variations in kit manufacture
While each kit lot of all FDA-licensed assays approved for blood donor screening undergoes strict evaluation for sensitivity (false negative rate, or ability to identify true positive samples), these kits may vary substantially in specificity (false positive rate, or ability to identify true negative samples). Thus, on rare occasions, the same sample may be tested in different kit lots and produce different results which are within the tolerance acceptable for manufacture. Most of these discrepancies have no clinical significance. However, in certain instances the variation may be relevant; for an example, consult the section on HTLV-I.

Evolution of test kits
In recent years, several assays have been replaced by newer versions which are more sensitive or more specific. For example, in March,1992 a second-generation kit for HCV became available. HCV 2.0 is much more sensitive than the first-generation kit. Many samples which were negative with the first-generation kit tested positive in the new assay. An improved HCV 3.0 is even more sensitive, although it is not sufficiently improved to be a third-generation test. Similarly, the new combined test for HIV-1/HIV-2 has the ability to detect IgM and is more sensitive than previous assays. Unfortunately, while the sensitivity has increased and benefitted the safety of recipients, new sources of non-specificity were created, and new sources of false positive results introduced.

MANUFACTURER

Assay kits from different manufacturers may produce discordant test results for additional reasons:

Assay configuration
This is a very important source of discordant results. Not infrequently, living donors are notified about a test result and consult their physician, who has the test repeated in a reliable clinical laboratory and obtains discordant results. In most instances, clinical laboratories use test kits more adapted to clinical testing and substantially different from assays used in donor screening. Test kits for antibodies to the core of hepatitis B are one of the most important examples. Blood screening laboratories frequently use direct EIA assays (Ortho, Genetic Systems) and bead EIA (Abbott). Many clinical laboratories use radioimmunoassay (Abbott RIA).

Support materials
Test kits from different manufacturers use different plastics, reagents and instruments, and have varying specificity and sensitivity.