Pathology Associates Of Lexington, P.A.
Pathology Associates Of Lexington, P.A.
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        Many Reasons for Serological False Positives and False Negatives
      

AXIOM as to Test Reliability as well as Weakness in ALL tests: The layperson public and even a high percentage of practicing physicians have a dangerous tendencity to demand and order disease diagnosis tests when there is a very low probability of the patient actually having the disease. As a function of the mathematics of statistics, a positive-result lab test ordered for "flu" NOT during the flu season or a flu outbreak has a high likelihood of being a false positive test result! A way to see this is: "In South Carolina, if you hear hoof-beats on the street, don't bet you money that a Zebra is the hoofed animal making the noise". Check the "Limitations" and "See Also" sections HERE4 for beginning of the scientific explanation. In ADDITION, all test types have methodology-specific factors that produce false positives and false negatives (including molecular tests such as PCR!). Finally, the beginning paragraph on the PSA page gives an illustration of another problem as to statistics, HERE.

ADDITIONAL Serology-Specific Confounders:
Heterophile, Background "Noise", and the Low/Low Problem False Positives:
Interpretative warning: There are natural, heterophile, and pathological antibodies; and there are "interferences". And most antibodies lag a certain amount of morphological disease activity prior to detectability (morphological change is apparent before serological change, especially in autoimmune disorders). 
Serological tests (direct or indirect antigen-antibody tests) have such a high prevalence of false positivity (background reactive "noise") in undiluted serum that it is standard practice to do the testing on serum diluted to at least 1 part of serum to 10 parts of diluent (expressed 1:10). By general medico-scientific agreement, a test is non-reactive ("negative" [for practical purposes]) if it fails to signal "positive" at the low dilution of 1:10. The serum sample is tested at increasingly higher double dilutions of 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280. Many results testing "positive" at dilutions of less than 1:80 are not specifically diagnostic. Clinical judgment is necessary to evaluate the meaning of these "low-titer-positive" results. For example, as to autoantibodies such as ANA, it is estimated that 25% of non-rheumatic and healthy persons carries positivity for ANA at low titer.
As the body begins to produce antibodies (Ab)  against an offending antigen (Ag) (an organism, or pollen, or mite dust,  or against normal components of the body..."auto-antibodies", all of which are antigens), the IgM class Ab (acute-phase Ab)  rises to detectability early, followed by the IgG class Ab (immune-phase Ab). The patient's very initial Ab production is (1) absorbed out of the blood onto organisms (or other antigens) until those targets are saturated.  Following saturation, serum antibody levels (2) rise into the range of detectability and (3) then into higher diagnostic levels.  Therefore, there is a window of nondetectability (from the time of Ag challenge to the body until the time of test's  initial detection of Ab...or a rise from a stable baseline titer) of days to weeks (briefest with re-infection).
Because the various possible antigens can be chemically complex, various tests (such as for two different organisms) can have cross-reactive "positivities" at dilutions of 1:20 to 1:80 (sometimes much higher as in case of HSV I  & HSV II). So, "positive" tests must be carefully interpreted in view of all other clinical information. One must guard against over-interpretation of test results. And this is especially true in infectious-disease testing  when there are "low/low" positivities of IgM/IgG (such as IgM, 1:20 with IgG of 1:40 in the same sample). 
Non-serological tests: Many non-serological tests are performed by modern immunological-reaction-based  test systems which makes those tests subject to immunological interferences, most of which are circulating in the blood of some patients. These could produce false positives resulting in diagnoses which don't exist in that patient. False-negatives are perhaps more obscure and less understood. In these cases, an antibody saturates the capture antibody and saturates to some extent the conjugate in solution. So there is no real possibility of forming any real complexes at all. No immuno-reagent "sandwiches" are created in complete form, and you get very low detection-signal results...passing for "non-reactive", "undetectable", or "negative".

Other False Positives:

Rheumatoid Factor False Positives: this is an IgM "anti-human-IgG" that interferes in test systems in which reagent antibodies seek patient antigens or biochemical molecules. As RF ties up the reagent antibodies, the test system interprets the result as increased presence of the target antigen/molecule.

Heterophile antibodies: One should also keep in mind the possibility that an unexpected positive is due to the patient having produced circulating serum antibodies [more detail] against the animal (anti-animal...anti-mouse, anti-goat, etc...antibodies: HA) which produced the reagents or tissues for the test system. And, there have been vaccinations & disease therapies that involved injection of your patient with animal-serum-based or generated reagents to which the patient can have developed anti-animal antibodies. In such instances, the patient asymptomatically carries the HA in his/her serum and the HA reacts against the test-system's animal-generated reagent antibodies to tie them up (consume them) as if they were being consumed by the target antigen/molecule in patient's serum.
Of the antibodies that have been found in human serum that have an animal specificity, human anti-mouse antibody (HAMA) is probably the best known; but there are many others, including human anti-rabbit, anti-goat, anti-chimera (for the chimeric antibodies that are being used now in therapy), anti-pig, even anti-human, anti-bovine, rat, and horse.
Animal husbandry and keeping pets have also been implicated as mechanisms to generate anti-animal antibodies in humans. “For example, if you’re looking for people who have got anti-mouse antibodies circulating, probably the best place to go to is your local mouse colony to see if the keeper of the mouse colony will give you some blood.”
Since animal-based reagents are also used extensively in pharmaceuticals, these can be another source of human anti-mouse antibody, human anti-rat antibody, or antibodies against other species.
In our lab, this problem has most often come to light in pregnancy testing where discordant sero-clinical details are so readily notable. But we have recently also investigated a recent thyroid question [CP10-5] with test pattern discordance and celiac question [S10-3259] with sero-biopsy (CD3-amplified VTS) discordance.
Medications, Chinese medicine, herbal drinks, and paraproteins: can interfere.

Bacteria: such as E. coli can cause false positives.

Poorly defined other interferences: ??.

False Negatives:

Serological tests, prozone type reaction: the circulating, sought antibody is present in such high titer that it saturates both the antibody capture component of the test system AND at least partially saturates the conjugate in solution so that no real complexes are formed & the test looks negative or only low reactive. This phenomenon is the basis of the prolactin "hook effect" (a rising level unexpectedly "hooks back" to lower levels) in instances of pituitary adenoma which seems product negative (that is a prolactin producing adenoma with serum level "not elevated" (pituitary lesion 100 mm or larger ought to produce a product at a level greater than 250ng/ml3

Poorly defined other interferences: ??.

References:

  1. Tozzoli R, et. al., "Guidelines for the Lab. Use of Autoantibody Tests in the Diagnosis and Monitoring of Autoimmune Rheumatic Disease", [The Italian Soc. of Lab. Med. Study Group...], AJCP 117(2):316-324, Feb. 2002.
  2. CAP Today October 2003, p. 76-78. Or Cap website , "Facing up to anti-animal antibody interference", Oct. 2003.
  3. NewsPath, July 2010, a periodical letter from the LMC Lab (KRM).
  4. The effect of prevalance of disease on likelihood that a test result is a true positive.
  5. Another source concerning the "false positive" & "false negative" problem HERE.

(posted 2001; latest addition 23 May 2012)

 
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