Thursday, March 3, 2016

Western Blots in perspective


Doctor, can you help me understand my Lyme test, Western Blot?
Many patients spend a lot of time on the internet in efforts to understand the significance of mysterious numbered bands. What does it all mean?

As a starting point: lower your expectations. Lyme Western Blots, like all Lyme tests, lack accuracy and should be taken with a grain of salt.

A Western Blot is a second test, (IDSA/CDC)  a confirmatory test, used to confirm positive results from the initial test an ELISA - according to the IDSA/CDC. The CDC requires the second test because they believe the ELISA is inaccurate, leading to too many false positives, whereas, those in the ILADS camp believe the opposite. On the other side it if felt the ELISA test in nearly completely worthless because of false negative: it misses the majority of cases. Therefore the ELISA is skipped and the Western Blot is ordered directly. In addition, the Western Blot, although more helpful, is still inaccurate in many cases.
The two views are so divergent that mutual understanding and compromise, for the present time, is impossible.
For purposes of this discussion, all of these tests attempt to measure an immune response mounted by a patient who has been exposed to Lyme, or Borrelia burgdorferi. These tests look for antibodies made by an infected person's immune system. These antibodies are tiny proteins, also called immunoglobulins.
These discrepancies are rooted in the early days of the disease. The Lyme bacteria first described in 1982. Soon thereafter scientist went to work developing new diagnostic tests.  Lyme, viewed as a new and emerging disease was thought to be rare and geographically confined. This assumption may have lead to problems with test development from the outset.
The Lyme tests observe reactions between antibodies (from found in patient's blood) and antigens  derived from the Lyme spirochete. This sounds straightforward but it isn't. When a Lyme patient's blood is combined with chopped up pieces of Lyme, an antigen--antibody reactions occur which may be seen as clumping. Many antibodies, ( and there are many thousands), unrelated to Lyme, may still adhere to antigen in the laboratory. This is the nonspecific reactivity or background noise that always occurs with such tests. Scientist are charged with the task of removing the nonspecific clumping, leaving behind the true Lyme-antibody clumping. Not so easy. This call is something of an educated guess. The decision as where to set the bar is made by a committee of experts. To do a good job the experts have to know who has the disease. The experts need control sample taken from truly non-infected persons. If the disease is rampant and difficult to diagnosis and if many control samples are from non-symptomatic persons infected with Lyme the test will perform poorly - or not at all.

The scientist working on the tests found that many supposed negative control groups reacted strongly to the ELISA. This was unexpected and surprising. It was assumed that there was something unique about Lyme antigens leading them to elicit such a high degree of nonspecific, therefore falsely positive, ELISA reactivity. The notion that the high degree of reactivity might be due to a high incidence of infection in the community was not considered. Scratching their collective beards, the test developing scientist decided a second level of testing was needed to confirm the diagnosis. The Western Blot.

The Western Blot is used clinically for only one other infection I am aware of: HIV. This should raise a red flag.

A Western Blot is more sophisticated. Homogenized Lyme bacteria can be placed on a special strips and passed through an electric current. This process separates out the Lyme antigens based by their respective weights. The heaviest proteins (antigens) migrate to the bottom of the strip, the lightest stay on the top. When these strips are incubated with patient serum, lines form on the strip, representing individual antigen-antibody reactions called bands. From the start there was confusion about the correct use of this second tier test.

In the early 1990s it was clear to Lyme researchers that Lyme testing was disaster. Labs used different techniques and got very different results. A conference was set up in Dearborn Michigan in 1994. Researchers, lab directors and the CDC showed up. The goal was to come up with a standard metric so that labs everywhere would be reading from the same sheet of music when discussing Lyme test results. Lyme Western Blots are divided two tests. Two distinct classes of immunoglobulins (antibodies) are produced by our immune system in response to infection: IgM and IgG.  Western Blot criteria for these two classes of antibodies were discussed at the meeting. One group (Dressler) presented an IgM criteria of 2/8 bands for a positive result.  A second group, (Krupp) suggested a criteria of 2/4. Quite different. Each lab had used a different strain. N40 and B31 respectively. The researchers threw out the N40 test because of poor 39 band reactivity. It was agreed to use the 2/4 criteria. One of the 4 bands (37) was discarded at the last moment so the adopted criteria became the well known 2/3.  Strangely, IgG criteria for positivity, the well known 5/10, came from Dressler's research. In other words, one strain was used for IgM criteria and another for IgG criteria. Certain key bands were intentionally overlooked because of impact on the long discarded Lyme vaccine.

The researchers had one agenda, the CDC another. The researchers sought a uniform, consistent standard so they could reasonably communicate with each other. The CDC wanted a surveillance standard: a test that they could use to track the disease in different locations over time.
The conference never pretended to be in the business of finding an accurate diagnostic test for Lyme: rather a standard for research purposes only. The test that resulted from this meeting can be fairly called a CDC surveillance test. This test continues to be used erroneously for purposes of diagnosis. To makes matters worse, many clinicians believe the test can be used to reliably diagnose the disease. 

The Lyme bacteria possess numerous surface structures against which antibodies can be made. Bands show up if a particular antibody directed against a particular antigen is present in the serum in adequate numbers.

Multiple labs use different bands and different numbers of bands. Here I will get a total by adding IgM and IgG results together.

CDC tests looks at 13 total bands
IgeneX test looks at 28 total bands
Stony Brook test looks at 52 different bands.

In theory IgM bands precede IgG bands. IgM bands are associated with new infection. IgG bands are associated with older infection. Not always.  An effort to apply this principal to Lyme bands has led to endless confusion. In the case of Lyme the rule does not apply.  Lyme infection leads to strange antibody responses. IgM and IgG are jumbled up. The presence of one or the other or both usually has no special meaning.

As alluded to, some antibody responses are nonspecific (background responses) and some are very specific. Some are somewhat specific. For example the 41 band represents a spirochete infection but is not highly specific for Lyme. Some highly specific bands include: 23-25, 31, 34. 37, 39, 66, 83-93. Whether or not bands are reported positive or not varies from lab to lab. Some labs use computers and measures pixels. With this configuration a band is reported positive if it has at least 60% the pixels of control strip. Other labs do the eyeball test. An IND from IgeneX means a reaction, less than the positive cut off point was present. The word indeterminate from Stony Brook means something completely different. It means that at least one of the CDC bands is present.

These test results may or may not be helpful. Western blot results are a lot more helpful when clearly positive. Each clinician may interpret results differently because there is no standard, widely accepted criteria. Based on the state of the art and limitations of the test it should stay that way for now.

The diagnosis of Lyme is clinical. The Western Blot is a tool which may or may not be helpful.















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