INTERFERENCES
If a test result does not correlate with the clinical picture, false-positive or false-negative test results can be suspected, but when false results are subtle and/or plausible, the results could be misleading. For exam- ple, a “normal” result may truly be “abnormal,” and a
disease state of a patient could be missed due to inter- ference in an immunoassay by heterophilic antibodies.
Use of Bayesian logic based on the prevalence of a dis- ease may help identify false results in the diagnosis of a disease [41]. Various ways of detecting heterophilic antibody and correcting interferences due to the pres- ence of heterophilic antibody in the specimen are sum- marized inTable 6.2.
Assay development scientists incorporate steps such as sample blanking and robust assay design to mini- mize interferences, including matrix effects arising from protein and other nonspecific constituents in the specimen. Thus, during the development of multi- plexed cytokine assays, researchers screened for and added appropriate blockers in the reagents to reduce heterophilic interference [42]. When suspected, the interfering substance may be removed from the speci- men by specific agents, ultrafiltration, gel filtration chromatography, precipitation, or centrifugation prior to reanalysis. Alternatively, the specimen may be ana- lyzed by a different method for the same analyte, which is known to be free from such interference.
If a discordant result is suspected to be caused by interference from some endogenous antibody, the best practices to confirm such interference include (1) dilu- tion linearity study with the specimen; (2) examination of the patient history (exposure to immunogenic ani- mals or animal products and history of hyperactive immune system); (3) assaying the sample, if possible, using a different immunoassay utilizing different anti- bodies/reagents; and (4) treating the sample to block the interference or remove the interfering antibody, and repeating the assay. This strategy is exemplified in a false-positive TSH result leading to thyroxin over- dose. The incorrect result was traced to RF interference because the sample showed nonlinear dilution.
The interference was removed by treating the sample with a heterophile blocking reagent. In addition, a correct result was also obtained using a different immunoassay [43]. An example of nonlinear dilution for specimens with heterophilic antibody interference is shown in Figure 6.1, which shows the effect of successive dilutions of a HAMA-containing sample (spiked with 32μg/mL of theophylline) versus those
TABLE 6.2 Different Sources of Heterophilic Interference, their Detection, and Reduction
Antibody Detection Reduction
Heterophilic antibody (Weak Interference)
Serial dilution producing non-linear results
1. Non-specific animal serum ‘Cocktail’ of animal sera
2. Blocking agent changes result Serial dilution or blocking agent Heterophilic antibody*
(Strong interference)
Serial dilution producing non-linear results
1. Serial dilution but preferably by using blocking agent.
Complement Proteins (Specific, strong interference)
Complement assay 1. Heat inactivation
2. Use Fab or F(ab’) antibodies in assay design Rheumatoid Factor (RF)* Test for RF Treat with anti-RF antibody
*For small non-protein bound analytes, use ultrafiltration or solid phase conjugated to Protein A or Protein G to remove interfering antibodies.
0 10 20 30 40 50 60 70
0 10 20 30 40 50 60 70
Expected Theophylline (àg/ml)
Observed Theophylline (àg/ml)
Non-discordant HAMA
FIGURE 6.1 HAMA interference detected by sample serial dilution.
70 6. IMMUNOASSAY DESIGN AND MECHANISMS OF INTERFERENCES
of a serum-based calibrator for the assay (60μg/mL) in a theophylline immunoassay using a mouse anti- theophylline antibody. The HAMA sample interferes with the assay and reads 59μg/mL when assayed undiluted (false-positive value). After successive dilu- tions (1.3-, 2-, 4-, 6-, and 12-fold with the assay dilu- ent), the interfering antibody is diluted enough so as not to cause any interference in the assay. The slope of a line fitted through the lowest three dilutions indi- cates a theophylline concentration of 31.1μg/mL, close to the original spike value (Datta et al., unpublished data). However, dilutions do not always provide the correct analyte value in the sample because of increased imprecision in the low end of the assay and because of the “matrix effect” between the calibrator matrix and a patient sample.
As described previously, a patient history of any exposure to animal antibodies, illness, or exposure to animals should also alert for heterophilic antibodies or HAAA as possible sources for inaccurate results. At that time, the assay insert should be examined for the types of antibodies and heterophilic antibody blockers used in the assay.
There are various types of commercial or home- brew blockers for heterophilic antibody or HAAA [44,45]. The blocker can be nonimmune animal serum, polyclonal antibody, polymerized IgG, nonimmune mouse monoclonals, or a mixture of monoclonal anti- bodies or fragments of IgG [Fc, Fab, or F(antibody’)2] preferably from the same species used to raise the reagent antibodies. As expected, although the nonspe- cific heterophilic interference can be mitigated by addi- tion of nonimmune serum to the reagents, purified IgG, preferably of the same subtype as used in assay, is better than serum in reducing the more specific and stronger binding HAAA.
Several blocking agents are commercially available:
Immunoglobulin Inhibiting Reagent (IIR;
Biorecalamation), Heterophilic Blocking Reagent (HBR;
Scantibodies), Heteroblock (Omega Biologicals), and MAB 33 (monoclonal mouse IgG1) and Poly MAB 33 (polymeric monoclonal IgG1/Fab; Boehringer Mannheim). IIR is a proprietary formulation of high- affinity anti-animal antibody, and HBR is monoclonal mouse anti-human IgM. A suspected discordant sam- ple (e.g., a sample giving false-positive hCG results) may be separately incubated with the blocker and then re-assayed [44]. Reinsberg [45] studied the efficacy of various blocking reagents in eliminating HAMA inter- ference. In another example, a clinically discordant false-positive serum myoglobin result (where another cardiac marker concentration, such as troponin I, was negative) was attributed to HAMA interference. The interference could be removed by the use of HBR[46].
Most commercial assay reagents include such blockers,
but due to the heterogeneous nature of the interfering antibodies, no blocker can guarantee success in all samples.
Nonspecific and weaker heterophilic antibody inter- ferences can even be mitigated by incubating the sam- ple with any nonimmune animal serum (even one different from the source of the assay antibody). Use of a “cocktail of animal sera” to reduce heterophilic inter- ference has been suggested [31]. Thus, heterophilic interference in a lutropin immunoassay was reduced equally by adding mouse, sheep, or goat sera to the sample [47]. If the interference is complement medi- ated involving Fc parts of the interfering antibodies, heat inactivation of the sample (56C for 30 min) may remove the interference [48]. A limitation of this method is that not all analytes can survive such an antibody denaturing process.
As previously mentioned, a general concept of pru- dent assay design is to use Fab or F(ab’)2fragments of the analyte-specific antibody, thereby reducing inter- ference from human anti-isotype antibodies [48].
Kuroki et al. [49] used human/mouse chimeric anti- body in a carcinoembryonic antigen assay to reduce HAMA interferences. Another interesting concept is to use chicken antibodies in the assay because to date no heterophilic antibody interference against assays using chicken antibodies has been reported in the literature [50]. By changing the reaction temperature and allow- ing a longer time to achieve equilibrium, it may be possible to reduce heterophilic antibody interference but not HAAA interference [51]. However, with mod- ern autoanalyzers, changing reaction temperature or reaction time is mostly not possible.
If the previously described methods to correct the interference do not work or cannot be applied, the interference can be resolved by removing the interfer- ing substance and re-assaying the clean specimen.
A simple solution to remove antibody interference is selective removal of the antibodies from a sample. This can be achieved by selective adsorption of human IgG by a solid phase containing protein A or protein G [52]. However, this does not work if the majority of the interfering antibodies are of the IgM type. Alternately, the antibody fraction in the sample may be precipi- tated out with a polyethylene glycol reagent (prefera- bly PEG 6000) [35]. Low-molecular-weight analytes, if they are not highly protein bound, may be extracted away from the interfering immunoglobulins by prepa- ration of protein-free filtrates via ultrafiltration or pro- tein precipitation (using trichloroacetic acid, sulfosalicylic acid, or ammonium sulfate). The centrifu- gal ultrafiltration is a fast and relatively easy method that uses 10- or 30-kDa cutoff filter membrane ultra- centrifugal cartridges (e.g., Amicon’s Microcon or Centricon). For digoxin assays, this step may not only
remove antibody interference but also remove interfer- ence from endogenous digoxin-like immunoreactive substances (B95% protein bound) or Digibind[53]. On the other hand, the harsh conditions of acid precipita- tion may damage many analyte molecules.
CONCLUSIONS
Immunoassays on automated systems are widely used in today’s clinical laboratories. Various types of immunoassays have been developed to analyze all types and sizes of antigens. The assays use photomet- ric, luminometric, or fluorometric signals and homoge- neous or heterogeneous reaction types. Serum and plasma are the main specimen types used for immu- noassays. Other types of specimens have also been used. Immunoassays are used not only on the central laboratory analyzers but also on the patient bedside point-of-care systems. Despite the excellent sensitivity and specificity of the immunoassays, they suffer from interferences: serum constituents, cross-reactants, or endogenous antibodies. There are various ways to detect such interferences and obtain accurate results.
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C H A P T E R
7
Effect of Herbal Remedies on Clinical Laboratory Tests
Amitava Dasgupta
University of Texas Health Sciences Center at Houston, Houston, Texas
INTRODUCTION
According to the Dietary Supplement Health and Education Act of 1994, herbal remedies sold in the United States are classified as food supplements.
Manufacturers of herbal remedies are not allowed by law to claim any medical benefit from these products, but at the same time they are not under surveillance of the U.S. Food and Drug Administration (FDA). In Germany, however, German Commission E has some control over marketing of herbal supplements because the commission publishes monographs prepared by an interdisciplinary committee using historical informa- tion; chemical, pharmacological, clinical, and toxicolo- gical study findings; case reports; epidemiological data; and unpublished manufacturers’ data. If an herbal supplement has an approved monograph, it can be marketed. European Directive 2004/24/EC, released in 2004 by the European Parliament and also by the Council of Europe, provides the basis for regulation of herbal supplements in the European market. This direc- tive requires that authorization be obtained from the national regulatory authorities of each European coun- try in which herbal medicines are to be released in the market and that these products must be safe. The safety of a supplement is established based on published sci- entific literature, and when the data on safety are not sufficient, this is communicated to consumers. In Europe, there will be two kinds of herbal supplements in the future: (1) herbal supplements with well- established safety and efficacy and (2) traditional herbal supplements that do not have a recognized level of effi- cacy but are relatively safe [1]. The Australian govern- ment also created a Complementary Medicine Evaluation Committee in 1997 to address regulatory
issues regarding herbal remedies. In Canada, the fed- eral government implemented a policy in 2004 to regu- late natural health products and naturopaths; many traditional Chinese medicine practitioners, homeopaths, and Western herbalists are concerned that this policy will eventually affect their access to the products they need to practice effectively[2].
In the United States, the sale of herbal remedies sig- nificantly increased from $200 million in 1988 to more than $3.3 billion in 1997. Within the European commu- nity, sales of herbal remedies are also widespread, with estimated annual sales of $7 billion in 2001[3]. Sales of herbal supplements were estimated to be $15.7 billion in 2000, and in 2003 sales increased to an estimated
$18.8 billion [4]. Currently, the global annual sales of herbal remedies are estimated to be $60 billion, repre- senting almost 20% of the overall pharmaceutical mar- ket[5]. The popularity of herbal supplements is steadily increasing among the general population in the United States. It is estimated that approximately 20,000 herbal products are available in the United States, and in one survey, approximately one out of five adults reported using an herbal supplement within the past year. The 10 most commonly used herbal supplements are echi- nacea, ginseng, ginkgo biloba, garlic, St. John’s wort, peppermint, ginger, soy, chamomile, and kava [6]. In general, higher use of herbal supplements is found among more educated, higher-income, white, and older females [7]. Use of herbal supplements can affect labo- ratory test results by various mechanisms:
• Physiological effects: When an herbal remedy alters normal physiological functions of the body or causes organ damage, the unexpected laboratory test may provide the first indication of toxicity of