2 IMMUNOLOGIC PROCEDURES Flashcards
(1) The interaction between individual antigen and antibody molecules depends on several types of bonds, such as ionic bonds, hydrogen bonds, hydrophobic bonds, and van der Waals forces. How is the strength of this attraction characterized?
A. Avidity
B. Affinity
C. Reactivity
D. Valency
B. Affinity
Affinity refers to the strength of a single antibody antigen interaction. Avidity is the strength of interactions between many different antibodies in a serum against a particular antigen (i.e., the sum of many affinities).
(2) A laboratory is evaluating an enzyme-linked immunosorbent assay (ELISA) for detecting an antibody to cyclic citrullinated peptide (CCP), which is a marker for rheumatoid arthritis (RA). The laboratory includes serum from healthy volunteers and from patients with other connective tissue diseases in the evaluation. These specimens determine which factor of the assay?
A. Sensitivity
B. Precision
C. Bias
D. Specificity
D. Specificity
Specificity is defined as a negative result in the absence of the disease. The non–RA specimens would be expected to test negative if the assay has high specificity. Precision is the ability of the assay to repeatedly yield the same results on a single specimen. Both bias and sensitivity calculations would include specimens from RA persons. Although those specimens would be included in the evaluation, they are not listed in the question.
(3) The detection of precipitation reactions depends on the presence of optimal proportions of antigen and antibody. A patient’s sample contains a large amount of antibody, but the reaction in a test system containing antigen is negative. What has happened?
A. Performance error
B. Low specificity
C. A shift in the zone of equivalence
D. Prozone phenomenon
D. Prozone phenomenon
Although performance error and low specificity should be considered, if a test system fails to yield the expected reaction, excessive antibody preventing a precipitation reaction is usually the cause. Prozone occurs when antibody molecules saturate the antigen sites, preventing cross-linking of the antigen–antibody complexes by other antibody molecules. Because antigen and antibody do not react at equivalence, a visible product is not formed, leading to a false-negative result.
(4) The positive and negative control values for an ELISA procedure are below their acceptable ranges. What is the most likely cause?
A. Decay of the positive and negative controls
B. Incomplete washing following specimen addition
C. Overly long incubation times
D. Decay of the antibody–enzyme conjugate
D. Decay of the antibody–enzyme conjugate
The antibody–enzyme conjugate is very sensitive to storage conditions and is easy to dissociate. Control specimens are unlikely to decay, and the other options would lead to higher values.
(5) What is the interpretation when an Ouchterlony plate shows crossed lines between wells 1 and 2 (antigen is placed in the center well and antisera in wells 1 and 2)?
A. No reaction between wells 1 and 2
B. Partial identity between wells 1 and 2
C. Nonidentity between wells 1 and 2
D. Identity between wells 1 and 2
C. Nonidentity between wells 1 and 2
Crossed lines indicate nonidentity between wells 1 and 2. The antibody from well 1 recognizes a different antigenic determinant than the antibody from well 2.
(6) A weight lifter taking many supplements is tested monthly for thyroid-stimulating hormone (TSH) in a direct capture assay, which uses a streptavidin biotin indicator system. She has had normal TSH levels for the past 3 months on specimens collected in the late evening. This month she comes in right after breakfast for her blood draw. The TSH level is three times her previous level. What may be the cause of this difference?
A. Diurnal variation in TSH levels
B. Exogenous biotin in her system from a supplement taken that morning
C. Reduced thyroid function caused by an unidentified pathology
D. Pipetting error
B. Exogenous biotin in her system from a supplement taken that morning
High levels of exogenous biotin may be present in blood up to 10 to 15 hours after ingestion and will usually cause falsely elevated values in avidin–biotin direct ELISA assays and competitive immunoassays but lower values in sandwich (immunometric) assays. Monthly variation in TSH should not approach the level seen in this situation.
(7) What comprises the indicator system in an indirect ELISA for detecting antibody?
A. Enzyme-conjugated antibody + chromogenic substrate
B. Enzyme conjugated antigen + chromogenic substrate
C. Enzyme + antigen
D. Substrate + antigen
A. Enzyme-conjugated antibody + chromogenic substrate
ELISA measures antibody by using immobilized reagent antigen. The antigen is fixed to the walls of a tube or bottom of a microtiter well. Serum is added (and incubated) and the antibody binds, if present. After washing, the antigen–antibody complexes are detected by adding an enzyme labeled antiimmunoglobulin (anti-Ig). The unbound enzyme label is removed by washing, and the bound enzyme label is detected by adding chromogenic substrate. The enzyme catalyzes the conversion of substrate to a colored product.
(8) What outcome results from improper washing of a tube or well after adding the enzyme–antibody conjugate in an ELISA system?
A. Result will be falsely decreased
B. Result will be falsely increased
C. Result will be unaffected
D. Result is impossible to determine
B. Result will be falsely increased
If unbound enzyme-conjugated anti-Ig is not washed away, it will catalyze the conversion of the substrate to a colored product, yielding a falsely elevated result.
(9) What would happen if the color reaction phase is prolonged in one tube or well of an ELISA test?
A. Result will be falsely decreased
B. Result will be falsely increased
C. Result will be unaffected
D. Impossible to determine
B. Result will be falsely increased
If the color reaction is not stopped within the time limits specified by the procedure, the enzyme will continue to act on the substrate, producing a falsely elevated test result.
(10) The absorbance of a sample measured by ELISA is greater than the highest standard. What corrective action should be taken?
A. Extrapolate an estimated value from the highest reading
B. Repeat the test using a standard of higher concentration
C. Repeat the assay using one half the volume of the sample
D. Dilute the test sample
D. Dilute the test sample
Usually, when a test sample reads at a value above the highest standard in an ELISA, the sample is diluted and measured again. In those instances where no additional clinical value can be obtained by dilution, the result may be reported as greater than the highest standard (citing the upper reportable limit of the assay).
(11) A patient was suspected of having a lymphoproliferative disorder. After several laboratory tests were completed, the patient was found to have an IgMκ paraprotein. In what sequence should the laboratory tests leading to this diagnosis have been performed?
A. Serum protein electrophoresis (SPE) followed by immunofixation electrophoresis (IFE)
B. Ig levels, followed by SPE
C. Total lymphocyte count, followed by Ig levels
D. Ig levels, followed by urine protein electrophoresis
A. Serum protein electrophoresis (SPE) followed by immunofixation electrophoresis (IFE)
SPE should be performed initially to detect the presence of an abnormal Ig that demonstrates restricted electrophoretic mobility. A patient producing only monoclonal light chains may not show an abnormal serum finding because the light chains may be excreted in urine. A positive finding for either serum or urine should be followed by IFE on the positive specimen. This is required to confirm the presence of monoclonal Ig and to identify the heavy and light chain types.
(12) An IFE performed on a serum sample showed a narrow dark band in the lanes containing anti-γ and anti-λ. How should this result be interpreted?
A. Abnormally decreased IgG concentration
B. Abnormal test result demonstrating monoclonal IgGλ
C. Normal test result
D. Impossible to determine without densitometric quantitation
B. Abnormal test result demonstrating monoclonal IgGλ
A narrow dark band formed in both the lane containing anti-γ and anti-λ indicates the presence of a monoclonal IgG-λ. A diffuse dark band would indicate a polyclonal increase in IgG that often accompanies chronic inflammatory disorders, such as systemic lupus erythematosus (SLE)
(13) Which type of nephelometry is used to measure immune complex formation almost immediately after reagent has been added?
A. Rate
B. Endpoint
C. Continuous
D. One-dimensional
A. Rate
Rate nephelometry is used to measure the formation of small immune complexes as they are formed under conditions of antibody excess. The rate of increase in the photodetector output is measured within seconds or minutes, and the rate increases with increasing antigen concentration. Antigen concentration is determined by comparing the rate for the sample with that for standards by using an algorithm that compensates for nonlinearity. In endpoint nephelometry, reactions are read after equivalence. Immune complexes are of maximal size but may have a tendency to settle out of solution, thereby decreasing the amount of scatter.
(14) An antinuclear antibody (ANA) test was performed by using immunofluorescence microscopy assay (IFA), and a clinically significant pattern and titer were reported. Positive and negative controls performed as expected. However, the clinical evaluation of the patient was not consistent with the reported pattern. What is the most likely explanation for this situation?
A. The clinical condition of the patient changed since the sample was tested
B. The pattern of fluorescence was misinterpreted
C. The control results were misinterpreted
D. The wrong cell line was used for the test
B. The pattern of fluorescence was misinterpreted
In an IFA for antinuclear antibodies, the fluorescence pattern must be correlated correctly with the specificity of the antibodies. Both pathological and nonpathological antibodies can occur, and antibodies may be detected at a significant titer in a patient whose disease is inactive. Failure to correctly identify subcellular structures may result in misinterpretation of the antibody specificity or a false-positive result caused by nonspecific fluorescence.
(15) What corrective action should be taken when a specific pattern cannot be identified in a specimen with a positive ANA IFA?
A. Repeat the test using a larger volume of sample
B. Call the physician
C. Have another medical laboratory scientist read the slide
D. Dilute the sample and retest
D. Dilute the sample and retest
An unexpected pattern may indicate the presence of more than one antibody. Diluting the sample may help to clearly show the antibody specificities, if they are found in different titers. If the pattern is still atypical, a new sample should be collected and the test repeated or the specimen should be tested by an alternative method, such as ELISA or multiplex.
