Medical Laboratory Review Harr. - 3.2 Immunology and Serology: Immunologic Procedures
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 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.
The directions for a slide agglutination test instruct that after mixing the patient's serum and latex particles, the slide must be rotated for 2 minutes. What would happen if the slide were rotated for 10 minutes? A. Possible false-positive result B. Possible false-negative result C. No effect D. Depends on the amount of antibody present in the sample
A Failure to follow directions, as in this case where the reaction was allowed to proceed beyond the recommended time, may result in a false-positive reading. Drying on the slide may lead to a possible erroneous positive reading.
Which statement best describes immunophenotyping? A. Lineage determination by detecting antigens on the surface of the gated cells using fluorescent antibodies B. Identification of cell maturity using antibodies to detect antigens within the nucleus C. Identification and sorting of cells by front and side-scatter of light from a laser D. Analysis of cells collected by flow cytometry using traditional agglutination reactions
A Immunophenotyping refers to classification of cells (lineage and maturity assignment) using a panel of fluorescent-labeled antibodies directed against specific surface antigens on the cells. Antibodies are referred to by their CD (cluster of differentiation) number. Monoclonal antibodies having a common CD number do not necessarily bind to the same epitope but recognize the same antigen on the cell surface. Reactivity of the selected cells with a panel of antibodies differentiates lymphoid from myeloid cells and identifies the stage of cell maturation.
What has happened in a titer, if tube Nos. 5-7 show a stronger reaction than tube Nos.1-4? A. Prozone reaction B. Postzone reaction C. Equivalence reaction D. Poor technique
A In tubes Nos.1-4, insufficient antigen is present to give a visible reaction because excess antibody has saturated all available antigen sites. After dilution of antibody, tubes Nos.1-4 have the equivalent concentrations of antigen and antibody to allow formation of visible complexes.
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 nephelometry is used to measure formation of small immune complexes as they are formed under conditions of antibody excess. The rate of increase in photodetector output is measured within seconds or minutes and increases with increasing antigen concentration. Antigen concentration is determined by comparing the rate for the sample to that for standards 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.
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 A The ELISA test measures antibody 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 anti-immunoglobulin. 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 colored product.
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. Immunoglobulin levels followed by SPE C. Total lymphocyte count followed by immunoglobulin levels D. Immunoglobulin levels followed by urine protein electrophoresis
A. Serum protein electrophoresis should be performed initially to detect the presence of an abnormal immunoglobulin that demonstrates restricted electrophoretic mobility. A patient producing only monoclonal light chains may not show any abnormal serum finding because the light chains may be excreted in the 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 immunoglobulin and to identify the heavy and light chain type
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 A narrow dark band formed in both the lane containing anti-γ and anti-λ indicates the presence of a monoclonal IgG λ immunoglobulin. A diffuse dark band would indicate a polyclonal increase in IgG that often accompanies chronic inflammatory disorders such as systemic lupus erythematosus (SLE).
The interaction between an individual antigen and antibody molecule depends upon 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 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).
What effect does selecting the wrong gate have on the results when cells are counted by flow cytometry? A. No effect B. Failure to count the desired cell population C. Falsely elevated results D. Impossible to determine
B Gating is the step performed to select the proper cells to be counted. Failure to properly perform this procedure will result in problems in isolating and counting the desired cells. It is impossible to determine if the final result would be falsely elevated or falsely lowered by problems with gating.
An immunofluorescence microscopy assay (IFA) was performed, and a significant antibody titer was reported. Positive and negative controls performed as expected. However, the clinical evaluation of the patient was not consistent with a positive finding. What is the most likely explanation of 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 In an IFA, for example, an antinuclear antibody (ANA) test, 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 caused by nonspecific fluorescence.
A flow cytometry scattergram of a bone marrow sample shows a dense population of cells located in-between normal lymphoid and normal myeloid cells. What is the most likely explanation? A. The sample was improperly collected B. An abnormal cell population is present C. The laser optics are out of alignment D. The cells are most likely not leukocytes
B Lymphoid cells and myeloid cells display in predictable regions of the scatterplot because of their characteristic size and density. Lymphoid cells cause less forward and side scatter from the laser than do myeloid cells. A dense zone of cells in between those regions is caused by the presence of a large number of abnormal cells, usually blasts. The lineage of the cells can be determined by immunophenotyping with a panel of fluorescent-labeled antibodies
What is the titer in tube No. 8 if tube No. 1 is undiluted and dilutions are doubled? A. 64 B. 128 C. 256 D. 512
B The antibody titer is reciprocal of the highest dilution of serum giving a positive reaction. For doubling dilutions, each tube has one half the amount of serum as the previous tube. Because the first tube was undiluted (neat), the dilution in tube No. 8 is (1/2)7 and the titer equals 27 or 128.
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 B If unbound enzyme conjugated anti immunoglobulin is not washed away, it will catalyze conversion of substrate to colored product, yielding a falsely elevated result
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 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.
Which part of the radial immunodiffusion (RID) test system contains the antisera? A. Center well B. Outer wells C. Gel D. Antisera may be added to any well
C In an RID test system, for example, one measuring hemopexin concentration, the gel would contain the antihemopexin. A standardized volume of serum containing the antigen is added to each well. Antigen diffuses from the well into the gel and forms a precipitin ring by reaction with antibody. At equivalence, the area of the ring is proportional to antigen concentration.
Which outcome indicates a negative result in a complement fixation test? A. Hemagglutination B. Absence of hemagglutination C. Hemolysis D. Absence of hemolysis
C In complement fixation, hemolysis indicates a negative test result. The absence of hemolysis indicates that complement was fixed in an antigen-antibody reaction and, therefore, that the specific complement binding antibody was present in the patient's serum. Consequently, it was not available to react in the indicator system.
Why is a chemiluminescent immunoassay (CIA) or enzyme immunoassay (EIA) the method of choice for detection of certain analytes, such as hormones, normally found in low concentrations? A. Because of low cross reactivity B. Because of high specificity C. Because of high sensitivity D. Because test systems may be designed as both competitive and noncompetitive assays
C The sensitivity of EIA methods producing visible color change, and fluorescent and chemiluminescent products approaches nanogram levels of antibody. These methods are easily automated.
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 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 the antigen and antibody do not react at equivalence, a visible product is not formed, leading to a false-negative result.
What corrective action should be taken when an indeterminate pattern occurs in an indirect 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 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.
Which statement best describes passive agglutination reactions used for serodiagnosis? A. Such agglutination reactions are more rapid because they are a single-step process B. Reactions require the addition of a second antibody C. Passive agglutination reactions require biphasic incubation D. Carrier particles for antigen such as latex particles are used
D Most agglutination tests used in serology employ passive or indirect agglutination where carrier particles are coated with the antigen. The carrier molecule is of sufficient size so that the reaction of the antigen with antibody results in formation of a complex that is more easily visible.
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. The laboratory includes serum from healthy volunteers and 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 is defined as a negative result in the absence of the disease. The non-rheumatoid arthritis 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 rheumatoid arthritis specimens. Although those specimens would be included in the evaluation, they are not listed in the question.
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 Usually when a test sample reads at a value above the highest standard in an ELISA test, it 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).
