Immunologic Procedures HARR

Question 1

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

Answer 1

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).

Question 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. 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

Answer 2

Specificity

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.

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

Answer 3

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 the antigen and antibody do not react at equivalence, a visible product is not formed, leading to a false-negative result.

Question 4

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

Answer 4

Gel

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.

Question 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

Answer 5

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.

Question 6

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

Answer 6

Because of high sensitivity

The sensitivity of EIA methods producing visible color change, and fluorescent and chemiluminescent products approaches nanogram levels of antibody. These methods are easily automated.

Question 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

Answer 7

Enzyme-conjugated antibody + chromogenic
substrate

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

Question 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

Answer 8

Result will be falsely increased

If unbound enzyme-conjugated anti-immunoglobulin is not washed away, it will catalyze conversion of substrate to colored product, yielding a falsely elevated result

Question 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

Answer 9

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.

Question 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

Answer 10

Dilute the test sample

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).

Question 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. Immunoglobulin levels followed by SPE
C. Total lymphocyte count followed by
immunoglobulin levels
D. Immunoglobulin levels followed by urine protein electrophoresis

Answer 11

Serum protein electrophoresis (SPE) followed by immunofixation electrophoresis (IFE)

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

Question 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

Answer 12

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 immunoglobulin. A diffuse dark band would indicate a polyclonal increase in IgG that often accompanies chronic inflammatory disorders such as systemic lupus erythematosus (SLE).

Question 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

Answer 13

Rate

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.

Question 14

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

Answer 14

The pattern of fluorescence was misinterpreted

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

Question 15

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

Answer 15

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

Question 16

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

Answer 16

Carrier particles for antigen such as latex particles are used

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.

Question 17

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

Answer 17

Prozone reaction

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.

Question 18

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

Answer 18

128

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.

Question 19

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

Answer 19

Possible false-positive result

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.

Question 20

Which outcome indicates a negative result in a
complement fixation test?
A. Hemagglutination
B. Absence of hemagglutination
C. Hemolysis
D. Absence of hemolysis

Answer 20

Hemolysis

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

Question 21

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

Answer 21

Failure to count the desired cell population

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.

Question 22

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

Answer 22

Lineage determination by detecting antigens on the surface of the gated cells using fluorescent antibodies

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

Question 23

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

Answer 23

An abnormal cell population is present

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.