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SECTION 11 Immunology

SECTION EDITORS: Steven D. Douglas and Michele E. Paessler


ASSOCIATE SECTION EDITOR: Thomas N. Denny
[Updated March 2007]

11.1. Immunology Introduction ...................................................... 11.1.1.1


11.1.1. Introduction • Steven D. Douglas ..................................... 11.1.1.1
11.1.2. Immunologic Assays Used in the Serologic Diagnosis
of Infectious Diseases • William M. Janda ................................ 11.1.2.1
11.1.3. Emerging Immunological Assays • Ambrosia Garcia ........... 11.1.3.1
11.2. Serologic Diagnosis of Group A Streptococcal Infections
Stanford T. Shulman and Elia M. Ayoub ........................................... 11.2.1.1
11.2.1. Introduction .............................................................. 11.2.1.1
11.2.2. Anti-Streptolysin O .................................................... 11.2.2.1
11.2.3. Anti-DNase B Test ..................................................... 11.2.3.1
11.3. Detection of Legionella Antigen by Direct
Immunofluorescence
Paul H. Edelstein ....................................................................... 11.3.1

11.4. Urinary Antigen Detection for Legionella spp.


Paul H. Edelstein ....................................................................... 11.4.1

11.5. Laboratory Diagnosis of Syphilis


Victoria Pope .......................................................................... 11.5.1.1
11.5.1. Introduction .............................................................. 11.5.1.1
11.5.2. Direct Fluorescent-Antibody Test
for Treponema pallidum ..................................................... 11.5.2.1
11.5.3. Rapid Plasma Reagin Test ........................................... 11.5.3.1
11.5.4. Serodia Treponema pallidum Particle
Agglutination Test ............................................................ 11.5.4.1
11.6. Detection of Borrelia burgdorferi Antibodies
Maria E. Aguero-Rosenfeld ............................................................ 11.6.1

11.7. Serodiagnosis of Rickettsial Infections


J. Stephen Dumler .................................................................... 11.7.1.1
11.7.1. Introduction .............................................................. 11.7.1.1
11.7.2. Indirect Fluorescent-Antibody Test ............................... 11.7.2.1
11.7.3. Solid-Phase (Dot Blot) EIA .......................................... 11.7.3.1
11.7.4. Latex Agglutination .................................................... 11.7.4.1
11.8. Immunoassay Detection of Shiga Toxin-Producing
Escherichia coli
Karen C. Carroll ....................................................................... 11.8.1

11.9. Serodiagnosis of Helicobacter pylori


James Versalovic ....................................................................... 11.9.1
(continued)

11.0.1
11.0.2 Immunology

11.10. Total Viable Cell Counting Procedure


Ambrosia Garcia ....................................................................... 11.10.1

11.11. Peripheral Blood Mononuclear Cell Cryopreservation Method


Raul Louzao ............................................................................ 11.11.1

11.12. Lymphocyte Proliferation Assay


Richard Tustin III, Frank I. Scott IV, and Nancy B. Tustin ....................... 11.12.1

11.13. Natural Killer Cell Assays .....................................................11.13.1.1


11.13.1. Introduction • Michele Paessler ....................................11.13.1.1
11.13.2. Natural Killer Cell Flow Cytometry Assay
• Michele Paessler ...............................................................11.13.2.1
11.13.3. Natural Killer Cell Assay • Nancy E. Raftery
and Nancy B. Tustin .............................................................11.13.3.1

11.14. Quantitation of Human Interleukin 4, Interleukin 6,


and Gamma Interferon
Marie T. Wilson and Nancy B. Tustin ............................................... 11.14.1

11.15. Flow Cytometry Whole-Blood Intracellular-Cytokine Assay


Using Phorbol Myristate Acetate, Ionomycin, and Brefeldin A
Dana Stein and Zenaida Garcia ..................................................... 11.15.1

11.16. Whole-Blood Lymphocyte Immunophenotyping Using Cell


Surface Markers by Flow Cytometry
Dana Stein .............................................................................. 11.16.1

11.17. Neutrophil Function Whole-Blood Flow Cytometric Test


for Leukocyte Adhesion Deficiency
Dana Stein and Zenaida Garcia ..................................................... 11.17.1

11.18. Flow Cytometric Test for Chronic Granulomatous Disease


Dana Stein and Zenaida Garcia ..................................................... 11.18.1
11.1 Immunology Introduction

11.1.1 Introduction

Since the previous edition of CMPH, there detection, indirect fluorescent-antibody cells. For other details on laboratory im-
have been remarkable advances in the testing for antigen and antibody detection, munology procedures, please refer to the
clinical immunology laboratory. Labora- immunoblot procedures, complement fix- Manual of Clinical Laboratory Immunol-
tory directors in clinical microbiology and ation, hemagglutination inhibition, and ogy, 6th ed. (2), as well as to Clinical and
clinical immunology interact with all immunodiffusion testing. In addition, a Diagnostic Laboratory Immunology. Sec-
fields of medicine. The intent of section procedure has been added listing emerging tion 11 provides specific details for utili-
11 is to highlight procedures for major as- immunological assays (11.1.3). This ad- zation of immunologic tests in the micro-
says which are called upon in the clinical dition provides unique outlines for flow biologic setting as well as for cellular
microbiology and clinical immunology cytometry-based assays for apoptosis, assays to determine the immunocompe-
laboratories. This field has expanded into chemokine detection, dendritic-cell detec- tence of the host. The tables provided by
many domains of diagnostic testing. Al- tion, and tetramer assays. Diagnostic pro- W. M. Janda (procedure 11.1.2) and A.
though the section cannot cover the full cedures added include basic serologic as- Garcia (procedure 11.1.3) cover both basic
spectrum of laboratory immunology test- says (procedures 11.2 to 11.9). New tests concepts and concepts for emerging im-
ing, a table from EPCM (1) provides have been added for cellular immunology munologic assays. Our thrust has been to
generic descriptions of assays used in se- (procedures 11.10 through 11.18). Several add the important immunological ap-
rologic diagnosis, including latex aggluti- flow cytometric assay procedures are pro- proaches that define the host response
nation for antigen and antibody detection, vided (procedures 11.15 through 11.18). against microbial agents to the armamen-
staphylococcal coagglutination, EIAs for Methods are also provided for the assay of tarium of clinical microbiologists, thereby
antibody and antigen detection, direct human cellular cytokines and studies of to broaden the assessment of clinically
fluorescent-antibody testing for antigen lymphocyte proliferation and natural killer overt infectious disease.

REFERENCES 1. Janda, W. M. 1998. Immunology, p. 561–


577. In H. D. Isenberg (ed.), Essential Pro-
cedures for Clinical Microbiology. ASM
Press, Washington, D.C.
2. Rose, N. R., R. G. Hamilton, and B. Detrick
(ed.). 2002. Manual of Clinical Laboratory
Immunology, 6th ed. ASM Press, Washington,
D.C.

11.1.1.1
11.1.2 Immunologic Assays Used in the
Serologic Diagnosis of Infectious
Diseases

The following table is a summary of vari-


ous tests that can be used in clinical mi-
crobiology laboratories for the serologic
diagnosis of infectious diseases.
Table 11.1.2–1 Generic description of immunologic assays used in the serologic diagnosis of infectious diseasesa,b
Test Basic principle Specimen type evaluated Basic materials Basic QC
Latex agglutination for Latex spheres coated with CSF, urine, or serum 1. Latex beads coated Positive and negative
antigen detection specific immunoglobu- with specific antibod- controls should be per-
lins agglutinate in the ies formed with each batch
presence of antigen. 2. Positive and negative of patient specimens.
antigen controls
3. Mixing sticks
4. Reaction slides
5. Heat block or boiling
water bath
6. Centrifuge

Latex agglutination for Latex spheres coated with Serum or plasma 1. Latex beads coated Inclusion of positive and
antibody detection specific antigens agglu- with specific antibod- negative controls each
tinate in the presence ies time test is performed
of serum containing the 2. Mixing sticks
corresponding specific 3. Glass agglutination
antibody. slides
4. Appropriate positive
and negative controls

Staphylococcal coagglu- Staphylococcal cells are Suspension of the organ- 1. Staphylococcal cells Inclusion of positive and
tination sensitized with anti- ism to be identified sensitized with specific negative controls each
body against specific prepared in PBS, buf- antibody time the test is per-
antigens by the ability fered saline, etc., de- 2. Nonsensitized staphy- formed
of staphylococcal pro- pending on the kit lococcal cells (nega-
tein A to bind immuno- (usually used for bacte- tive control reagent)
globulin to the bacterial rial identification) 3. Reaction slides or
cell surface by the Fc cards
regions of the antibody 4. Mixing sticks
molecules.

11.1.2.1
Serologic Diagnosis of Infectious Diseases 11.1.2.2

Basic procedure Expected results Basic limitations Generic examples


Mix specimen with reagent on a 1. Positive control must yield a 1. Tests should not be used to Several commercial kits for de-
glass slide and inspect for strong agglutination reaction replace culture (CSF antigen tection of bacterial antigens
specific agglutination. within 10 min. detection). (group B streptococcus, Hae-
2. Negative control must show 2. Urine and serum specimens mophilus influenzae type b,
no agglutination. require additional processing Streptococcus pneumoniae,
3. Specimens showing aggluti- to prevent uninterpretable re- and Neisseria meningitidis
nation are positive. sults. serogroups A, C, Y, and
4. Specimens showing no ag- 3. Low antigen levels may re- W135) in CSF; also kits for
glutination are negative. sult in false-negative tests. detection of Cryptococcus
neoformans capsular polysac-
charide in CSF

1. Mix serum specimen on a 1. Positive control must pro- 1. Low levels of antibody may Latex agglutination tests for de-
glass slide. duce appropriate specific ag- not be reliably detected. tection of rubella virus and
2. Rock or rotate slide for spec- glutination reaction. 2. Some kits may not be able to CMV antibodies
ified period. 2. Negative control must not produce results that correlate
3. Inspect for agglutination. show agglutination. with traditional antibody ti-
3. Patient specimens showing ters.
agglutination contain specific
antibody.
4. Negative specimens show no
agglutination.

1. Standardized, properly pro- 1. Positive control suspension 1. Isolates that show no aggluti- Coagglutination tests for the
cessed bacterial cell suspen- must produce at least 2Ⳮ ag- nation must be identified by identification of N. gonor-
sion (1 drop) is reacted with glutination with the sensi- other methods. rhoeae (e.g., Phadebact
antibody-sensitized staphylo- tized cells and no agglutina- 2. Not all strains of an organism OMNI, Gonogen, Meritec)
coccal cells (1 drop) and tion with the control cells. may be identified by a partic-
cells that are not sensitized in 2. Negative control suspension ular test. For example, not all
circles or walls of a reaction must show no agglutination strains of Neisseria gonor-
slide. with either the sensitized or rhoeae are identified by com-
2. Mix and rock or rotate the re- the control staphylococcal mercially available coagglu-
action slide for specified pe- cells. tination tests due to lack of
riod. 3. Agglutination reactions of appropriate monoclonal anti-
3. Inspect for specific agglutina- 2Ⳮ or greater with the sensi- bodies on the surfaces of the
tion. tized cells and no agglutina- sensitized staphylococcal
tion with the nonsensitized cells.
cells on patient isolates are
positive.
(continued)
11.1.2.3 Immunology

Table 11.1.2–1 Generic description of immunologic assays used in the serologic diagnosis of infectious diseasesa,b (continued)
Test Basic principle Specimen type evaluated Basic materials Basic QC
EIA for antibody detec- Antigen is bound to a Serum or plasma 1. Microtiter walls/beads Inclusion of sera known
tion solid phase (bead or coated with antigen to be positive and neg-
microtiter well). Anti- 2. PBS with Tween 80 ative for antibody be-
body present in the for serum dilution ing detected; diluent
specimen binds to the 3. Detection antibody-en- buffer control
antigen on the solid zyme conjugate
phase. This complex is 4. Chromogenic enzyme
then reacted with a sec- substrate
ond antibody that is en- 5. Spectrophotometer
zyme labeled and also 6. Incubator
directed against the an- 7. Pipettes
tigen (the conjugate is
often an anti-human
immunoglobulin raised
in goats). Subsequent
exposure of this com-
plex to the enzyme
substrate results in the
generation of a colored
end product that is de-
tected spectrophotomet-
rically.

EIA for antigen detection In this case, antibody Serum, plasma, other 1. Antibody-sensitized Known positive and nega-
(e.g., capture antibody) body fluids (e.g., stool microtiter wells, beads, tive control samples
instead of antigen is for detection of Giar- or membrane filters
bound to a solid phase. dia lamblia antigen, 2. Second enzyme-conju-
Antigen present in a respiratory tract secre- gated antibody di-
specimen binds to the tions for detection of rected against other
capture antibody on the RSV, etc.) epitopes of the antigen
solid phase. Following 3. Positive and negative
wash steps, this com- controls that contain
plex is reacted with a antigen or lack the an-
second enzyme-labeled tigen, respectively
antibody that is di- 4. Some antigen detec-
rected against another tion EIAs contain cali-
epitope of the captured brators and standards
antigen. After washing, to allow semiquantita-
enzyme substrate is tion of antigen levels
added, resulting in the present in certain spec-
generation of a colored imens.
end product that is de- 5. Spectrophotometric
tected visually or spec- detection of positive
trophotometrically. results is used with
some kits, although
many rely on visual
reading for detection
of positive and nega-
tive tests.
Serologic Diagnosis of Infectious Diseases 11.1.2.4

Basic procedure Expected results Basic limitations Generic examples


1. Diluted serum specimen is Positive results are determined 1. Sera obtained during the EIA kits for detection of anti-
incubated in antigen-sensi- by comparison with negative acute phase of infection may bodies are available for a
tized wall or with antigen- control; generally absorbance contain only antibodies of the wide variety of agents, par-
sensitized bead. values 2- to 3-fold greater IgM class and will not be ticularly certain bacteria (e.g.,
2. Incubation and wash stage than the mean absorbance of a identified in IgG-specific as- Borrelia burgdorferi) and vi-
3. Addition of enzyme-labeled group of negative controls are says; the converse is also ruses (HIV-1, CMV, rubella,
conjugate directed against an- considered positive. Some true. HSV, measles, mumps,
tibody (e.g., anti-human IgG kits may include low- and 2. Contaminated, icteric, li- HIV-1/2)
raised in goats and linked to high-titer positive controls, pemic, heat-inactivated, or
HRP) calibrations, etc. hemolyzed sera may produce
4. Incubation and wash steps erroneous results.
5. Addition of enzyme substrate 3. Departure from specific pro-
and generation of signal cedure may affect test results.
6. Addition of stop solution
7. Generated color assessed
spectrophotometrically

1. The specimen is incubated 1. Positive results are deter- 1. Tests must be used exactly as Immunoassays for detection of
in a microtiter well or tube mined visually by the pres- described in package inserts. group A streptococci directly
containing the capture anti- ence of a colored endpoint. 2. Low levels of antigen may in throat swabs; immunoas-
body. This may also be done with a not be detected. says for detection of RSV and
2. Incubate for specified pe- spectrophotometer. 3. Antigens already present in influenza A virus in respira-
riod. 2. Semiquantitative results may serum specimens as antigen- tory tract specimens; assays
3. Wash steps be determined by standard antibody complexes will not for detection of antigen in
4. A second enzyme-labeled curves with cutoffs deter- be detected unless immune stool of patients with G. lam-
antibody that is also di- mined by negative controls complexes are first dissoci- blia and Cryptosporidium
rected against epitopes of and regression analysis of ated. parvum; assays for detection
the antigen is added. standards. of HIV p24 antigen in serum
5. Incubate for specified pe-
riod.
6. Wash steps
7. Enzyme substrate is added
and allowed to incubate for
a specified period.
8. Addition of stop solution
9. Color reaction is read visu-
ally or with a spectropho-
tometer.
10. In semiquantitative assay,
standard curves are prepared
from calibrators run in the
same assay.

(continued)
11.1.2.5 Immunology

Table 11.1.2–1 Generic description of immunologic assays used in the serologic diagnosis of infectious diseasesa,b (continued)
Test Basic principle Specimen type evaluated Basic materials Basic QC
Direct fluorescent- Specific antibody conju- Specimens may include 1. Appropriate clinical Positive and negative
antibody test for anti- gated to a fluorescent smears made directly specimens controls performed
gen detection tag (e.g., FITC) is re- from clinical specimens 2. Teflon-coated slides along with clinical
acted directly with a (e.g., endocervical used for immunofluo- specimens
specimen on a glass smears for detection of rescence (i.e., with
slide or with a smear of Chlamydia trachoma- specimen wells)
an organism prepared tis) or from cultures, 3. Specific antibodies
from a culture plate. where this procedure conjugated directly to
After incubation and may be used for identi- a fluorophore (e.g.,
washing, the slide is fication (e.g., identifica- FITC)
examined under a fluo- tion of Legionella spp. 4. Humidor for slide in-
rescence microscope from cultures grown on cubation
and examined for spe- BCYE medium, identi- 5. Pipettes
cific immunofluores- fication of respiratory
cence. viruses growing in
shell vials, etc.)

IFA test for antigen de- A specimen fixed on a IFA tests can be used for 1. Appropriate clinical Positive and negative
tection slide is overlaid with detection of antigens in specimens control slides prepared
an excess of unlabeled various clinical speci- 2. Unlabeled specific an- from previously tested
immune serum directed mens (e.g., IFA test for tibodies directed patient specimens are
against the antigen and Pneumocystis carinii in against the antigen of processed through the
incubated. After a wash bronchoalveolar lavage interest procedure along with
step, the specimen-anti- specimens). 3. Fluorochrome-conju- unknown patient speci-
body complex is re- gated antibodies di- mens.
acted with a fluoro- rected against the unla-
chrome-labeled beled antibodies (e.g.,
antibody directed FITC-labeled anti-goat
against the species of antibodies raised in
the first unlabeled anti- mice)
body (e.g., goat anti- 4. Teflon-coated immu-
human immunoglobu- nofluorescence slides
lin conjugated to with multiple wells
FITC). After a wash 5. Humidor for incuba-
step, the specimen is tion
examined under a fluo- 6. Pipettes
rescence microscope
fitted with the appropri-
ate barrier filters for
detection of specific
immunofluorescence.
Serologic Diagnosis of Infectious Diseases 11.1.2.6

Basic procedure Expected results Basic limitations Generic examples


1. Specimen is applied to a well Positive control and positive pa- Usually used only for antigen Direct fluorescent-antibody test
on a Teflon-coated immuno- tient specimens show specific detection; while more rapid for C. trachomatis
fluorescence slide and fixed. apple-green immunofluores- and less nonspecific, direct
2. Specimen is overlaid with la- cence (FITC); negative con- fluorescence procedures may
beled conjugate and incu- trol and negative patient sam- be less sensitive and produce
bated in humidor for 15- to ples show no fluorescence. duller fluorescence than indi-
30-min period. rect immunofluorescence anti-
3. Slide is washed to remove gen detection methods.
unbound conjugate.
4. Air dry specimen and apply
mounting fluid and coverslip.
5. Examine under a microscope
outfitted with a UV light
source and appropriate bar-
rier filters for assay under
consideration.

1. Appropriate clinical speci- 1. Positive control material re- 1. Positive and negative con- P. carinii IFA test.
men and positive and nega- veals presence of specific im- trols of human origin should
tive control material are munofluorescence of the anti- be tested to verify reagent
placed in walls of Teflon- gen. performance.
coated immunofluorescence 2. No immunofluorescence 2. Care must be taken to mini-
slide. should be observed with neg- mize nonspecific immunoflu-
2. Slides are dried and fixed. ative control material. orescence caused by “trap-
3. The wells are overlaid with 3. Positive and negative patient ping” of the fluorochrome
unlabeled antibodies di- specimens are determined by (e.g., treatment of sputa with
rected against the antigen of comparison of unknowns mucolytic agents when look-
interest. with the immunofluorescence ing for P. carinii by IFA
4. Incubate slides for 15–30 observed in the positive con- test).
min in humidor. trol. 3. Test may not detect antigens
5. Wash and blot slide dry. present in low concentrations
6. Overlay wells with fluoro- or in inappropriately col-
chrome-conjugated anti- lected specimens.
body.
7. Incubate in humidor for 15–
30 min.
8. Wash and blot slide dry.
9. Add mounting medium and
place a coverslip over wells.
10. Read wells for specific im-
munofluorescence under a
fluorescence microscope
with appropriate filter sys-
tem for fluorochrome detec-
tion.
(continued)
11.1.2.7 Immunology

Table 11.1.2–1 Generic description of immunologic assays used in the serologic diagnosis of infectious diseasesa,b (continued)
Test Basic principle Specimen type evaluated Basic materials Basic QC
IFA test for antibody de- This test is performed as An antibody titer can be 1. Teflon-coated immu- 1. Negative, low-titer
tection described for the indi- determined with a sin- nofluorescence slides, positive, and high-titer
rect assay; however, gle serum. To docu- each well of which positive control sera
the first unlabeled anti- ment recent infection, contains the antigen of are included with each
body used in the test is an acute-phase speci- interest (e.g., cells in- test run performed on
that of the patient. Pa- men (collected early in fected with CMV and patient specimens.
tient serum is serially the illness) and a con- expressing CMV-spe- 2. Negative control se-
diluted and placed on a valescent-phase speci- cific antigens) rum is usually tested at
series of immunofluo- men (collected 2–3 2. Patient serum (acute titers of 4 and 16,
rescence slide wells weeks later) are needed and convalescent) while the low- and
containing the same an- to demonstrate an in- 3. Low- and high-titer high-titer positive con-
tigen. After an incuba- crease in titer of anti- positive and negative trols are run at 4 dilu-
tion and wash step, body against the anti- control sera tions, usually 1 two-
fluorochrome-labeled gen being tested. 4. Fluorochrome-labeled fold dilution below
goat anti-human anti- anti-human immuno- and 2 twofold dilu-
body is applied to each globulin raised in an- tions above the ex-
well. After another in- other animal species pected titer (e.g., if the
cubation-wash step, the (e.g., FITC-labeled low positive control
slide is observed with a anti-human immuno- has an expected titer
fluorescence micro- globulins raised in of 64, it should be
scope. The reciprocal goats) tested at titers of 32,
of the highest dilution 5. Humidor for incuba- 64, 128, and 256).
of the patient’s serum tions 3. A conjugate control
that shows specific im- 6. Pipettes using PBS is also in-
munofluorescence is cluded.
called the titer. Titers
may be determined for
both IgG and IgM. In
the latter determination,
the fluorochrome-la-
beled conjugate is di-
rected against human
IgM.
Serologic Diagnosis of Infectious Diseases 11.1.2.8

Basic procedure Expected results Basic limitations Generic examples


1. Prepare twofold serial dilu- 1. No immunofluorescence Rheumatoid factor present in pa- IFA tests are available for the
tions of the control sera and should be observed with ei- tient sera may cause false- detection of antibodies against
patients’ sera. Depending on ther dilution of the negative positive results in tests for several viral agents (e.g.,
individual lab procedures, pa- control or with the PBS con- both IgG and IgM. CMV, HSV, HIV-1) and
tient serum may be screened jugate control. toxoplasma.
at 1 dilution (e.g., 1:16) or at 2. Low- and high-titer control
2 dilutions (e.g., 1:16 and sera should agree with the
1:64). predetermined titers, or nei-
2. Place 0.10 ml of the diluted ther titer should be ⬎ Ⳳ1 di-
control and patient sera on lution off the expected titer.
appropriately labeled wells of 3. If the controls are correct, the
the immunofluorescence antibody titer is defined as
slide. the reciprocal of the highest
3. Incubate slide in humidor for dilution giving characteristic
30 min at 37⬚C. immunofluorescence.
4. Rinse off sera and gently blot
slide dry.
5. Place a drop of fluoro-
chrome-tagged, appropriately
diluted goat anti-human im-
munoglobulin on each of the
slide wells.
6. Incubate in humidor for 30
min at 37⬚C.
7. Rinse slides, and blot dry.
8. Place small drops of buffered
glycerol on each well and
place a coverslip on the slide.
9. Read for specific immunoflu-
orescence under the high-dry
objective of the fluorescence
microscope.

(continued)
11.1.2.9 Immunology

Table 11.1.2–1 Generic description of immunologic assays used in the serologic diagnosis of infectious diseasesa,b (continued)
Test Basic principle Specimen type evaluated Basic materials Basic QC
Immunoblot procedure In this modified EIA, a Serum or plasma 1. Nitrocellulose strips Negative and low- and
mixture of different an- containing electropho- high-titer controls
tigens (e.g., a lysate resed antigen (these should be performed
prepared from a virus may be prepared in- with each test.
growing in culture) is house or purchased
separated into its com- from a vendor if avail-
ponent proteins by mo- able)
lecular weight with 2. Plastic container with
electrophoresis. The troughs for holding
separated components and incubating nitro-
are then electrophoreti- cellulose blot strips
cally transblotted onto 3. Pipettes
a sheet of nitrocellulose 4. Positive and negative
paper. The sheet is cut control sera
into strips that are used 5. HRP-labeled goat anti-
as the solid phase for human antibodies (al-
reaction with patient ternatively, biotiny-
sera. Subsequently, the lated goat anti-human
strip is incubated with conjugate and avidin-
enzyme-labeled anti- HRP can be used in
human immunoglobu- place of the HRP-la-
lin. After wash steps, beled goat anti-human
enzyme substrate is immunoglobulin)
added. The presence of 6. HRP substrate
antibody in patient se-
rum that is reactive
with antigens separated
on the nitrocellulose
strip is indicated by the
appearance of a colored
band at the location of
that particular antigen
on the strip.
Serologic Diagnosis of Infectious Diseases 11.1.2.10

Basic procedure Expected results Basic limitations Generic examples


1. The nitrocellulose strip with The appearance of colored 1. Test must be performed ex- HIV-1 Western immunoblot
the electrophoresed antigens bands on the strip indicates actly as described in the supplemental test for detec-
is placed in an incubation that antibody against that par- package insert. tion of specific anti-HIV-1 an-
trough. ticular antigen was present in 2. Western blotting is as sensi- tibodies
2. A dilution of the serum to be the original serum specimen. tive as and more specific than
tested is placed in the trough standard colorimetric EIA.
along with the antigen strip.
3. After incubation and wash
steps, the strip is incubated
with goat anti-human IgG la-
beled with an enzyme. Alter-
natively, goat anti-human im-
munoglobulin conjugated to
biotin may be used as the
conjugate.
4. After incubation and wash
steps, the enzyme substrate is
added to the trough. The ap-
pearance of colored bands at
the locations of the various
antigens indicates the pres-
ence of antibody to that anti-
gen in the original serum
specimen. If a biotinylated
conjugate is used, then avi-
din-enzyme is added and, af-
ter incubation and wash
steps, the enzyme substrate is
added for color development.

(continued)
11.1.2.11 Immunology

Table 11.1.2–1 Generic description of immunologic assays used in the serologic diagnosis of infectious diseasesa,b (continued)
Test Basic principle Specimen type evaluated Basic materials Basic QC
CF Terminal components of Serum 1. Sheep erythrocyte sus- Known antibody-positive
the complement cas- pension and antibody-negative
cade (C7, C8, C9) 2. Hemolysin (commer- controls; serum control
damage cell mem- cially available) to detect anticomple-
branes in the presence mentary activity; anti-
of specific antibody, gen controls without
which fixes comple- serum to detect anti-
ment to the cell sur- complementary activ-
face. In the CF test, ity; tissue control (the
erythrocytes are used cells or tissue in which
as the target cells and the antigen was pre-
complement-induced pared); buffer control;
“leakiness” can be de- back titration of com-
tected colorimetrically mercial complement to
or visually as an in- ensure use of 5 CH50
crease in free (rather units in each well; cell
than cell-bound) hemo- control to demonstrate
globin. In the presence absence of hemolysis;
of specific antibodies to reference hemolysis
an infectious agent, any standards
complement added to
the test system be-
comes bound, leaving
no residual comple-
ment for reaction with
antibodies to the “indi-
cator” target erythro-
cytes. Therefore, the
presence of specific an-
tibody is indicated by
the absence of hemoly-
sis
Serologic Diagnosis of Infectious Diseases 11.1.2.12

Basic procedure Expected results Basic limitations Generic examples


Sensitization of erythrocytes Highest dilution of antibody All reagents must be free of an- The CF test is considered the
1. Serial dilutions of hemoly- providing 3Ⳮ to 4Ⳮ fixation ticomplementary activity, the reference method for detec-
sin incubated with 2.8% of complement (⬍30% he- correct quantity of comple- tion of antibodies against a
solution of sheep RBCs in molysis) is the endpoint. ment must be present, and wide variety of viral agents
buffer controls must produce ex- and Mycoplasma pneumoniae.
2. As hemolysin concentra- pected reactions. The CF test has been largely
tion increases, degree of supplanted by EIA methodol-
hemolysis increases, to ogy.
achieve a maximal “pla-
teau.”
Quantitation of complement
1. Various amounts of a
1:400 dilution of cold, re-
constituted guinea pig
complement are incubated
with sensitized RBCs.
2. Volume of complement
producing 50% lysis of
RBCs (CH50 unit) is calcu-
lated from graphed results
of the hemolysis.
CF test
1. Block titration of serial di-
lutions of both antigen and
antibody is performed in
presence of 5 CH50 units of
complement at 4⬚C for 16
h.
2. Patient sera are heated at
56⬚C for 30 min to inacti-
vate endogenous comple-
ment.
3. RBCs that have been sen-
sitized with the optimal di-
lution of hemolysin are
added and the mixture is
incubated at 37⬚C for 30
min.
4. Plates are centrifuged to
sediment unlysed erythro-
cytes.
(continued)
11.1.2.13 Immunology

Table 11.1.2–1 Generic description of immunologic assays used in the serologic diagnosis of infectious diseasesa,b (continued)
Test Basic principle Specimen type evaluated Basic materials Basic QC
Hemagglutination inhibi- Some viruses produce Serum or plasma 1. Erythrocytes from ap- Known positive and nega-
tion surface proteins that propriate species (usu- tive sera; back titration
agglutinate erythrocytes ally chicken) collected of antigen to ensure
from various species. in Alsever’s solution that the correct concen-
The presence of anti- or heparin tration of hemaggluti-
bodies to such viruses 2. Diluent of appropriate nating units was tested
in patient serum can be pH
determined by specific 3. Solutions to remove
inhibition of that he- nonspecific agglutinins
magglutination. from serum
4. Standardized viral an-
tigen
Serologic Diagnosis of Infectious Diseases 11.1.2.14

Basic procedure Expected results Basic limitations Generic examples


Determination of hemagglutinat- End point is the last well in Use of test is limited to detec-
ing titer which partial or complete in- tion of antibodies against
1. Diluted suspension of hibition of viral agglutination those viruses that possess sur-
RBCs is incubated at 4⬚C of RBCs occurs. face hemagglutinins (e.g., ru-
or at room temperature bella).
with serial dilutions of vi-
ral antigen until RBCs in
tubes lacking virus settle
as a button.
2. Highest antigen dilution
that produces partial or
complete agglutination
equals 1 HAU.
Treatment of sera
Treat serum by physical
means (kaolin), enzymes
(neuraminidase), etc., to re-
move nonspecific aggluti-
nins.
Inhibition test
Dilutions of antigen contain-
ing 4 HAU are mixed with
erythrocytes and twofold
dilutions of pretreated pa-
tient sera and incubated as
for the determination of
HAU titer.
(continued)
11.1.2.15 Immunology

Table 11.1.2–1 Generic description of immunologic assays used in the serologic diagnosis of infectious diseasesa,b (continued)
Test Basic principle Specimen type evaluated Basic materials Basic QC
Immunodiffusion test Double immunodiffusion Patient serum or plasma Materials listed are those For fungal immunodiffu-
is used to detect anti- for detection of anti- needed for fungal im- sion, control serum
gen or antibody. body. CIE has been munodiffusion. containing the antibod-
Known and unknown used with other body 1. Blastomyces dermatiti- ies being sought is re-
reactants are placed in fluids (e.g., CSF) pri- dis immunodiffusion quired to discern lines
adjoining wells of an marily for the detection antigen of identity that may
agarose matrix and are of bacterial antigens. 2. B. dermatitidis immu- form between the anti-
allowed to passively nodiffusion rabbit anti- gen, the known anti-
diffuse towards one an- sera sera, and the patient’s
other for 12–24 h. A 3. Coccidioides immitis specimen. In addition
precipitin line forms immunodiffusion anti- to homologous testing,
where optimal levels of gen each test serum and an-
antigen and antibody 4. C. immitis immunodif- tigen should also be
are present. Double dif- fusion rabbit antisera tested for immunodif-
fusion is usually used 5. Histoplasma capsula- fusion reactivity with
for the detection of an- tum immunodiffusion heterologous sera and
tibodies. CIE is similar antigen antigens.
to double diffusion, ex- 6. H. capsulatum immu-
cept that the migration nodiffusion rabbit anti-
of one or both reactants sera
is directed by an elec- 7. 1% Purified agar
trical field. (Cells to the 8. Immunodiffusion ma-
right describe materials trix pattern containing
and procedures for fun- a single central well
gal immunodiffusion and 6 peripheral wells
testing.) located equidistant
from the central well
and from adjacent
wells
9. Reading box contain-
ing an incandescent
light source with a flat,
black background
a
Reproduced from W. M. Janda. 1998. Immunology, p. 561–577. In H. D. Isenberg (ed.), Essential Procedures for Clinical Microbiology. ASM Press,
Washington, D.C.
b
Abbreviations: CMV, cytomegalovirus; PBS, phosphate-buffered saline; HIV-1, human immunodeficiency virus type 1; HSV, herpes simplex virus; IgM,
immunoglobulin M; IgG, immunoglobulin G; RSV, respiratory syncytial virus; BCYE, buffered charcoal-yeast extract; IFA, indirect fluorescent antibody;
HRP, horseradish peroxidase; CF, complement fixation; CH50, 50% hemolytic complement; HAU, hemagglutinating unit(s); CIE, countercurrent immuno-
electrophoresis; FITC, fluorescein isothiocyanate.
Serologic Diagnosis of Infectious Diseases 11.1.2.16

Basic procedure Expected results Basic limitations Generic examples


1. Each immunodiffusion grid The location and identity of pre- Low levels of antibodies may Fungal immunodiffusion for de-
can be used to detect anti- cipitin lines should be noted. not be detected by this tection of antibodies to the
bodies against a given anti- Reactions of identity (i.e., method. systemic molds and Aspergil-
gen for four patients. precipitin lines formed by the lus spp.
2. Using B. dermatitidis immu- test reagents which are con-
nodiffusion testing, as an ex- tinuous with precipitin lines
ample, B. dermatitidis anti- formed by reference reagents)
gen is placed in the central indicate that the patient serum
well (1), and reference anti- specimen contains antibodies
sera are placed in wells 2 and directed against the antigen in
3. the central well.
3. Patients’ sera to be tested for
B. dermatitidis antibodies are
placed in wells 4, 5, 6, and 7.

4. Similar plates are set up for


C. immitis and H. capsulatum
antigens and reference anti-
sera. The patient sera are
again placed in the four wells
as stated in step 3 above.
5. Incubate plates at room tem-
perature and read for lines of
precipitation after 24 and 48
h of incubation.
11.1.3 Emerging Immunological Assays
Table 11.1.3–1 Emerging immunological assays using flow cytometrya
Test Basic principle Specimen type
Sub-G0-G1 Apoptosis can be characterized as cells with subdiploid DNA content. These cells can be measured Whole blood, heparinized, is
utilizing PI staining and DNA software. preferred. Isolate PBMCs.

TUNEL The TUNEL method allows rapid phenotypic identification of individual apoptotic cells using flow Blood should be drawn in ACD.
cytometry. Cleavage of the genomic DNA during apoptosis is detected by labeling the free 3⬘ A total of 20 ml is used for
OH termini with fluorescein-labeled nuleotides. The reaction is catalyzed by terminal deoxynu- this assay.
cleotidyl-transferase in a template-independent fashion. The fluoresceinated DNA is then de-
tected on a single-cell level by flow cytometry. Cell phenotype, activation, or maturation state is
established by cell surface labeling with MAb prior to the TUNEL reaction.

Annexin V Apoptosis can be characterized by changes in cell membrane structure. During apoposis, the cell
membrane’s phospholipid asymmetry changes: PS is exposed on the outer membrane while
membrane integrity is maintained. Annexin V specifically binds PS, while PI or 7-AAD is a
DNA binding fluorochrome. When a cell population is exposed to both reagents, apoptotic cells
will stain positive for annexin V and negative for PI or 7-AAD, necrotic cells will stain positive
for both, and live cells will stain negative for both.
Caspase 3 Caspase 3 has been implicated as a key protease that is activated during the early stages of apopto-
sis. The caspases are synthesized as inactive proenzymes that are processed in cells undergoing
apoptosis by self-proteolysis and/or cleavage by another protease. The processed forms consist
of a large heterodimer of 17–21 kDa and a small one at 12 kDa, subunits which derive from the
32-kDa proenzyme. These subunits form an active enzyme. Active caspase 3 proteolytically
cleaves and activates the other caspases, as well as relevant targets in the cytoplasm and nu-
cleus. Caspases trigger a cascade of proteolytic cleavage events within the apoptotic cell, which
include numerous cytoplasm and nuclear proteins as well as other caspase family members.
Chemokines Chemokines are a new subset of cytokines, which participate in the activation of leukocytes and
play a critical role in controlling their movement during inflammation, infectious diseases, tumor
growth, and hematopoietic progenitor cell proliferation. Chemokines are 8 to 10 kDa proteins
sharing 20–70% homology in amino acids. According to the relative positions of their cysteine
residues, chemokines can be subdivided into 4 families, including CXC, CC, C, and CX3C.
Chemokines exert their function through a family of 7 G protein-coupled transmembrane recep-
tors.
DCs DCs are specialized for antigen uptake, processing and presentation to the T cells. DCs are distin- Whole blood collected in either
guished from the antigen-presenting cells, such as B cells and monocytes, by their ability to in- EDTA, ACD, or sodium hepa-
duce a primary immune response, such as activation of immunologically naive T cells. DCs are rin, or PBMCs. Use blood
present in low frequencies in peripheral lymphoid tissue and blood and in nonlymphoid organs. within 24 h of draw.
The two distinct peripheral dendritic subsets can be detected using 3- or 4-color flow cytometry,
CD123Ⳮ and CD11cⳭ HLA-DR lineage (CD3, -14, -16, -19, -20, and -56) dim or negative.

11.1.3.1
Emerging Immunological Assays 11.1.3.2

Basic materials Basic QC Basic procedure Flow analysis Basic limitations


1. PBMCs adjusted to 10 / 6
Set flow
ml. compen-
2. PI in H2O for fresh cells sation.
3. For fixed cells, use RNase
(incubate for 1–2 h at
37⬚C); then add PI in
PBS.
4. DNA analysis software

1. In situ cell death detection Set flow 1. Prepare and adjust 1. Apoptotic-cell number is reported as a DNA cleavage can be ab-
kit compen- PBMCs at 106/ml in cul- percentage of total CD4 and CD8 cells. sent or incomplete in
2. PBS sation. ture medium. 2. Look at dot plot: FSC vs SSC, FL2 vs some forms of apop-
3. FACS permeabilizing so- 2. Culture PBMCs for 24 h SSC, and FL1 vs SSC. Also look at FL1 totic cell death. Steri-
lution at 37⬚C. (TUNEL) histogram of CD4 (FL2) and cal hindrance such as
4. CD4 PE, CD8 cyto- 3. Add 100 ll of cells to CD8 (FL3) cells. extracellular matrix
chrome, CD8 FITC, CD8 tubes with MAb; incubate components can pre-
PE for 30 min at 4⬚C in the vent access of TdT to
5. DNase dark. DNA strand breaks.
6. Culture medium 4. Wash cells, fix with 2%
paraformaldehyde, and in-
cubate for 30 min or
overnight.
5. Follow manufacturer in-
structions for TUNEL
method.
6. Do flow analysis.
1. 10⳯ Binding buffer (BD
catalog no. 66121A)
2. PI
3. Annexin V-FITC (BD cat-
alog no. 65874⳯)
4. 1⳯ PBS

1. For a 3-color assay: line- Set flow Basic immunophenotyping: 1. Gate 1 ⳱ R1 ⳱ all cells minus debris DCs occur with low fre-
age cocktail 1 (lineage 1) compen- 1. 100 ll of whole blood and dead cells quency in peripheral
FITC, which contains sation. 2. Add MAb. 2. Gate 2 ⳱ R2 ⳱ all cells from gate 1 blood. Need to acquire
CD3, -14, -16, -19, -20, 3. Incubate for 15 min. and further selecting cells that are dim at least 50,000 events.
and -56. 4. Lyse for 10 min. and negative for lineage [(CD3, -14, -16,
2. CD123 PE 5. Wash twice. -19, -20, and -56) FITC and HLA-DR
3. HLA-DR PerCp 6. Fix and analyze. PerCp]
4. CD11c PE 3. Gate 3 ⳱ R3 ⳱ gate 1 Ⳮ gate 2. Using
5. Isotypes gate 3, create an HLA-DR/CD11c plot
6. Lyse and an HLA-DR/CD123 plot.
4. Draw regions to define basophiles as
[HLA DRⳮ/CD123Ⳮ] and DCs as [HLA-
DRⳭ/CD123Ⳮ] and [HLA-DRⳭ/
CD11cⳭ].
5. DCs are defined as lineage negative/
HLA-DRⳭ/CD123Ⳮ/CD11cⳭ.
(continued)
11.1.3.3 Immunology

Table 11.1.3–1 Emerging immunological assays using flow cytometrya (continued)


Test Basic principle Specimen type
Tetramer assay This assay is a peptide epitope-specific assay for antigen-specific T cells. Normally this is done to Whole blood, heparinized, is
detect virus-specific (influenza virus, HIV, CMV, EBV, etc.) CD8Ⳮ T cells, but it can be done preferred over PBMCs. Whole
with any antigen. Sample of blood needs to be prescreened for HLA-A2; it must be HLA-A2Ⳮ blood has a lower background,
in order for the assay to work using the tetramer. HLA-A2 forms a tetramer in solution when and the tetramer-positive
bound to a microglobulin and an epitope peptide. Once the class I chain-peptide-microglobulin population has a higher MFI
is ready, it is mixed with fluoresceinated streptavidin, thus forming a tetramer of peptide-loaded in whole blood. PBMC stain-
MHC class I molecules. The dominant epitope of the antigenic peptide is bound to the appropri- ing is adequate, and fresh and
ate class of HLA. Since this mimics how macrophages, B cells, or DCs present antigens, it can frozen PBMCs give similar re-
identify antigen-specific cells. The binding affinity of the complex to the T cell is near that of sults. Staining at room tem-
Ab-Ag complexes and is suitable for flow cytometry. perature is best.
a
Abbreviations: PI, propidium iodide; TUNEL, terminal deoxynucleotidyltransferase-mediated duTP-biotin nick end labeling; MAb, monoclonal antibody;
PS, phosphotidylserine; 7-AAD, 7-aminoactinomycin D; PBS, phosphate-buffered saline; PBMCs, peripheral blood mononuclear cells; FACS, fluorescence-
activated cell sorter; PE, phycoerythrin; FITC, fluorescein isothiocyanate; ACD, acid citrate dextrose; FSC, forward scatter; SSC, side scatter; TdT, terminal
deoxynucleotidyl transferase; DCs, dendritic cells; HIV, human immunodeficiency virus; CMV, cytomegalovirus; EBV, Epstein-Barr virus; MHC, major
histocompatibility complex; MFI, mean fluorescence intensity.
Emerging Immunological Assays 11.1.3.4

Basic materials Basic QC Basic procedure Flow analysis Basic limitations


1. Tetramers labeled with PE Set flow 1. Use MAb to identify a Due to the infrequency of antigen-specific Procedure is MHC class I
(FL2) less background or compen- tetramer-specific popula- cells, a minimum of 150,000 lymphocyte restricted like HLA-
antigen-presenting cells sation. tion included in the CD3 events in a flow cytometer must be col- A2Ⳮ
(FL4) CD8 population. lected to detect an antigen-specific popu-
2. FACS Lysing Solution 2. Gate on CD3Ⳮ lympho- lation of less than 0.1% or higher of T
3. CD8-, CD3-labeled MAb cytes and plot tetramer vs cells. To detect a lower frequency, one
4. 1% Fixative CD8. All tetramer-posi- will need to stain more cells and collect
5. Flow cytometer 4-color tive cells will be CD8Ⳮ. more events.
acquisition/analysis
11.2 Serologic Diagnosis of Group A Streptococcal Infections

11.2.1 Introduction

A number of extracellular antigenic prod- lows infection by nephritogenic serotypes A streptococcal infection, utilizing assays
ucts have been identified in cultures of (M1, M2, M4, M6, M12, M49, and M60). for serum antibodies against one or more
group A streptococci, primarily enzymatic Rheumatic fever follows untreated tonsil- of the antigenic streptococcal extracellular
proteins. These include streptolysin O, litis-pharyngitis. Glomerulonephritis oc- products or cellular components (Table
streptokinase, hyaluronidase, deoxyribon- curs in as many as 10 to 15% of individ- 11.2.1–1). The use of these assays for the
ucleases (DNases A, B, C, and D), and ad- uals involved in outbreaks of impetigo. diagnosis of acute group A streptococcal
enine dinucleotide glycohydrolase. These complications occur after a period infection is rarely indicated. Occasionally,
Infections by group A streptococci are of latency following the infection, during they may be used to confirm that an acute
unique because they can be followed by which the patient is asymptomatic. The la- illness is related to group A streptococcal
the serious nonpurulent complications tency period for acute glomerulonephritis infection, such as in a patient with sus-
acute rheumatic fever and glomerulo- is approximately 10 days, and that for pected scarlet fever. For these patients,
nephritis. Rheumatic fever is associated acute rheumatic fever is about 20 days. acute- and convalescent-phase sera ob-
with infection by certain M serotypes (M3 Serologic tests have been developed to tained 2 to 4 weeks apart can be assayed in
and M18), while glomerulonephritis fol- provide evidence for an antecedent group tandem so as to demonstrate a rise in titer.

TESTS FOR ANTIBODIES TO The anti-streptolysin O (ASO) test was devised by Todd (1) to measure serum
EXTRACELLULAR PRODUCTS OF neutralizing antibody against streptolysin O. Hemolysin activity is measured by the
GROUP A STREPTOCOCCI capacity of the enzyme to lyse erythrocyte membranes, and this activity is neutral-
ized by serum antibody. The ASO test was followed by the development of other
neutralizing-antibody tests for enzymes produced by group A streptococci. The
ASO test remains the best standardized, most reproducible, and most universal. The
use of other tests, such as the antihyaluronidase and antistreptokinase tests, has
declined in part because of difficulties in reproducibility and the limited availability
of some reagents. In contrast to these tests, the anti-DNase B test has gained usage.
DNase B is one of four deoxyribonuclease isozymes (A, B, C, and D) produced by
group A streptococci, and because the B isozyme is produced predominantly by
group A streptococci, the anti-DNase B test is highly specific for recent group A
streptococcal infection. The test is highly reproducible, and reagents are also avail-
able commercially.
Although the ASO test is quite reliable, the ASO response is not universal:
elevated serum ASO titers are found in only about 85% of individuals with acute

Table 11.2.1–1 Tests of antibodies to extracellular products of group A streptococci


Type of test Name Supplier Comment(s)
ASO Bacto-Streptolysin O reagent Difco, Detroit, Mich. Preferred, most accurate
ASO latex test Biotec, Ipswich, Suffolk, United Kingdom Rapid, qualitative
Colorcard ASO Wampole, Cranbury, N.J. Rapid, semiquantitative

Anti-DNase B Streptonase-B Wampole, Cranbury, N.J. Preferred

11.2.1.1
Introduction 11.2.1.2

TESTS FOR ANTIBODIES TO rheumatic fever. While the same holds true for other streptococcal antibody tests,
EXTRACELLULAR PRODUCTS OF a significant proportion of individuals with normal antibody titers for one test have
GROUP A STREPTOCOCCI elevated antibody titers for another test. Thus, the number of false negatives can
(continued)
be reduced by performing two or more antibody tests. In addition, skin infection,
in contrast to throat infection, is associated with a poor ASO response; thus, the
ASO is far less reliable than the anti-DNase B test in cases of postimpetigo nephritis.
The Streptozyme test is an agglutination test that detects antibodies against sev-
eral streptococcal antigens. Five products (streptolysin O, DNase B, streptokinase,
hyaluronidase, and adenine dinucleotide glycohydrolase) are used to coat a sensi-
tized erythrocyte. Antibody against these antigens is tested for by hemagglutination
of the coated erythrocytes on a slide. Discrepancies between the streptozyme test
and the ASO and anti-DNase B tests on the same serum have been reported in
several studies. While the streptozyme test may be used as a screening test, results
should be confirmed by the use of more specific tests.
Serum antibody responses to the extracellular products of group A streptococci
peak 2 to 3 weeks after the acute infection. Because the nonpurulent complications
present 2 to 3 weeks after the acute infection, a single serum sample obtained at
presentation of a patient with acute nephritis or acute rheumatic fever should reveal
elevated ASO and/or anti-DNase B titers in most patients. An upper limit of normal
titer must be established for these tests for proper interpretation.
We recommend that a laboratory establishing procedures for providing evidence
of group A streptococcal infection limit itself to two tests for which reagents are
readily available and which can be depended on to yield reproducible results. Be-
cause interpretation of a single titer depends on the upper limit of normal, it is
advisable to survey a healthy population to establish this value for populations of
different age groups within a geographic region. In addition, serum controls of
known low and high titers should be included in each assay. The ASO and anti-
DNase B tests are the most commonly utilized assays. Details of the performance
and interpretation of these tests are given in procedures 11.2.2 and 11.2.3.

REFERENCE 1. Todd, E. W. 1932. Antigenic streptococcal


hemolysis. J. Exp. Med. 55:267–280.

SUPPLEMENTAL READING Ayoub, E. M. 1982. Streptococcal antibody tests


in rheumatic fever. Clin. Immunol. Newsl. 3:107–
111.
Ferrieri, P. 1986. Immune responses to strepto-
coccal infections, p. 336–346. In N. R. Rose, H.
Friedman, and J. L. Fahey (ed.), Manual of Clini-
cal Laboratory Immunology, 3rd ed. American
Society for Microbiology, Washington, D.C.
11.2.2 Anti-Streptolysin O

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE Many individuals respond to respiratory rated by the bacteria. The presence of
infection by group A (and to a lesser de- ASO activity is indicated by the ability of
gree to group C or G) streptococci with diluted serum to neutralize the hemolytic
antibody to the hemolysin streptolysin O activity of the streptolysin O reagent.
(anti-streptolysin O [ASO]) that is elabo-

II. SPECIMEN COLLECTION Peripheral blood should be collected using aseptic techniques. Blood is stored pref-
erably at 4⬚C prior to serum separation. Storage for over 72 h may lead to hemolysis,
which can result in misinterpretation of the ASO test, as can grossly hemolyzed
serum of other causes. The blood sample should be kept refrigerated, on ice, or at
room temperature, but it should not be frozen. Shipped specimens should have
serum separated prior to shipment.
The serum is clarified by centrifugation of erythrocytes at 500 ⳯ g for 5 min,
and the serum is transferred to a sterile tube or vial for storage. Sera that will be
assayed within a week can be stored at 4⬚C. If longer storage is necessary, the serum
should be stored at ⳮ20 or ⳮ70⬚C. Repeated freezing and thawing should be
avoided.
Protective gloves are used during the handling of the blood samples and sub-
sequently during the dilution of the sera. Proper precautions for the disposal of
blood-contaminated material should be observed.

III. MATERIALS A. Equipment 7. Microtiter plate reading platform


1. Disposable 96-well, round-bottom with mirror (Dynatech Laboratories
microtiter plates (USA Scientific Inc., Alexandria, Va.)
Plastics, Ocala, Fla.) 8. Centrifuge with rotor and carriers
2. Micropipette, 100- to 1,000-ll ca- for microtiter plates
pacity (P1000 Rainin pipette, 9. Conical graduated centrifuge tubes
Rainin Instruments, Woburn, B. Reagents
Mass.) 1. Streptolysin O reagent, 10-ml vial
3. Micropipette, 1- to 100-ll capacity (Difco Laboratories, Detroit,
(P100 Rainin pipette) Mich.; Difco Laboratories, Grand
4. Multichannel pipette, 12 row, 25- Island, N.Y.; Fisher Scientific, Or-
to 100-ll variable capacity (Finn) angebury, N.Y.)
5. Plastic disposable tips for micropi- 2. Citrated fresh type O human eryth-
pettes rocytes (rabbit or sheep RBC, can
6. Disposable test tubes (13 by 100 be substituted)
mm), sterilized

11.2.2.1
Anti-Streptolysin O 11.2.2.2

III. MATERIALS (continued) 3. Two internal control sera of known


antibody titer, one with high titer
and one provided with the strepto-
lysin reagent by the manufacturer
4. ASO buffer concentrate (ns. 2679-
79; Fisher Scientific)

ANALYTICAL CONSIDERATIONS

IV. PROCEDURE The ASO assay can be performed by a macrodilution or a microdilution assay. The
microdilution assay has the advantage of requiring small amounts of serum and
reagents. Accurate micropipetting instruments allow for high test reproducibility.
The microdilution procedure described by Klein et al. (1) was adapted in our lab-
oratory to use a 0.1-log serum dilution scheme. This scheme has the advantage of
determining relatively small increments in antibody titers.
The major precautions for proper test performance include (i) the use of clean
instruments and utensils, (ii) the use of fresh streptolysin O reagent with active
sulfhydryl-reducing compounds to prevent oxidation and inactivation of the en-
zyme, (iii) the use of freshly collected (within 1 week) human type O or rabbit or
sheep erythrocytes, and (iv) the inclusion of proper controls.

V. ERYTHROCYTE SUSPENSION Citrate-treated human type O erythrocytes are collected in 3.8% sodium citrate
(2.5%) dihydrate in a ratio of 1.0 volume of blood to 1.2 volumes of citrate. Commercially
citrate-treated blood bank type O erythrocytes may be used. Pipette 3 to 4 ml of
citrate-treated blood into a 12- to 15-ml conical centrifuge tube. Add 2 to 3 volumes
of physiologic saline (0.9%) to 1 volume of blood. Mix gently by inverting the
tube. Centrifuge at 400 ⳯ g for 5 min. Remove the supernatant, and wash the cells
twice in physiological saline. After the second wash, suspend the cells in physio-
logical saline and centrifuge for 10 min. The supernatant should be clear. Note the
volume of packed erythrocytes, and make a 2.5% suspension by adding 3.9 ml of
phosphate-buffered saline (7.4 g of NaCl, 3.17 g of KH2PO4, and 1.8 g of Na2HPO4
per liter of distilled water; adjust to pH 6.5 to 6.7 with NaOH) to each 0.1 ml of
packed erythrocytes. The cell suspension can be stored at 4⬚C but should be dis-
carded at the first sign of hemolysis.

VI. STREPTOLYSIN O REAGENT The streptolysin O reagent is reconstituted by adding 10 ml of sterile distilled water
until the lyophilized preparation is dissolved. This should be done immediately
prior to use. Store at 2 to 8⬚C.

VII. SERUM DILUTIONS A. Stock dilution


Make a 1:10 serum dilution by adding 50 ll of serum to 450 ll of Bacto
streptolysin O buffer or phosphate-buffered saline described above. Cover this
dilution and inactivate it at 56⬚C for 30 min.
B. Serum dilution in the microtiter plate
Each serum is assigned three vertical columns on the microtiter plate; thus, four
sera can be assayed in each plate. The top row of wells is used for the initial
serum dilutions. We start with initial dilutions of 1:25, 1:30, and 1:40 in the
three top wells, but this can be altered to higher or lower starting dilutions. The
scheme outlined here is a 0.1-log dilution scheme. Top wells are not used in
the assay but only to initiate the dilution process. The bottom row of wells is
used for controls that lack serum or lack streptolysin O reagent.
11.2.2.3 Immunology

VII. SERUM DILUTIONS Make the initial dilutions as follows. To each of the three top wells, add 30
(continued) ll of the stock 1:10 serum dilution. Then add 45 ll of diluent phosphate-buffered
saline to the first of the three wells to obtain a 1:25 dilution, 60 ll of diluent to
the second well to obtain a 1:30 dilution, and 90 ll of diluent to the third well
to obtain a 1:40 dilution. To the remaining wells on the plate, add 50 ll of
dilution buffer. Then make twofold serial dilutions with the multichannel pipette
by transferring vertically 50 ll from the preceding horizontal row of wells to
the following row, mixing gently with the multichannel pipette (three or four
strokes) while avoiding the production of air bubbles, and discarding the 50 ll
removed from the next to last row of wells. Then add 25 ll of the freshly
reconstituted streptolysin O reagent to all wells except those in the top row.
Incubate the plate for 30 min at 37⬚C, followed by addition of 25 ll of the
erythrocyte suspension to all the wells. Reincubate plates at 37⬚C, for 45 min,
with careful shaking (by gently tapping the plate) at 15-min intervals. Centrifuge
the plates on the carriers at 400 ⳯ g for 5 min. Place the plate on the plate
reader, using a fluorescent lamp or daylight as a source of light. Cover the top
of the plate with clear white paper while reading to avoid light distortion.

POSTANALYTICAL CONSIDERATIONS

VIII. READING AND With the plate in the position described above, examine the wells for hemolysis.
INTERPRETATION OF TEST The ASO titer of a serum is equal to the inverse of the highest serum dilution that
inhibits the action of streptolysin, or the serum dilution in the last well that shows
no hemolysis. The titer may be expressed in Todd units if the streptolysin reagent
used was adjusted against the Todd standard serum, as the Bacto reagent is.
A test is considered uninterpretable if the streptolysin O control well (containing
streptolysin O reagent, RBCs, and buffer) does not show total hemolysis or if the
erythrocyte control well (without streptolysin O reagent) shows hemolysis. In ad-
dition, if the reference serum standards yield titers that differ by more than 1 dilution
from the designated titer for that serum, the test should be repeated.
To determine a rise in antibody between acute- and convalescent-phase sera,
both sera should be assayed in tandem on the same plate. A difference of at least
0.2 log unit (two wells if the 0.1-log unit serum dilution scheme is used) between
the titers of the two sera is considered to be a significant difference. When one
serum is assayed, an elevated titer should clearly exceed the upper limit of the
normal range as described above. In our laboratory, the upper limit of normal titer
is 240 for adults and at least 340 for children. An elevated ASO titer or other
streptococcal antibody titer is not diagnostic of acute rheumatic fever but provides
evidence for an antecedent group A streptococcal infection in the modified Jones
criteria (2). Performance of several streptococcal antibody tests may increase the
frequency of finding an elevated antibody titer indicative of a preceding strepto-
coccal infection.
In addition, falsely high titers may be obtained with sera that are contaminated
by certain bacterial organisms during shipment or storage or from and in patients
with liver disease in whom high serum lipoprotein concentrations may mimic an-
tibody activity by inhibiting the lytic action of the streptolysin on the erythrocyte
membrane. Preassay precipitation of these lipoproteins with dextran sulfate will
remove this nonspecific inhibitory activity (3).
Anti-Streptolysin O 11.2.2.4

REFERENCES 1. Klein, G. C., E. C. Hall, C. N. Baker, and 3. Winblad, S. 1966. Studies on non-specific an-
B. V. Addison. 1970. Antistreptolysin O test: tistreptolysin O titre. 1. The influence of serum
comparison of micro and macro techniques. beta-lipoproteins on the non-specific antistrep-
Am. J. Clin. Pathol. 53:159–162. tolysin O titre. Acta Pathol. Microbiol. Scand.
2. Special Writing Group of the Committee on 66:93–104.
Rheumatic Fever, Endocarditis, and Ka-
wasaki Disease of the American Heart As-
sociation. 1992. Guidelines for the diagnosis
of rheumatic fever: Jones criteria, 1992 up-
date. JAMA 268:2069–2073.
11.2.3 Anti-DNase B Test

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE The anti-DNase test evaluates the ability able for the performance of the test; one is
of diluted serum to neutralize the effect of described by Nelson et al. (1), and the
DNase B to digest a DNA substrate com- other is available as a commercial kit from
plexed to a colored dye and thus to block Wampole. The latter method is described
the loss of color expressed by the digested here.
substrate. Two microtechniques are avail-

II. SPECIMEN COLLECTION Specimen collection is as described above for the anti-streptolysin O (ASO) test.

III. MATERIALS A. Reagents B. Supplies


Anti-DNase B test kit (Streptonase-B; 1. Negative control serum (human se-
Wampole, Cranbury, N.J.) rum previously found to be nega-
1. DNase B enzyme reagent (stable tive for anti-DNase B antibodies)
for 3 months at 2 to 8⬚C after open- (stable at 2 to 8⬚C)
ing) 2. Disposable round-bottom 96-well
2. Calf thymus DNA with color indi- microtitier plates
cator (DNase substrate). Use within 3. Serological pipettes, 1.0, 0.05, and
5 days of preparation. 0.025 ml
3. Buffer concentrate (imidazole, cal- 4. Test tubes (10 by 75 mm) and racks
cium chloride, magnesium sulfate, 5. Incubator, 37 Ⳳ 2.0⬚C
and gelatin, containing 0.5% so- 6. Distilled water
dium azide) (stable at 2 to 8⬚C)
4. Anti-DNase B positive control se-
rum, titer of 1:240 (stable at 2 to
8⬚C)

ANALYTICAL CONSIDERATIONS

IV. PROCEDURE A. Preparation of reagents


1. Buffer
Bring buffer concentrate to room temperature. If buffer is lyophilized, mix
with appropriate amount of distilled water (usually 100 ml). Store at 2 to
8⬚C.
2. DNase B enzyme reagent
Prepare a stock solution of DNase B by reconstituting with the correct
amount of buffer (usually 2 ml) according to the manufacturer’s directions.
Stock DNase B is stable for 3 months at 4⬚C. On the day of the test, prepare

11.2.3.1
Anti-DNase B Test 11.2.3.2

IV. PROCEDURE (continued) working DNase B reagent by adding 0.1 ml of the stock to 6.9 ml of buffer.
Store at 2 to 8⬚C.
3. Calf thymus DNA substrate with color indicator
If lyophilized, add appropriate amount of distilled water (usually 12 ml) to
the contents of the vial at least 3 h before use. Allow substrate to dissolve
at room temperature, swirling the vial every 30 min. The dissolved substrate
must be used within 5 days. The substrate can be stored at 4⬚C but must be
at room temperature before use.
4. Positive control serum
Use the positive control supplied with the kit or a past positive specimen
with a known titer. Make an initial 1:10 dilution by adding 0.5 ml of buffer.
The positive control supplied with the kit is already at a 1:10 dilution. Final
dilution is 1:240.
B. Serum dilution and assay method
Make a 1:10 dilution of the patient serum by adding 50 ll of serum to 450 ll
of buffer. Inactivate serum by placing the Parafilm-covered tube in a 65⬚C water
bath for 30 min. Serum dilutions for titration are performed as described above
for the ASO test, except that after the initial dilutions are made, 25 ll of diluent
is added to the remaining wells. Using a 25-ll multichannel pipette, make two-
fold serum dilutions, leaving a 25-ll volume in each well.
Add 25 ll of the appropriate dilution of DNase B enzyme reagent to each
well. Incubate the plates at 37⬚C for 30 min. Remove the plates from the in-
cubator, and add 50 ll of DNA substrate to each well. Cover the plate carefully
with clear sealing tape, using the plate-sealing device. Incubate the plates at
37⬚C for 15 min, gently mix, and reincubate for 4 h at 37⬚C. Remove the plates
from the incubator, and read the endpoint of each serum titration. As with the
ASO test, the two internal control sera are included in the assay. In addition,
appropriate enzyme and substrate controls should be included.

V. READING AND DNase activity on the DNA substrate with color indicator is reflected by digestion
INTERPRETATION of the DNA and depolymerization of the colored complex. This results in a pink
or pinkish-violet color in the solubilized substrate. Thus, the endpoint of antibody
neutralization is reflected in a decrease or loss of the blue or blue-violet color in
the well. The antibody titer is the reciprocal of the highest dilution of serum showing
a bluish color. As with the ASO test, divergence of the control serum titers by more
than 1 dilution requires repetition of the assay. The negative control should read
⬍1:50. The serum control and substrate control wells should show no enzyme
activity (blue), and the enzyme control well must show enzyme activity (pink), for
valid results.

POSTANALYTICAL CONSIDERATIONS

VI. RESULTS AND Stability of the reagents provides a decided advantage in reproducibility of the anti-
CONCLUSIONS DNase B test over that of the ASO test. The DNase B enzyme can be stored for 3
months at 4⬚C without any appreciable loss of activity. In practice, we divide the
concentrated stock solution into aliquots and store them at ⳮ70⬚C. An aliquot is
thawed and diluted in the working buffer to 10 times the working dilution and
stored at 4⬚C for use over a period of about 3 months. Other advantages are that
the calf thymus DNA is available commercially and the substrate (the DNA-color
conjugate) is stable when stored at 4⬚C. This obviates a fresh source of erythrocytes
and the requirement for processing the cells before each ASO test.
11.2.3.3 Immunology

VI. RESULTS AND To ensure the reproducibility and specificity of the assay, certain precautions
CONCLUSIONS (continued) need to be considered. Group A streptococci as well as other streptococcal sero-
groups (B, C, and G) produce DNase isozymes (DNases A, C, and D). Although
the isozymes produced by other streptococci are antigenically distinct from the
group A streptococcal nucleases, assessment of the purity of the DNase B prepa-
ration by the availability of a specific antiserum with known titers for DNase B is
important in the standardization and performance of the test. The use of a pure
DNAse B preparation ensures specificity of the assay for group A streptococcal
infections.
In contrast to ASO titers, anti-DNase B titers are elevated following either strep-
tococcal pharyngitis or pyoderma.

REFERENCE 1. Nelson, J., E. M. Ayoub, and L. W. Wan-


namaker. 1968. Streptococcal antideoxyri-
bonuclease B: microtechnique determination.
J. Lab. Clin. Med. 11:868–873.
11.3 Detection of Legionella Antigen by
Direct Immunofluorescence

Legionnaires’ disease is a type of bacterial acquired Legionnaires’ disease, with L. other than the lungs and pleural space may
pneumonia caused by Legionella spp. (2, pneumophila serogroup 1 being by far the be infected by the bacterium, causing such
13). It is estimated that about 1 to 4% of most common causative agent of the dis- diseases as prosthetic heart valve endocar-
adults with pneumonia requiring hospital- ease. Immunosuppressed patients, and ditis, assorted soft tissue abscesses, and
ization have Legionnaires’ disease and those with nosocomial pneumonia, may systemic infection.
that about 10,000 adults with community- have infections caused by other L. pneu- The Legionella direct immunofluores-
acquired pneumonia have this disease in mophila serogroups and other Legionella cence antibody (DFA) test can be used for
the United States each year. About 5 to spp., in particular L. micdadei, L. longbea- two purposes in the clinical laboratory: (i)
20% of patients with Legionnaires’ dis- chae, L. bozemanii, and L. dumoffii. Dis- detection of the bacteria in clinical speci-
ease die of the disease, with major depen- tribution of the common serogroups and mens and (ii) identification and serotyping
dence on promptness of specific antibiotic species causing infection may be quite dif- of bacterial isolates suspected to be Le-
therapy and underlying health of the pa- ferent in various geographic regions. Le- gionella spp. The first use has fallen out
tient. Legionnaires’ disease occurs world- gionella bacteria are commonly found in of favor in many laboratories because of
wide, in both epidemic and sporadic form, the aqueous environment, including tap its requirement for expertise in interpre-
with sporadic cases being far more com- water and sometimes even distilled water. tation and performance, its low sensitivity,
mon. The disease is more prevalent in The preponderance of L. pneumophila as and under certain circumstances its low
some geographic regions than others, for the cause of Legionnaires’ disease has re- specificity. The DFA test is commonly
unclear reasons. The primary host risk fac- sulted in the development of reagents op- used by many laboratories for rapid and
tors for the disease include immuno- timized for detection of this species; de- relatively accurate confirmation of bacte-
suppression, cigarette smoking, and travel. tection of other Legionella species can be rial identity.
Legionella pneumophila is the cause of problematic, for reasons of both test sen-
more than 90% of cases of community- sitivity and specificity. Rarely, organs

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE A thin layer of sputum, tissue homoge- green color. The presence in clinical spec-
nate, or bacterial suspension is heat fixed imens of fluorescent bacteria of the proper
to a slide, which is then stained with anti- size and shape is diagnostic of Legion-
Legionella antibody conjugated with fluo- naires’ disease, providing that the test has
rescein isothiocyanate (FITC). The slide is been performed correctly. A positive test
then washed to eliminate unbound anti- of a bacterial suspension confirms the
body and examined microscopically using identity of the bacterium as a Legionella
UV light illumination. The FITC fluo- sp., again assuming proper performance of
resces under UV light, which causes the the assay and selection of the correct bac-
Legionella bacteria to appear a bright teria to test.

II. SPECIMEN TYPES, The DFA test has been used to detect the bacterium in a variety of tissues (Table
COLLECTION, AND TRANSPORT 11.3–1). Lower respiratory tract secretions, including expectorated sputum, are the
most frequently tested specimen types. The test can be quite valuable for detecting
Legionella bacteria in premortem or postmortem lung tissue. The bacteria are very
stable in refrigerated or frozen sputum and tissues, and the major determinants of
specimen handling are other requirements for the specimen, such as bacterial cul-

11.3.1
11.3.2 Immunology

Table 11.3–1 Suitable specimen types for L. pneumophila DFA testing


Sputum Vascular grafts
Endotracheal suction Kidney
Bronchoalveolar lavage fluid Liver
Transbronchial biopsy specimen Spleen
Lung biopsy specimen Soft tissue abscesses, including muscle
Pleural biopsy specimen or pleural fluid Brain
Bronchoscopic aspirates Heart valve
Bronchial brushings Pericardium

II. SPECIMEN TYPES, ture. If the specimen will be tested within 1 to 5 days, then store the specimen in
COLLECTION, AND TRANSPORT a tightly sealed sterile container at 3 to 5⬚C. Longer-term storage is best done at
(continued) ⳮ70⬚C. Since Legionella bacteria, and cross-reacting bacteria, are ubiquitous in
water, avoid contamination of the specimen with non-filter-sterilized water. Test
lots of empty specimen containers before buying them for intrinsic contamination,
as on rare occasion, false-positive results may be obtained because of water con-
tamination of the containers before they are sterilized. Preservation of tissues in
10% neutral Formalin obscures the antigen detected by the Bio-Rad monoclonal
antibody to L. pneumophila, making freezing at -70⬚C a preferred alternative for
long-term storage. However, polyclonal antibodies to the individual Legionella ser-
ogroups still react with Formalin-fixed materials, making possible examination of
Formalin-fixed specimens.
Identification of bacteria by DFA testing requires that the bacteria first be sus-
pended in Formalin. The Formalin concentration used depends on the type of an-
tibody used in the DFA test; polyclonal antibodies work correctly with bacteria that
have been suspended in 1 to 10% neutral Formalin, whereas the commercial mono-
clonal antibody works best with 1% Formalin, as higher Formalin concentrations
may cause false-negative reactions with prolonged fixation.

III. MATERIALS All commercial antibody preparations are ever, in many cases they are the only re-
available with the antibody conjugated to agents available for a broad range of spe-
FITC and with few exceptions are rabbit cies, and they are exceptionally useful in
polyclonal antibodies (Table 11.3–2). The serogroup and species identification of
polyclonal antibodies are generally, but culture isolates. Since these reagents have
not always, serogroup specific and may be not had extensive evaluations, unusual re-
available either as polyvalent pools or as sults obtained using them must be con-
monovalent reagents. A monoclonal anti- firmed by a reference laboratory.
body to all L. pneumophila serogroups is Microscope slides made specifically
also available. Many commercially avail- for immunofluorescent microscopy give
able antibodies have not been extensively the best results. The slides are ringed or
evaluated in terms of their analytical or masked by a hydrophobic material, which
clinical specificity and sensitivity. The prevents the antibody from running off the
Bio-Rad monoclonal antibody reagent has slide or onto an adjacent well. The slides
had the most extensive clinical evaluation must be cleaned in alcohol and manufac-
(3). Only Food and Drug Administration tured in such a way that dead Legionella
(FDA)-cleared reagents, and preferably bacteria are not present on the slides. One
the Bio-Rad monoclonal product, should reliable manufacturer is Cel-Line (http://
be used to detect L. pneumophila in clini- www.cel-line.com).
cal specimens.
Evaluations of the non-FDA-cleared
reagents have not been published. How-
DFA for Legionella 11.3.3

Table 11.3–2 Commercial DFA reagents


Manufacturer Antibody type Notes FDA cleared?
Bio-Rad, Monoclonal Reacts with all L. pneumophila Yes
http://www.bio-rad.com serogroups. Formerly known
as Genetic Systems or Sanofi
Pasteur monoclonal antibody
M-Tech, Polyclonal, monovalent, Very wide range of antibodies No
http://www.4m-tech.com and polyvalent available
Trinity Biotech, Polyclonal, polyvalent Four polyvalent reagents are Only the L. pneumophila
http://www.trinitybiotech.com only available: L. pneumophila ser- serogroup 1–6 reagent
ogroups 1–6; L. pneumophila is FDA cleared
serogroups 1–14; and two re-
agents reacting with non-L.
pneumophila spp. Formerly
known as MarDx reagents
and, before that, BioDx.
SciMedx, Polyclonal, polyvalent, L. pneumophila serogroup 1–6 Yes
http://www.scimedx.com and monovalent monovalents and pooled; a
pooled reagent and monova-
lent reagents to non-L. pneu-
mophila spp. Formerly known
as BioDx reagents.
Wampole Laboratories, Polyclonal, monovalent, Individual antibodies to L. pneu- Yes
http://www.wampolelabs.com and polyvalent mophila serogroups 1–6 and
L. micdadei, as well as a sin-
gle pooled antibody. Formerly
known as Zeus reagents.
Remel, Polyclonal, polyvalent Pooled antibody preparation to a Yes
http://www.remelinc.com/ number of species

ANALYTICAL CONSIDERATIONS

IV. QUALITY ASSURANCE Each time the DFA test is performed, positive and negative controls should be
performed. A good negative control is a Formalin suspension of Escherichia coli
ATCC 25922. This preparation is stable for years, if not decades. A positive control
is either one recommended by the manufacturer, or the type strain of the L. pneu-
mophila serogroup, or the Legionella spp., detected by the antibody; type strain
information and the bacteria themselves can be obtained from the ATCC (http://
w.atcc.org). Many of the commercially available kits provide a set of QC strains.
If at all possible, known positive and negative clinical specimens should be used
as an additional QC, especially when training new personnel. Sputum or lung ho-
mogenates can be suspended in Formalin and a small amount can be used for QC
purposes.
In addition to verifying the proper performance of the antibody being used,
specimen containers should be tested by examination of known negative sputum
specimen placed in the containers. Several different lots of new specimen containers
should be tested before wide-scale purchase of the containers. Similarly, although
much less of a problem, plastic ware used to prepare bacterial isolate suspensions
should be similarly tested.
11.3.4 Immunology

IV. QUALITY ASSURANCE At least 75% of all DFA-positive clinical specimens should be culture positive
(continued) for the same serogroup or species. In the case of L. pneumophila serogroup 1 DFA-
positive specimens, the L. pneumophila serogroup 1 urinary antigen test should be
positive at least 90% of the time. Lower culture or urine antigen positivity rates
may indicate a problem with false-positive DFA tests, which are usually due to
operator inexperience or cross-contamination of the specimen during processing.

V. PROCEDURE Instructions for use of the FDA-cleared products are available from the manufac-
turer, but in other cases, few or no instructions are provided. All of the rabbit
polyclonal antibodies use a protocol developed by Cherry and McKinney for the
detection of L. pneumophila serogroup 1 in clinical specimens and from culture,
and the procedure used by any of the manufacturers can be used for any of the
polyclonal antibodies made by other manufacturers (reference 1 and references
therein). The Bio-Rad monoclonal antibody uses a slightly different protocol, which
must be followed exactly to obtain accurate results. Since the CDC protocol may
not be readily available, a modified abbreviated version of it follows.

A. Sample application onto a slide


1. For clinical specimens such as expectorated sputum or lower respiratory tract
specimens, a very thin layer of the sample is smeared onto a Teflon-ringed
or masked slide. The best way to do this is to use a wooden applicator stick
to squeegee the sample onto the slide. Fresh lung specimens are best prepared
by making a thin impression smear directly onto the slide, and Formalin-
fixed lung specimens are best prepared by scraping the lung with a scalpel
and then applying the sample to the slide in a very thin layer. It is very
important to make the material on the slide one cell layer thick, as thick
smears cannot be reliably read using this procedure. Only one clinical spec-
imen should be placed on a slide. All slides must be individually processed
to avoid slide-to-slide cross-contamination. The slide is air dried and then
heat fixed at 50 to 60⬚C for 2 to 10 min on a heating block. Methanol fixation
has not been studied, and is not recommended as a substitute for heat fixation.
Formalin (10% neutral) is added to the slide and the sample is fixed on the
slide for 10 min in a moisture chamber at room temperature; this can be
omitted for previously Formalin-fixed specimens.
2. Bacterial colonies to be tested must first be suspended in 10% neutral For-
malin to approximate the density of a no. 1 MacFarland barium sulfate tur-
bidity standard (⬇3 ⳯ 108 cells/ml). Then the suspension is pipetted onto
the slide and immediately sucked back into the pipette. Emulsifying bacteria
directly onto the slide can cause false-negative results due to a prozone phe-
nomenon. Formalin fixation is not crucial for antigen exposure, so if only a
single very small bacterial colony is available for testing, then the suspension
can be made in sterile water to facilitate subculture and simultaneous DFA
testing; the slide is heat fixed on a heating block to kill the bacteria. Proce-
dures involving the manipulation of live bacterial suspensions must be per-
formed under appropriate biosafety conditions.
B. Slide staining
1. The slides are washed with filter-sterilized distilled water and air dried. It is
important to use filter-sterilized water to avoid false-positive results.
2. Antibody is added to the slide, which is incubated at room temperature in
individual moisture chambers for 15 to 30 min.
DFA for Legionella 11.3.5

V. PROCEDURE (continued) C. Removal of excess antibody


1. Wash the slide with filter-sterilized phosphate-buffered saline (PBS), pH 7.4,
using a syringe. Perform an additional 5-min soak in PBS, which need not
be filter sterilized.
2. Rinse the slides in distilled water, and then air dry.
D. Mounting of coverslips
Alkaline buffered glycerol (Bio-Rad) is applied to the slide, and a coverslip is
mounted. Antifade mounting fluid (Molecular Probes, Eugene, Oreg.) can be
used but is usually not necessary.
E. Slide examination
1. The slide is examined microscopically using a microscope equipped with the
appropriate filters and illumination for FITC microscopy. Optimal total mag-
nification ranges from ⳯200 to ⳯1,000. The lower magnification can be
used when screening culture isolates and by very experienced technicians
when screening clinical samples. Confirmation of positive samples should
be at ⳯500 to ⳯1,000.
2. Clinical specimens containing more than one brightly staining coccobacillus
are considered positive. Some authorities require up to 25 fluorescent bac-
terial cells to be present to designate a sample as positive, and others require
5. However, both of these criteria are too conservative and will result in an
unacceptably high false-negative rate. Only those with months or years of
experience in reading these sorts of slides can reliably use ⱖ2 cells/specimen
as a cutoff, whereas those with less experience are advised to use the higher
cutoff. Positive and negative controls must perform acceptably.
3. Organism suspensions are considered positive when large numbers of
brightly fluorescent bacteria are seen. Rare fluorescent bacteria are consid-
ered negative.

POSTANALYTICAL CONSIDERATIONS

VI. INTERPRETATION Properly performed by experienced laboratorians, the DFA test for detecting L.
pneumophila in clinical specimens is highly specific but only moderately sensitive
(3, 6, 9, 12, 15, 16). With training and experience, the sensitivity of the DFA test
for L. pneumophila in clinical specimens ranges from 50 to 75%, and the specificity
ranges from 94 to 99%. The best performance of the DFA test is with open-lung
biopsy specimens or necropsy specimens; providing that the biopsied specimens
are from areas with acute pneumonia, the sensitivity and specificity of the DFA test
are more than 95 and 99%, respectively, if the antibody being used reacts with the
Legionella species and serogroup causing the infection.
Use of the test by inexperienced laboratorians may result in a very insensitive
and nonspecific test. Several pseudoepidemics of Legionnaires’ disease have been
reported when nonfiltered Formalin, PBS, or water was used in the test or when
there was cross-contamination between clinical specimens and positive controls (8,
10). A number of fluorescent artifacts and cross-reactive bacteria may be present
in clinical specimens, which the expert can discriminate from L. pneumophila by
morphologic appearance. The cross-reactive bacteria are generally gram-positive
coccal bacteria, such as staphylococci and streptococci; gram-positive bacilli, such
as Bacillus spp. and lactobacillus-like bacteria; and Candida-like yeasts. In addition,
there may be cross-reactions with gram-negative bacilli, including Pseudomonas
aeruginosa, Pseudomonas fluorescens, and Bacteroides fragilis; a number of these
cross-reactive bacteria are available from the ATCC, for those interested in devel-
oping new reagents (ATCC strain numbers 49266 to 49269, 43935 to 43937, and
49270 and 49271). Using polyclonal antibodies, even experts may not be able to
11.3.6 Immunology

VI. INTERPRETATION reliably discriminate between cross-reactive B. fragilis, or P. aeruginosa, and L.


(continued) pneumophila bacteria. The Bio-Rad monoclonal antibody does not cross-react with
other gram-negative bacteria, although it may stain some Bacillus spores, or some
gram-positive cocci (7, 14).
More than 60 different Legionella serogroups have been reported, many with
little or no cross-reactivity with one another. This means that the DFA test of clinical
specimens will be negative if the antibody being used does not react with the
Legionella bacterium in the clinical specimen. Fortunately, more than 90% of all
cases of community-acquired Legionnaires’ disease are caused by L. pneumophila,
most by L. pneumophila serogroup 1. However, the sensitivity and specificity of
the DFA test for the detection of other seragroups and species in clinical specimens
have not been established. One polyvalent polyclonal reagent cross-reacted with a
number of non-Legionella bacteria and stained non-Legionella bacteria in clinical
specimens, mirroring the problems observed with some monovalent polyclonal re-
agents (4). Use of multiple polyvalent polyclonal pools to screen clinical specimens
leads to a high false-positive rate (P. Edelstein, unpublished data). Unfortunately,
no genus-specific monoclonal antibody is commercially available. Because of the
problems with cross-reactions observed with polyvalent pools, it is inadvisable to
use them for testing clinical specimens.
One key for proper interpretation of DFA testing of clinical specimens is that L.
pneumophila is a small coccobacillus in clinical specimens, not the long filamentous
rod sometimes found in positive control reagents. This is in contrast to the appear-
ance of the bacterium when isolated from culture, in which case it varies from a
short to a very long filamentous bacillus but is almost never coccobacillary.
Identification of Legionella bacteria isolated from culture with DFA reagents can
be very useful, but it does have some pitfalls. False-negative results can be obtained
if the bacterial suspension put on the slide is too dense (4) or if the serogroup of
the bacterium does not match that of the antibody being used. False-positive tests
can be obtained if the antibody is allowed to dry on the slide during incubation, if
nonfluorescent bacteria are misinterpreted as staining with the antibody, or if the
organism cross-reacts with the antibody being used. If is important to view isolated
bacterial cells to be certain that they are brightly fluorescent, as sometimes bacterial
clumps will be fluorescent because of trapped antibody. Bacteria stained with poly-
clonal antibodies stain very brightly around their periphery but not at all in the
central portion, creating a donut-like appearance. In contrast, the Bio-Rad mono-
clonal antibody stains L. pneumophila bacteria uniformly but less intensely than do
the polyclonal antibodies. Since there can be considerable cross-reactivity between
some serogroups and species, the DFA test may incorrectly assign a Legionella
bacterium to the wrong species or serogroup. Identification of L. pneumophila ser-
ogroup 1 with polyclonal antibodies, and of L. pneumophila with the Bio-Rad
monoclonal antibody, is quite reliable. However, some L. pneumophila serogroup
1 strains may fail to react with the polyclonal antibody made to the Philadelphia
strain but will react with antibody made to the Bellingham or Knoxville strain.
Rarely, a non-Legionella bacterium will stain with the DFA reagent; however, with
the exception of Francisella tularensis, none of these bacteria have a nutritional
requirement for L-cysteine (5, 7, 11). In the case of F. tularensis and several other
cross-reacting bacteria, review of the colony morphology and Gram stain of the
bacterium by an experienced microbiologist quickly discriminates between the
cross-reacting and Legionella bacteria. Molecular methods are sometimes the only
means by which some Legionella species may be differentiated and identified.
DFA for Legionella 11.3.7

REFERENCES 1. Cherry, W. B., B. Pittman, P. P. Harris, G. 9. Ramirez, J. A., and J. T. Summersgill. 1994.
A. Hébert, B. M. Thomason, L. Thacker, Rapid tests for the diagnosis of Legionella in-
and R. E. Weaver. 1978. Detection of Le- fections. J. Ky. Med. Assoc. 92:62–65.
gionnaires disease bacteria by direct immu- 10. Ristagno, R. L., and L. D. Saravolatz. 1985.
nofluorescent staining. J. Clin. Microbiol. A pseudoepidemic of Legionella infections.
8:329–338. Chest 88:466–467.
2. Edelstein, P. H. 1993. Legionnaires’ disease. 11. Roy, T. M., D. Fleming, and W. H. Ander-
Clin. Infect. Dis. 16:741–749. son. 1989. Tularemic pneumonia mimicking
3. Edelstein, P. H., K. B. Beer, J. C. Sturge, A. Legionnaires’ disease with false-positive di-
J. Watson, and L. C. Goldstein. 1985. Clini- rect fluorescent antibody stains for Legionella.
cal utility of a monoclonal direct fluorescent South. Med. J. 82:1429–1431.
reagent specific for Legionella pneumophila: 12. Saravolatz, L. D., G. Russell, and D. Cvit-
comparative study with other reagents. J. Clin. kovich. 1981. Direct immunofluorescence in
Microbiol. 22:419–421. the diagnosis of Legionnaires’ disease. Chest
4. Edelstein, P. H., and M. A. C. Edelstein. 79:566–570.
1989. Evaluation of the Merifluor-Legionella 13. Stout, J. E., and V. L. Yu. 1997. Legionel-
immunofluorescent reagent for identifying and losis. N. Engl. J. Med. 337:682–687.
detecting 21 Legionella species. J. Clin. Mi- 14. Tenover, F. C., P. H. Edelstein, L. C. Gold-
crobiol. 27:2455–2458. stein, J. C. Sturge, and J. J. Plorde. 1986.
5. Edelstein, P. H., R. M. McKinney, R. D. Comparison of cross-staining reactions by
Meyer, M. A. C. Edelstein, C. J. Krause, Pseudomonas spp. and fluorescein-labeled
and S. M. Finegold. 1980. Immunologic di- polyclonal and monoclonal antibodies directed
agnosis of Legionnaires’ disease: cross-reac- against Legionella pneumophila. J. Clin. Mi-
tions with anaerobic and microaerophilic or- crobiol. 23:647–649.
ganisms and infections caused by them. J. 15. Winn, W. C., Jr., W. B. Cherry, R. O.
Infect. Dis. 141:652–655. Frank, C. A. Casey, and C. V. Broome.
6. Edelstein, P. H., R. D. Meyer, and S. M. Fi- 1980. Direct immunofluorescent detection of
negold. 1980. Laboratory diagnosis of Le- Legionella pneumophila in respiratory speci-
gionnaires’ disease. Am. Rev. Respir. Dis. mens. J. Clin. Microbiol. 11:59–64.
121:317–327. 16. Zuravleff, J. J., V. L. Yu, J. W. Shonnard,
7. Flournoy, D. J., K. A. Belobraydic, S. L. Sil- B. K. Davis, and J. D. Rihs. 1983. Diagnosis
berg, C. H. Lawrence, and P. J. Guthrie. of Legionnaires’ disease. An update of labo-
1988. False positive Legionella pneumophila ratory methods with new emphasis on isola-
direct immunofluorescent monoclonal anti- tion by culture. JAMA 250:1981–1985.
body test caused by Bacillus cereus spores.
Diagn. Microbiol. Infect. Dis. 9:123–125.
8. Johnson, D. A., K. F. Wagner, J. Blanks,
and J. Slater. 1985. False-positive direct fluo-
rescent antibody testing for Legionella. (Let-
ter.) JAMA 253:40–41.

APPENDIX 11.3–1 Buffered Glycerin Mounting Medium


I. MATERIALS
A. 250-ml beaker or flask
B. Magnetic stir bar
II. REAGENTS
glycerin, neutral .................................10 ml
NaCO3 ...........................................5.3 g
NaHCO3 .........................................4.2 g
distilled water ................................. 200 ml
III. PREPARATION
A. Add the carbonate and bicarbonate powders to separate flasks, each containing 100
ml of water. Stir to dissolve. This will make 0.5 M solutions of each salt.
B. Mix 4.4 ml of 0.5 M Na2CO3 with 100 ml of 0.5 M NaHCO3. The final pH should
be 9.0; if it is not, adjust by addition of appropriate salt.
C. Add 1 part buffer, pH 9.0, with 9 parts glycerin.
IV. STORAGE
Store in a small-volume tightly stoppered plastic container at room temperature. Discard
if cloudy or if the pH is ⬍9. The shelf life is weeks to months, depending on the amount
of CO2 gas that is absorbed into the buffer.
11.4 Urinary Antigen Detection for
Legionella spp.

Patients with Legionnaires’ disease ex- pneumophila serogroup 1 infections, and mophila serogroups and other Legionella
crete soluble serogroup-specific Legion- they were initially thought capable of de- species will be negative. Tang and col-
ella antigen into their urine. Urinary anti- tecting only this serogroup. It is now leagues developed a polyvalent test for an-
gen tests to detect Legionnaires’ disease known that the L. pneumophila serogroup tigenuria produced by multiple Legionella
were developed by several groups soon af- 1 antigenuria test will detect an unknown spp., but this test has never been commer-
ter the 1976 Philadelphia, Pa., outbreak of fraction of infections caused by other L. cialized (30, 31).
Legionnaires’ disease (3, 32) and then fur- pneumophila serogroups and possibly The Kohler L. pneumophila serogroup
ther refined by Kohler et al. and other some other species (2, 10). Regardless, the 1 assay was first commercialized by
groups (5, 13, 21–24, 27–29). The antigen analytical sensitivity of all commercially DuPont, which sold the assay to Binax.
being detected has never been extensively available test kits is 100- to 1,000-fold Binax subsequently developed an immu-
purified, but it is known that it is resistant greater for the detection of L. pneumophila nochromatographic version of the test,
to boiling, is trypsin resistant, has a mo- serogroup 1 antigen than for other L. pneu- which has had a major impact on the lab-
lecular mass of approximately 10 kDa, and mophila antigens (2, 8, 18). The vast ma- oratory diagnosis of Legionnaires’ dis-
is most likely a lipopolysaccharide (23, jority of positive tests will be for patients ease. Several manufacturers other than
34). with L. pneumophila serogroup 1 infec- Binax now sell a urinary antigen detection
The first urinary antigen tests devel- tions, and most specimens from patients kit, all of them based on a microwell EIA
oped were designed to detect Legionella with infections caused by other L. pneu- method.

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE
Two different methods are used in these detection antibody, and an enzyme sub- is also applied to an inert fibrous support.
tests: EIA antigen capture and immunoch- strate and chromogen are added. If the an- Urine is applied to a spot on the fibrous
romatographic assay. In the EIA method, tigen is present in the specimen, the detec- support, and then a citrate-phosphate buf-
microtiter plates containing antibody to L. tion antibody bound to it will cleave the fer is applied to the same fibrous support.
pneumophila serogroup 1 (or other Le- enzyme substrate, which in turn changes The urinary antigen and the rehydrated de-
gionella species and serogroups, depend- the color of the chromogen. A strong acid tection antibody migrate to the filter areas
ing on the kit) are provided by the manu- is added to stop the reaction, and a signifi- containing the capture antibody and anti-
facturer. Urine is added to the microwells cant color change is detected either visu- rabbit antibody by capillary action. The
and allowed to incubate for a short period ally (for one kit) or by using a microplate detection antibody binds to the antigen
of time, and the urine is then removed by spectrophotometer (for other kits). The while both are being eluted onto the filter
washing with buffer. If L. pneumophila test is interpreted based on the intensity of membrane. The detection antibody-anti-
serogroup 1 antigen is present in the urine, the color change. gen complex binds to the capture antibody
it binds to the antibody in the well. The In the immunochromatographic assay, in an easily visible colored line, as well as
antigen-antibody complex bound to the capture rabbit antibody to L. pneumophila to the anti-rabbit antibody, producing a
well is detected by the addition of the serogroup 1 antigen is bound in a line to second line. If the urine contains no anti-
same antibody as used to capture the an- a nitrocellulose filter. On a second line on gen, then the detection antibody is eluted
tigen. This detection antibody is supplied the filter is bound goat anti-rabbit anti- past the capture antibody, binding only to
already conjugated to an enzyme, such as body. Dried encapsulated detection rabbit the control anti-rabbit antibody, which
peroxidase or alkaline phosphatase. The antibody, conjugated to colored particles, produces only a single line.
well is again washed to removed unbound

11.4.1
Urinary Antigen Detection for Legionella spp. 11.4.2

II. SPECIMEN COLLECTION Only a few milliliters of freshly collected urine is required for the assay. The urine
AND TRANSPORT should be collected in a clean screw-top container and does not require refrigeration
during short (hours) transport periods. For longer transport, refrigeration of the
specimen at 3 to 5⬚C may be beneficial, although this has never been studied.
Addition of boric acid (1% final concentration) preserves the antigen for longer
than a week when stored at room temperature and for up to many years when stored
at 3 to 5⬚C. The use of evacuated tubes containing preservative that are designed
for preserving urine for bacterial culture has not been studied for the transport of
urine used for the antigen test. Freezing the specimen should be avoided if possible,
as freeze-thawing can very rarely cause false-positive results. Long-term (months
to years) storage of urine at ⳮ70⬚C has been reported to rarely cause false-negative
results (6, 26).

III. MATERIALS Four manufacturers sell kits for urinary controls. Some kits require making buffer
antigen detection, three of which are solutions from supplied stock solutions,
cleared for marketing by the Food and wash bottles, or plate washers. All of the
Drug Administration (FDA) (Table 11.4– EIA kits, except the Bartels kit, require the
1). The kit manufacturers provide needed use of a microplate spectrophotometer.

ANALYTICAL CONSIDERATIONS

IV. QUALITY ASSURANCE Proper performance of the negative and positive kit controls gives assurance of
proper test performance. One cost-saving measure for use of the Binax NOW assay
is to perform a negative control test only when there is a positive patient sample
result, since the vast majority of all tests performed are negative. The positive
control should be tested with every test run of the Binax NOW test, or at least once
each day the test is performed. The EIA kits should include positive and negative
controls with every run. Current Clinical Laboratory Improvement Act (CLIA)
regulations and CAP guidelines specify that tests such as the Binax NOW assay
have QC testing performed with every test run, or at least once per day if multiple
runs are performed on the same day. However, the rationale for this is unclear, as
the assay appears to be very stable. My laboratory has never documented a Binax
NOW test kit failure after performing this test for many years. Ascertaining the
correct performance of each lot of kits on arrival in the laboratory should be suf-
ficient, but this currently does not comply with CAP or CLIA guidelines. In contrast,
QC failures with the Binax EIA have been frequent enough to warrant QC testing
with each test run, aside from the fact that test interpretation depends on the results
of the negative control provided in the kit.
An external QC scheme exists for the urinary antigen test in some countries,
including the United States (Medical Laboratory Evaluation, http://

Table 11.4–1 Urinary antigen kits


FDA
Company Test kit name Note(s)
cleared?
Trinity Biotech, http:// Bartels Prima EIA Formerly known as Yes
www.trinitybiotech.com Itracel/Bartels
Binax, http://www.binax.com NOW Legionella Immunochromato- Yes
graphic assay
Biotest, http:// EIA No
www.biotestusa.com
Wampole, http:// Binax Legionella uri- EIA, manufactured by Yes
www.wampolelabs.com nary antigen EIA Binax
11.4.3 Immunology

IV. QUALITY ASSURANCE www.acponline.org/mle/index.html) and European countries (http://


(continued) www.ewgli.org/links.html). These should be subscribed to if available. If they are
not available in a particular country or region, trading blinded specimens with other
regional laboratories can help to detect testing problems not detected with the kit
control reagents.
Since urine antigen testing may be more sensitive than other means of laboratory
diagnosis of Legionnaires’ disease, it may be difficult to use the results of other
laboratory tests for Legionnaires’ disease to determine the proper performance of
the urine test, especially for a single case of the disease. However, the majority of
patients with a positive urine test should have a positive sputum culture or sero-
convert to L. pneumophila serogroup 1. Every effort should be made to confirm the
laboratory diagnosis of the disease, if at all possible. Also, it should be determined
if the patients have an illness compatible with Legionnaires’ disease.

V. PROCEDURE Use of the test kits is straightforward and should be familiar to any laboratorian
experienced with EIAs. As with all immunoassays, particular attention must be paid
to equilibration of reagents to room temperature before use, to use of proper in-
cubation temperatures, to adequate plate washing, and to use of a calibrated spec-
trophotometer. The Binax NOW kit is very simple to use, requiring relatively little
training or prior experience.
Three modifications of the sample preparation procedure for all of the test kits
can enhance test sensitivity and specificity. Test sensitivity can be increased by up
to 30% by concentrating urine prior to testing (9, 11). This is done by concentrating
the urine 25-fold using an Amicon Minicon B15 concentrator. Concentrating the
urine does not appear to affect test specificity. The other modification is to boil
urine for 10 min to inactivate rheumatoid-like factors that can rarely cause false-
positive tests (about 1 to 5% of positive tests). In addition, clarification of urine by
brief low-speed centrifugation can also reduce the frequency of false-positive tests.
Whether and when to perform these specimen modifications are dependent some-
what on the test format being used. All urine samples giving a positive result in the
assay should be retested after clarification and boiling, in parallel with retesting of
the original specimen. Users of an EIA format assay will probably find it most cost-
effective and expeditious to boil and clarify all urine specimens prior to testing, as
repeating the assay requires several hours and can be quite costly.
Immunochromatographic card assay users will probably find it easiest to boil
and clarify only the urine samples giving a positive result, since the vast majority
of urine specimens tested will give negative results and because repeating a test is
quick and relatively inexpensive. Since concentrating urine samples is expensive
because of the cost of the Amicon concentrators, some concentrate only those giving
an equivocal result in an EIA (e.g., a ratio between 2 and 3 in the Binax EIA). No
one has offered guidelines for when to concentrate urine specimens giving a neg-
ative result in the Binax NOW assay. In investigation of an outbreak of Legion-
naires’ disease, it may be wise to concentrate all urine samples tested.

POSTANALYTICAL CONSIDERATIONS

VI. INTERPRETATION The urine antigen tests are very specific and sensitive for the detection of L. pneu-
mophila serogroup 1. The levels of performance of all kits have been roughly
equivalent in published and unpublished studies (http://www.fda.gov/cdrh/pdf/
k991074.pdf) (1, 2, 6, 8–10, 12, 15–18, 28). One recent retrospective comparison
of the Bartels and Binax EIAs showed that the clinical and analytical sensitivity of
the Bartels kit was significantly greater than its comparator if nonconcentrated sam-
Urinary Antigen Detection for Legionella spp. 11.4.4

VI. INTERPRETATION ples were tested and that the tests were roughly equivalent when testing concen-
(continued) trated urine samples (8). The sensitivity of the kits for the detection of L. pneu-
mophila serogroup 1 is estimated to be somewhere between 75 to 95%, depending
on the severity of the illness (1, 23). Test sensitivity is about 80 to 99% for urine
specimens taken from patients with L. pneumophila serogroup 1 pneumonia severe
enough to require hospitalization, especially those with respiratory failure due to
pneumonia. Of those patients with culture-proven L. pneumophila serogroup 1
pneumonia, 95% have positive urine antigen tests, and 100% have positive tests
after urine concentration (1). About 75% of patients with milder disease, especially
those with L. pneumophila serogroup 1 pneumonia not requiring hospitalization,
have a positive test. The test has had its greatest utility in the investigation of
outbreaks of Legionnaires’ disease, providing rapid diagnoses for far more patients
than does culture diagnosis (14, 20, 33).
The specificity of the Binax EIA is extraordinarily high, somewhere in the range
of 99.9%, if urine boiling and clarification are performed for initially positive sam-
ples (P. Edelstein, unpublished data). The specificities of the other test kits appear
to be similar (8, 10, 17). A recent study reported that the specificity of the Binax
NOW assay was only 97% but that this could be enhanced by reincubation of the
test cards for 60 min, after which very faint lines that were false positive became
negative (18). My experience with the NOW assay is that its specificity is ⱖ99%,
using the specimen clarification and boiling protocol detailed above. The specificity
of the Bartels kit may be slightly lower when the test wells are read visually rather
than with a spectrophotometer, leading the authors of the paper to recommend that
visual reading not be used for this test (17). The very high specificity of these assays
means that a patient with a compatible clinical illness has a 75 to 90% chance of
truly having Legionnaires’ disease.
False-negative tests can occur for several different reasons. Patients with severe
renal failure may not concentrate the antigen in the urine, and for those patients
with oliguric renal failure, simply obtaining an adequate urine specimen can be
difficult. Overly dilute urine may mask the presence of a positive test, especially
after a fluid load or administration of diuretics. Concentrating urine or testing an-
other urine specimen may alleviate this problem. Patients with Legionnaires’ dis-
ease caused by a Legionella bacterium other than L. pneumophila serogroup 1 may
have a negative test with any of the kits. It is claimed, but unproven, that the Biotest
kit is more sensitive in this circumstance. In fact the analytical and clinical sensi-
tivities of the Biotest and Binax kits appear to be very similar for non-L. pneumo-
phila serogroup 1 Legionella antigens, although some claim that the Biotest kit is
more sensitive (2, 10, 19). Some patients with very early pneumonia may have a
negative urine assay, a cause for repeating negative tests for patients with pneu-
monia present for less than 2 days (4, 22, 23). Finally, improper test performance
or inherent limitations of the assay may also cause false-negative tests.
False-positive tests are usually the result of presence of rheumatoid-like factors
in the urine, which can be eliminated by boiling the urine. Freeze-thawing of urine
has also been reported to be rarely responsible for false-positive tests (1). A unique
case of a false-positive test was reported for a patient with anti-rabbit antibodies,
after immunotherapy for kidney transplant rejection with rabbit antibody (7). Al-
though not determined by the authors of the report, boiling of the urine should have
eliminated this false-positive reaction. Inherent errors in device performance may
also be responsible for some false-positive tests, although this occurs very infre-
quently. Improper test performance or interpretation of results has been reported to
be the cause of a pseudo-outbreak of Legionnaires’ disease (25). Patients with very
severe pneumonia may excrete detectable urinary antigen for months after recovery
from Legionnaires’ disease (22). Such patients usually have multilobar pneumonia,
accompanied by respiratory failure, and have been hospitalized in intensive care
11.4.5 Immunology

VI. INTERPRETATION units because of the severity of their illness. Such prolonged urinary antigen excre-
(continued) tion is not seen in patients with mild pneumonia (Edelstein, unpublished). The
concentration of urinary antigen excreted in such severe cases of Legionnaires’
disease diminishes fairly quickly, so that if testing both acute- and convalescent-
phase urine samples using a EIA method, it is easy to distinguish the two. However,
for a patient with two episodes of pneumonia spaced weeks to months apart, it may
be difficult to determine if a positive urinary antigen test is due to the first, second,
or both episodes of pneumonia, especially if the first episode was severe and no
semiquantitative antigen assay was performed with the first pneumonia episode.
There is often a good correlation between the apparent concentration of antigen
excreted and the severity of Legionnaires’ disease, at least for the Binax EIA.
Patients with urine samples testing positive with a sample-to-negative ratio greater
than 30 are likely to have severe disease (Edelstein, unpublished).

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Clin. Microbiol. 20:605–607. 1989. Detection of urinary antigens of Legion-
23. Kohler, R. B., S. E. Zimmerman, E. Wilson, ella pneumophila serogroup 12 by broad-spec-
S. D. Allen, P. H. Edelstein, L. J. Wheat, and trum enzyme-linked immunosorbent assay. J.
A. White. 1981. Rapid radioimmunoassay di- Clin. Microbiol. 27:783–784.
agnosis of Legionnaires’ disease: detection 32. Tilton, R. C. 1979. Legionnaires’ disease an-
and partial characterization of urinary antigen. tigen detected by enzyme-linked immunosor-
Ann. Intern. Med. 94:601–605. bent assay. Ann. Intern. Med. 90:697–698.
24. Lebrun, L., C. Tram, F. Lapierre, L. Gran- 33. Wever, P. C., E. P. Yzerman, E. J. Kuijper,
geot-Keros, and J. Pillot. 1983. Detection of P. Speelman, and J. Dankert. 2000. Rapid
Legionella pneumophila antigen by ELISA in diagnosis of Legionnaires’ disease using an
urine or [sic] experimentally infected guinea immunochromatographic assay for Legionella
pigs. Ann. Microbiol. (Paris) 134A:155–161. pneumophila serogroup 1 antigen in urine dur-
25. Regan, C. M., Q. Syed, K. Mutton, and B. ing an outbreak in The Netherlands. J. Clin.
Wiratunga. 2000. A pseudo community out- Microbiol. 38:2738–2739.
break of Legionnaires’ disease on Merseyside: 34. Williams, A., and M. S. Lever. 1995. Char-
implications for investigation of suspected acterisation of Legionella pneumophila anti-
clusters. J. Epidemiol. Community Health gen in urine of guinea pigs and humans with
54:766–769. Legionnaires’ disease. J. Infect. 30:13–16.
11.5 Laboratory Diagnosis of Syphilis

11.5.1 Introduction

Syphilis is a sexually transmitted disease most people receive adequate antimicro- nosis is usually a presumptive one based
that is caused by the organism Treponema bial therapy for some other reason, such on the mother’s serology and lack of his-
pallidum subsp. pallidum. The disease as an upper respiratory infection. Neuro- tory of adequate treatment. If immuno-
goes through several stages if untreated (2, syphilis, which is the most common man- globulin M (IgM) antibodies against T.
13). The primary chancre occurs at the site ifestation of late syphilis, can occur during pallidum can be detected in the neonate’s
of inoculation approximately 3 to 4 weeks any stage of syphilis, including as a late serum, a stronger case can be made for a
(range, 10 to 90 days) after the initial ex- manifestation of the disease, and is char- diagnosis of congenital syphilis since IgM
posure. Treponemes may be visualized in acterized by meningitis, peripheral neu- antibodies do not cross the placenta as IgG
lesion exudates using either dark-field mi- ropathy, meningovascular brain lesions, antibodies do. Treponemes are frequently
croscopy or direct fluorescent antibody for and/or psychiatric illness. Neurosyphilis demonstrable using Steiner stain, DFA-
T. pallidum (DFA-TP). About 7 to 10 days may be asymptomatic, with the only in- TP, or immunohistochemical staining of
after the chancre appears, antibodies to T. dication being elevated levels of WBCs the placenta or umbilical cord of neonates
pallidum are detectable using the routine (⬎5 WBCs per ml) and protein (⬎50 mg with congenital syphilis. The best preven-
serologic tests for syphilis. The chancre per dl) in the CSF. The Veneral Disease tion for congenital syphilis is identifica-
spontaneously heals after 1 to 4 weeks. Research Laboratory (VDRL) CSF test tion and treatment of pregnant women
The symptoms of secondary syphilis ap- may or may not be reactive for persons who have syphilis.
pear about 6 weeks later (range, 2 weeks with asymptomatic neurosyphilis, but the In the United States, several states
to 6 months). All serologic tests are gen- fluorescent treponemal antibody absorp- (Alabama, Alaska, California, Connecti-
erally reactive during secondary syphilis. tion (FTA-ABS) test with CSF should be cut, Georgia, New Jersey, Oklahoma,
The most common symptoms are a gen- nonreactive for those who do not have Pennsylvania, West Virginia) and the Dis-
eralized or localized maculopapular rash syphilis (4). Persons coinfected with T. trict of Columbia still require premarital
that occurs on the palms of the hands and pallidum and human immunodeficiency testing for syphilis (3). Screening for
soles of the feet (palmar plantar rash) or virus (HIV) should be evaluated for neuro- syphilis is carried out in sexually trans-
on the trunk of the body, mucosal mem- syphilis. mitted disease clinics, detention centers,
brane lesions, generalized lymphadenop- Syphilis serologies can be complicated and outreach programs. All blood dona-
athy, and condylomata lata. These symp- by a positive HIV status. For some patients tions are screened for syphilis. Tradition-
toms will resolve without treatment. The who are coinfected, syphilis tests may be ally, the serologic diagnosis of syphilis has
patient then enters a period of latency nonreactive (2), while for others the non- been made using a nontreponemal test to
when there are no symptoms. In about 20 treponemal titers may be higher (10). For screen for the disease and a treponemal
to 25% of individuals, secondary symp- patients with HIV infection only, nontre- test to confirm the results of the nontre-
toms may reoccur during the early part of ponemal tests may be falsely reactive (5), ponemal test. The rationale for this algo-
this latent period. In early latency (⬍1 perhaps due to polyclonal activation of B rithm is that confirmation of original test
year) the results for the serologic tests for lymphocytes. This may also be the cause results should be done using the more spe-
syphilis are reactive. As patients progress of higher nontreponemal test titers in HIV- cific test. The most commonly used non-
into late latency, the nontreponemal tests and syphilis-coinfected individuals. treponemal tests are the rapid plasma re-
may become nonreactive, but the trepo- Syphilis is one of the few diseases that agin (RPR) test or the VDRL test to screen
nemal tests will remain reactive. About can be transmitted from a mother to her and the T. pallidum particle agglutination
65% of persons with untreated syphilis unborn child. Rates of transmission to the test (TP-PA) or FTA-ABS to confirm.
will remain in this stage for life (13). In fetus are dependent on the stage of syphilis Outside the United States, screening is fre-
the remaining 35%, late manifestations of in the mother and when in the pregnancy quently done using a treponemal test such
syphilis will occur. the mother became infected with T. palli- as the T. pallidum hemagglutination assay
Late syphilis can be divided into be- dum (12). Syphilis contracted in utero may followed by a nontreponemal test such as
nign syphilis, cardiovascular syphilis, and result in fetal death, low birth weight, pre- the RPR or VDRL test to distinguish ac-
neurosyphilis. Benign syphilis and cardio- mature delivery, or various symptoms at tive syphilis from latent or late syphilis.
vascular syphilis are rarely seen since birth, or it may be asymptomatic. Diag- This algorithm is being used more in the

11.5.1.1
Introduction 11.5.1.2

Table 11.5.1–1 List of suppliers for syphilis diagnostic tests


Supplier Product(s)
Ampcor Technologies, Inc., Denville, N.J. RPR card test kits and control sera
http://www.ampcordx.com

Arlington Scientific Inc. (ASI), Spring- RPR card test reagents


ville, Utah
http://www.ArlingtonScientific.com

Avanti Polar Lipids, Inc., Alabaster, Ala. VDRL test kits


http://www.avantilipids.com

Baxter Healthcare Corp., Miami, Fla. RPR card test reagents, FITC-labeled rab-
http://www.baxterhealthcare.com bit or human anti-T. pallidum globulin

Beacon Biologicals, Inc., Boca Raton, Fla. TRUST kit


http://www.beaconbiologicals.com

Becton Dickinson Microbiology Systems, RPR card test reagents, FITC-labeled rab-
Cockeysville, Md. bit or human anti-T. pallidum globulin,
http://www.bd.com FTA-ABS antigen

Biologic Products Section, CDC, Atlanta, FITC-labeled monoclonal antibody to T.


Ga. pallidum

Bio-Rad Laboratories, Hercules, Calif. Syphilis-G EIA kits


http://www.bio-rad.com

Cenogenics Corp., Morganville, N.J. VDRL control sera, VDRL test kits
http://www.cenogenics.com

Fisher, Pittsburgh, Pa. RPR card test kit and components, VDRL
http://www.fishersci.com test control sera

Fujirebio America, Fairfield, N.J. TP-PA kit


http://www.fujirebioamerica.com

Hardy Diagnostics, Santa Maria, Calif. Distributor for most reagents


http://www.hardydiagnostics.com

Hemagen Diagnostics, Inc., Columbia, FTA-ABS kits, antigen slides, sorbent


Md.
http://www.hemagen.com

Lee Laboratories, Grayson, Ga VDRL test kit, unheated-serum reagin an-


http://www.leelabs.com tigen, RPR card test kit, FTA-ABS sor-
bent and antigen

New Horizons Diagnostics Corp., Colum- TRUST kits


bia, Md.
http://www.nhdiag.com

Phoenix Bio-Tech Corp., Mississauga, Trep-Chek EIA (IgG and IgM) kit
Ontario, Canada
http://www.phoenixbiotech.com

Pulse Scientific Inc., Burlington, Ontario, TRUST kit


Canada
http://www.worldexport.com/pulse/

Remel Microbiology Products, Lenaxa, RPR card test kit


Kans.
http://www.remel.com
(continued)
11.5.1.3 Immunology

Table 11.5.1–1 List of suppliers for syphilis diagnostic tests (continued)


Supplier Product(s)
SciMedx Corp., Denville, N.J. FTA-ABS and FTA-ABS DS kits and
http://www.scimedx.com components

Sigma Chemical Co., St. Louis, Mo. Trep-Chek (IgG and IgM) EIA kit
http://www.sigma-aldrich.com

Trinity Biotech, Bray, Ireland Syphilis-G EIA kits and FTA-ABS kit
http://www.trinitybiotech.com

ViroStat, Inc., Portland, Maine Polyclonal anti-T. pallidum antibody and


http://www.virostat-inc.com conjugate

Wampole Laboratories, Cranbury, N.J. Syphilis-G EIA kits, FTA-ABS and FTA-
http://www.wampolelabs.com ABS DS kits, RPR card test compo-
nents and kits

Zeus Scientific, Inc., Raritan, N.J. FTA-ABS and FTA-ABS DS kits


http://www.zeusscientific.com

United States, especially in blood banks There are two types of serologic tests munizations, or mechanical damage to the
where the screening is usually done with for syphilis: the nontreponemal tests, cardiovascular system as occurs in intra-
either a modified hemagglutination test or which use a cardiolipin-based antigen, and venous drug use.
an EIA. Unfortunately, by screening with the treponemal tests, which use either dis- Four treponemal tests are in common
a treponemal test, treated cases of syphilis rupted T. pallidum or cloned antigens. use: the FTA-ABS test, FTA-ABS double
will also be detected, since about 85% of Nontreponemal tests, which detect anti- staining test (FTA-ABS DS), Serodia TP-
persons with treated syphilis retain trepo- bodies produced against cardiolipin in re- PA, and EIA. All are available commer-
nemal test reactivity for life. The 15% that sponse to lipids in the outer membrane of cially (Table 11.5.1–1). The TP-PA is
lose treponemal test reactivity are usually the syphilis spirochete or against host car- probably the most commonly used test in
those who had adequately treated primary diolipin, are either microscopic or macro- clinical laboratories in the United States.
syphilis and no history of syphilis (1, 11). scopic flocculation tests. The microscopic The EIA format tests are gaining in pop-
Early syphilis is best diagnosed with an tests, the VDRL test and unheated-serum ularity, especially in situations where large
antigen detection test to demonstrate the reagin (USR) test, require a microscope to numbers of tests are performed. The FTA-
presence of treponemes in the primary read the test results. The USR test uses a ABS, FTA-ABS DS, and TP-PA are used
chancre (ulcer). The most frequently used stabilized VDRL antigen, which allows only for confirmation of nontreponemal
method is dark-field microscopy to detect unheated serum to be used in the test. The test results. The EIA (IgG only) can be
living, motile spirochetes in lesion exu- macroscopic tests, the RPR test and tolu- used for confirmation of nontreponemal
dates (6, 7). This does require a light mi- idine red unheated-serum test (TRUST), test results or as a screening test. The IgM
croscope equipped with a dark-field con- also utilize stabilized VDRL antigen as EIA is used primarily in diagnosing con-
denser. Smears also must be read within well as colored particles which get caught genital syphilis. There is also a Western
minutes of collection to ensure the motility in the flocculation matrix and make the re- blot test commercially available outside
of the spirochetes in the sample (7, 8). Al- action visible to the naked eye. The RPR the United States. It can be used to detect
ternatively, one can use the DFA-TP tech- test uses sized charcoal particles and the either IgG or IgM antibodies. A micro-
nique on an air-dried smear (7). This does TRUST uses an azo red paint pigment. hemagglutination test for treponemal an-
not require living, motile spirochetes but There was an EIA based on VDRL antigen tibodies (MHA-TP) had been available in
does require a monoclonal or polyclonal (Reagin II test), but manufacture of that the United States until the end of 1998,
antibody against T. pallidum conjugated to test has recently been discontinued. It is when it was replaced by the TP-PA. The
fluorescein isothiocyanate (FITC) and a their reactivity to the host cardiolipin, or TP-PA has been shown to be slightly more
properly working fluorescent microscope. as one researcher terms it, “Nontrepone- sensitive in detecting primary syphilis
A modification of the DFA-TP can also be mal-infection associated phospholipid an- than the MHA-TP (9). There is a T. pal-
used to look for T. pallidum in tissues (7). tibodies” (14), that gives the nontrepone- lidum hemagglutination assay available
This modification is especially useful in mal tests their nonspecificity. These outside the United States.
testing for congenital syphilis, with the antibodies may be produced in response to Table 11.5.1–1 lists the manufacturers
umbilical cord being one of the easiest tis- damage by the spirochete, autoimmune of various reagents for syphilis diagnosis
sue samples to obtain. disease, acute viral infection, recent im- and their websites. The tests that are dis-
Introduction 11.5.1.4

cussed below are the DFA-TP, RPR test, the nontreponemal tests have similar pro- test performance meets the laboratory’s
and TP-PA. The RPR test and the TP-PA cedures and measure the same type of an- requirements.
are the most commonly used tests based tibody. However, because they do have 䊓 NOTE: Because of variations in ship-
on the number of laboratories reporting slightly different sensitivities, the tests are ping and storage conditions, the results ob-
proficiency testing results. The procedures not interchangeable and the same test as tained by the manufacturer or by CDC in
described here are not detailed; readers was used for the baseline titer should be the premarket evaluation should not be re-
should refer to the Manual of Tests for used to monitor treatment efficacy. Re- garded as the definitive QC results. The
Syphilis (7) and the manufacturers’ prod- gardless of the test used, it is the respon- end user must determine acceptability of
uct inserts for detailed instructions. All of sibility of the end user to verify that the the reagents.

REFERENCES 1. Augenbraun, M., R. Rolfs, R. Johnson, R. 9. Pope, V., M. B. Fears, W. E. Morrill, A.


Joesoef, V. Pope, and the Syphilis and HIV Castro, and S. E. Kikkert. 2000. Comparison
Study Group. 1998. Treponemal specific tests of the Serodia Treponema pallidum particle
for the serodiagnosis of syphilis. Sex. Transm. agglutination, Captia Syphilis-G, and
Dis. 25:549–552. SpiroTek Reagin II tests with standard test
2. Centers for Disease Control. 1988. Recom- techniques for diagnosis of syphilis. J. Clin.
mendation for diagnosing and treating syphilis Microbiol. 38:2543–2545.
in HIV-infected patients. Morb. Mortal. Wkly. 10. Rolfs, R. T., M. R. Joesoef, E. F. Hender-
Rep. 37:600–602, 607–608. shot, A. M. Rompalo, M. H. Augenbraun,
3. Hough, M. K., and J. A. Poppe. 1988. Sex- M. Chiu, G. Bolan, S. C. Johnson, P.
ually transmitted diseases; a policymaker’s French, E. Steen, J. D. Radolf, and S. Lar-
guide and summary of state laws. National sen for the Syphilis and HIV Study Group.
Conference of State Legislatures, Denver, 1997. A randomized trial of enhanced therapy
Colo. for early syphilis in patients with and without
4. Jaffe, H. W., S. A. Larsen, M. Peters, D. F. human immunodeficiency virus infection. N.
Jove, B. Lopez, and A. L. Schroeter. 1978. Engl. J. Med. 337:307–314.
Tests for treponemal antibody in CSF. Arch. 11. Romanowski, B., R. Sutherland, G. H. Fick,
Intern. Med. 138:252–255. D. Mooney, and E. G. Love. 1991. Serologic
5. Joyanes, P., M. V. Borobio, J. M. Arquez, response to treatment of infectious syphilis.
and E. J. Perea. 1998. The association of Ann. Intern. Med. 114:1005–1009.
false-positive rapid plasma reagin results and 12. Singh, A. E., and B. Romanowski. 1999.
HIV infection. Sex. Transm. Dis. 25:569–571. Syphilis: review with emphasis on clinical, ep-
6. Larsen, S. A., B. E. McGrew, E. F. Hunter, idemiologic, and some biologic features. Clin.
and E. T. Creighton. 1984. Syphilis serology Microbiol. Rev. 12:187–209.
and dark-field microscopy, p. 875–888. In K. 13. Venereal Disease Program. 1968. Syphilis: a
K. Holmes, P.-A. Mårdh, P. F. Sparling, and synopsis. U.S. Department of Health, Educa-
P. J. Wiesner (ed.), Sexually Transmitted Dis- tion and Welfare. National Communicable
eases. McGraw-Hill Book Co., New York, Disease Center, Atlanta, Ga.
N.Y. 14. Wicher, K., H. W. Horowitz, and V.
7. Larsen, S. A., V. Pope, R. E. Johnson, and Wicher. 1999. Laboratory methods of diag-
E. J. Kennedy, Jr. (ed.). 1998. A Manual of nosis of syphilis for the beginning of the third
Tests for Syphilis, 9th ed. American Public millennium. Microbes Infect. 1:1035–1049.
Health Association, Washington, D.C.
8. Larsen, S. A., B. M. Steiner, and A. H. Ru-
dolph. 1998. Laboratory diagnosis and inter-
pretation of tests for syphilis. Clin. Microbiol.
Rev. 8:1–21.
11.5.2 Direct Fluorescent-Antibody Test for
Treponema pallidum

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE
The direct fluorescent-antibody test for area. Polyclonal antibodies can be ab- clonal conjugate cross-reacts with non-
Treponema pallidum (DFA-TP) (1) can be sorbed with nonpathogenic treponemes to pathogenic treponemes, it cannot be used
used in conjunction with dark-field mi- remove cross-reacting antibodies. Ab- on oral or rectal lesions where other spi-
croscopy or in place of it. The test utilizes sorbed polyclonal conjugates and the rochetes are part of the normal microbiota.
either monoclonal antibody or polyclonal monoclonal conjugate can be used for the The FITC-labeled antibody reacts with T.
antibody conjugated to fluorescein iso- same specimens. Unabsorbed polyclonal pallidum antigen present in the samples.
thiocyanate (FITC). The monoclonal con- conjugate can be used on genital ulcer Examination of the smear with a fluores-
jugate is specific for the pathogenic trep- samples with a fair degree of confidence cence microscope reveals fluorescing trep-
onemes and is the conjugate of choice for that the organism identified is a pathogenic onemes with typical morphology.
ulcers that occur in the oral cavity or rectal treponeme. Because the unabsorbed poly-

II. SPECIMEN COLLECTION Body fluids, lesion or ulcer exudates, tissue impressions, or suspensions of tissue
which have been macerated using either scissors to finely mince or a tissue grinder
are appropriate samples for DFA-TP. Samples are collected in the same manner as
for dark-field microscopy (1, 2). Standard precautions should be observed when
collecting samples and preparing slides. Smears are air dried and may be shipped
at ambient temperature to an off-site laboratory for staining. However, slides that
have been stored frozen should be shipped on dry ice.

III. MATERIALS A commercial kit is not available, but the smears made using infected rabbit testic-
conjugates are available commercially ular tissue (1). Negative slides can be
(Table 11.5.1–1). In addition, positive and made using smears of washed nonpatho-
negative control slides are required. Posi- genic Reiter cultures (1). A fluorescent mi-
tive control slides can be made from fluo- croscope in good working order with the
rescent treponemal antibody absorption proper filters for FITC conjugates is also
test (FTA-ABS) antigen or impression required.

ANALYTICAL CONSIDERATIONS

IV. QUALITY CONTROL A. The conjugate must be checked for acceptable performance. The nonreactive
control should be negative at 2 doubling dilutions below the working dilution
specified by the manufacturer. Treponemes in the reactive control should stain
with 3 to 4Ⳮ intensity when used at the working dilution recommended by the
manufacturer.

11.5.2.1
DFA-TP 11.5.2.2

IV. QUALITY CONTROL B. The pH of the mounting medium and phosphate-buffered saline (PBS) must be
(continued) within the acceptable ranges.
C. Positive and negative controls should be included daily.

V. PROCEDURE Prepare smears and allow them to air dry. If the smears will be stained with poly-
clonal antibody, they should be fixed in acetone for 10 min and air dried prior to
staining. If the monoclonal antibody conjugate is going to be used, the slides need
to be fixed with 1 or 2 drops of 100% methanol for 10 s, drained, and allowed to
air dry. Cover each smear with 30 ll of properly diluted conjugate and incubate in
a moist chamber at 35 to 37⬚C for 30 min. Rinse the slides in PBS and then place
on a staining rack, cover with PBS, and allow to sit for 10 min. Rinse off the PBS
with distilled water. Blot the areas outside the smear with bibulous paper to remove
any residual water. Do not allow the smears to completely dry. Add a small amount
of mounting fluid and place a coverslip over the smear. Scan the controls and the
test smear using the 45⳯ or 63⳯ objective and the proper filters for FITC conju-
gates (BG12 or KP490 with K510 or K530 for transmitted light; BG38, K480,
KP490, TK510, or K515 for incident light) on the fluorescent microscope. Confirm
any treponemes by examining the smear with the 100⳯ oil immersion lens. Ensure
that the control slides are satisfactory before interpreting the test results.

POSTANALYTICAL CONSIDERATIONS

VI. INTERPRETATION Observation of treponemes showing 2Ⳮ or greater fluorescence and morphology


typical of pathogenic treponemes (2) is considered a positive test result. The absence
of treponemes is considered a negative test result; however, a negative result does
not rule out syphilis.

VII. LIMITATIONS OF THE A. If polyclonal antibody is used and it is not preabsorbed with nonpathogenic
PROCEDURE treponemes, the test cannot be performed on oral or rectal lesions or ulcers
because of other treponemes that are part of the normal microbiota.
B. If there is precipitate in the conjugate, artifacts may interfere with reading the
results.
C. If the pH of the mounting medium or PBS is incorrect, fluorescence may be
quenched, leading to false-negative results.
D. If smears are too thick, material may wash off the slide, treponemes may not
stain, or there may be too much background fluorescence to see treponemes.
E. Care must be taken to ensure that treponemes from the positive control slide do
not contaminate patient samples.
F. If the lesion is healing, the sample was collected incorrectly, or the sample
contains very few treponemes, one may not be able to demonstrate the presence
of treponemes.
G. The test cannot distinguish the treponemes that cause yaws and pinta from those
that cause syphilis.

REFERENCES 1. Larsen, S. A., V. Pope, R. E. Johnson, and


E. J. Kennedy, Jr. (ed.). 1998. A Manual of
Tests for Syphilis, 9th ed. American Public
Health Association, Washington, D.C.
2. Larsen, S. A., B. M. Steiner, and A. H. Ru-
dolph. 1995. Laboratory diagnosis and inter-
pretation of tests for syphilis. Clin. Microbiol.
Rev. 8:1–21.
11.5.3 Rapid Plasma Reagin Test

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE
The rapid plasma reagin (RPR) 18-mm syphilis. In the test, unheated serum or produced in response to tissue damage
circle card test is a macroscopic nontre- plasma is spread within the confines of a from other causes such as autoimmune
ponemal test intended as a screening test circle on a plastic-coated card. A drop of disease or intravenous drug use. A four-
for syphilis (4). The antigen is Venereal antigen is added, and the card is placed on fold (2 doubling dilutions) decline in titer
Disease Research Laboratory (VDRL) an- a rotator at a specified speed for a specified following syphilis treatment indicates that
tigen that has been stabilized with EDTA length of time. If antibodies are present, treatment has been successful; a fourfold
and has had choline chloride added to the flocculation reaction takes place. The increase indicates either treatment failure
eliminate the need for heat inactivation of RPR test measures immunoglobulin G or reinfection. Failure of the titer to de-
the serum sample. Sized charcoal particles (IgG) and IgM antibodies. These antibod- cline following treatment, in the absence
have been added to make the reaction visi- ies may be in response to lipoproteins or of a rise in titer, does not necessarily in-
ble. Charcoal particles get caught in the cardiolipin of the treponeme itself or to dicate a treatment failure and must be con-
lattice formed by the stabilized VDRL an- host lipoidal antigens exposed by damage sidered along with the clinical presenta-
tigen and antibodies to cardiolipin that are from the infection with Treponema palli- tion of the patient.
present in the blood of someone who has dum. Antilipoidal antibodies may also be

II. SPECIMEN COLLECTION When handling serum or plasma, standard precautions must be observed (3). It is
recommended that persons who handle blood and body fluids be vaccinated against
hepatitis B virus (2).

A. Serum is the preferred sample, especially if testing is going to be delayed for


more than 48 h. However, as the name of the test implies, plasma (with EDTA
anticoagulant) is a suitable sample if testing is to take place soon after the sample
is collected.
B. Blood samples should be collected and processed in such a way as to minimize
hemolysis or contamination. Samples that are too hemolyzed, extremely lipemic,
or contaminated or that contain particulate matter should not be used since these
may interfere with the test.

III. MATERIALS RPR card test reagents, cards, and control B. Diluent
sera are all available commercially (Table A 1:50 dilution of serum nonreactive
11.5.1–1). In addition to the reagents in for syphilis prepared in 0.9% NaCl.
the kits, persons performing the test will This diluent should be used in the
also need the following reagents and sup- quantitative procedure for dilutions of
plies. 1:32 and higher. A calibrated dropper
that delivers 50 ll should be used.
A. 0.9% Sodium chloride (0.9 g of NaCl C. Safety pipetting device with dispos-
added to 100 ml of distilled water) able tip that delivers 50 ll

11.5.3.1
RPR Test 11.5.3.2

III. MATERIALS (continued) D. Rotator, either fixed speed or ad- F. Disposable latex or nitrile gloves,
justable to 100 ⴣ 2 rpm, circum- safety glasses, and protective cloth-
scribing a 3/4-in.-diameter circle ing
E. Discard containers and disinfectants

ANALYTICAL CONSIDERATIONS

IV. QUALITY CONTROL It is the responsibility of the laboratory to adhere to Clinical Laboratory Improve-
ment Amendments of 1988 regulations and guidelines. This includes annual cali-
bration of pipettors; daily monitoring of room, water bath, and refrigerator tem-
peratures; calibration of any thermometers used for water baths, etc.; participation
and satisfactory results in an approved proficiency testing program; and running
and recording controls for new lots of reagents and for daily testing. If the daily
controls do not produce expected results, patient results are not valid and cannot
be reported.
For lot-to-lot QC, 20 sera of graded reactivity, including minimally reactive,
should be tested over a 2-day period using the kit currently in use along with the
new kit. For quantitative testing, 6 serum samples of various titers should be tested
in parallel on two consecutive work days. For a detailed description of the proce-
dure, refer to A Manual of Tests for Syphilis (4).
The revolutions per minute of the rotator, room temperature, and reactivity of
the controls should be determined each day that testing is done. In addition, the
accuracy of the timer used, whether on the rotator or as a separate timer, should be
validated. The needle should be checked each time a new needle is used, when the
needle has been dropped or wiped, or when the control pattern is not met.
If the temperature of the room is too warm or cool, or if the reagents and patient
samples have not reached room temperature, the accuracy of the test will be com-
promised (1). Likewise, if the samples or reagents are contaminated, the results may
be inaccurate.

V. PROCEDURE For complete instructions on the test procedure, refer to the manufacturer’s product
insert or to A Manual of Tests for Syphilis (4).

A. Qualitative test
1. Allow all reagents, controls, and samples to reach room temperature.
2. Place 50 ll of control or patient sample (serum or plasma) onto appropriately
labeled circle of the plastic test card. Using the flat end of the Dispenstir or
a toothpick, spread the sample to fill the entire circle, taking care not to go
outside the circle.
3. Gently shake the antigen-dispensing bottle to resuspend the particles.
4. Holding the dispensing bottle and needle upside down in a vertical position,
dispense several drops to clear the needle of air. Add exactly 1 free-falling
drop to each circle containing either controls or patient sample. Do not mix.
5. Place the card on the rotator and cover with a humidifying cover. Rotate the
card for 8 min at 100 Ⳳ 2 rpm.
6. Remove the card from the rotator. Briefly rotate and tilt the card by hand to
facilitate differentiation between nonreactive, negative rough, and minimally
reactive results.
7. Perform the quantitative test on any samples that had any clumping or rough-
ness.
B. Quantitative test
1. Dilute to endpoint all samples with rough nonreactive and reactive results
in the qualitative test.
11.5.3.3 Immunology

V. PROCEDURE (continued) 2. Place 50 ll of 0.9% saline in circles 2 through 5 in one row of an RPR test
card. Do not spread the saline.
3. Using a safety pipetting device, place 50 ll of patient sample (serum or
plasma) in circle 1 and 50 ll in circle 2. Mix the saline and sample in circle
2 by drawing the mixture up and down eight times in the safety pipette.
Avoid forming bubbles.
4. Transfer 50 ll from circle 2 to circle 3 and mix. Continue making dilutions
through circle 5. Discard 50 ll from circle 5.
5. Using the flat end of the Dispenstir or a toothpick, and starting with circle
5, the highest dilution (1:16), spread the serum dilution to fill the circle.
Using the same Dispenstir or toothpick, repeat for circles 4 (1:8), 3 (1:4),
2 (1:2), and 1 (undiluted).
6. Gently shake the dispensing bottle to resuspend the antigen and charcoal
particles. Hold the dispensing bottle in an inverted vertical position and
dispense 1 or 2 drops to clear the needle of air. Add exactly 1 free-falling
drop to each circle. Do not mix.
7. Place the card on the rotator. Cover with a humidifying cover and rotate at
100 Ⳳ 2 rpm for 8 min.
8. Remove the card from the rotator and gently rotate and tilt the card by hand
to and fro several times to aid in differentiating minimally reactive results
from nonreactive results.
9. If the 1:16 dilution is still reactive, the sample needs to be diluted further,
continuing as follows.
a. Prepare a 1:50 dilution of nonreactive serum in 0.9% saline to be used
for making the 1:32 and high dilutions of the sample to be tested.
b. Prepare a 1:16 dilution of the test sample by adding 0.1 ml of serum to
1.4 ml of 0.9% saline. Mix well.
c. Place 50 ll of the 1:50 nonreactive serum dilution in circles 2 through
5 of the test card.
d. Use a safety pipette to dispense 50 ll of the 1:16 dilution of the test
sample in circles 1 and 2.
10. Using the same pipette tip, make twofold serial dilutions. Complete the test
as described in steps V.B.4 through 9 above. Use a new pipette tip for each
patient sample.
11. At the completion of the day’s tests, remove the needle from the dispensing
vial, rinse with distilled water, and allow to air dry. Do not wipe the needle,
as this removes the silicon coating, which will affect the size of the antigen
drop.
12. Recap the dispensing bottle containing the antigen suspension and refrig-
erate at 2 to 8⬚C. Do not freeze. Antigen stored in the dispensing bottle will
retain its reactivity for 3 months or until the expiration date, whichever
occurs first.

POSTANALYTICAL CONSIDERATIONS

VI. INTERPRETATION A. Qualitative test


1. Read the reaction immediately under a high-intensity incandescent lamp. Do
not allow the reaction to start to dry out, which may lead to misinterpretation
of results.
2. Any serum or plasma sample that has characteristic clumping ranging from
marked and intense (reactive) to slight but definite (minimally to moderately
reactive) is considered reactive.
3. Slight roughness or an absence of clumping is considered nonreactive.
RPR Test 11.5.3.4

Table 11.5.3–1 Reporting quantitative RPR test resultsa


Result with Result at serum dilution of:
Report
undiluted serum (1:1) 1:2 1:4 1:8 1:16
Rm or R N N N N Reactive, undiluted (1:1)
R R N N N Reactive, 2 dilutions or 1:2 or R2
R R R Rm N Reactive, 8 dilutions or 1:8 or R8
a
Abbreviations: N, nonreactive; R, reactive; Rm, reactive minimal.

VI. INTERPRETATION B. Quantative test


(continued) 1. Read the reaction immediately under a high-intensity incandescent lamp as
for the qualitative test. Do not allow the reaction to start to dry out, which
may lead to misinterpretation of results.
2. Report the results of the test as the highest dilution showing any reactivity,
including minimally reactive, as shown in Table 11.5.3–1.

VII. LIMITATIONS OF THE TEST A. False-positive test results can occur in serum or plasma samples from patients
who abuse drugs; who have diseases such as lupus erythematosus, human im-
munodeficiency virus, mononucleosis, malaria, leprosy, viral pneumonia, or
hepatitis; or who have recently received antiviral immunizations. Anecdotally,
aspirin is thought to cause false-positive reactions, but there is no documented
evidence to this effect.
B. Persons with late latent syphilis or late syphilis may have a nonreactive RPR
test. Persons who were treated in the late latent or late stage of syphilis or
persons who have a history of syphilis infections may maintain low levels of
reactivity for life.
C. Prozone reactions may occur in undiluted serum samples. This occurs when
antibody levels are high and flocculation cannot occur because of antibody
excess. These samples will appear either as rough negatives or as weakly re-
active. All samples giving this type of reaction must be quantitated. Addition-
ally, if the clinician suspects syphilis and the nontreponemal test is nonreactive,
it should be quantitated to rule out a prozone reaction.
D. The RPR test cannot be used to test CSF for the diagnosis of neurosyphilis.
Only the VDRL CSF test can be used for this purpose.
E. The test has to be performed with all reagent, serum sample, and room tem-
peratures between 23 and 29⬚C (73 to 85⬚F). Temperatures below this range
will cause false negatives and lower titers, while temperatures above have the
opposite effect (1).

REFERENCES 1. Bossak, H. N., A. Harris, and S. Olansky. 4. Larsen, S. A., V. Pope, R. E. Johnson, and
1955. Effect of room temperature on serologic E. J. Kennedy, Jr. (ed.). 1998. A Manual of
tests for syphilis. Br. J. Vener. Dis. 31:33–36. Tests for Syphilis, 9th ed. American Public
2. Centers for Disease Control. 1987. Update Health Association, Washington, D.C.
on hepatitis B prevention. Morb. Mortal Wkly.
Rep. 36:353–360, 366.
3. Centers for Disease Control. 1988. Update:
universal precautions for prevention of trans-
mission of human immunodeficiency virus,
hepatitis B virus, and other blood-borne path-
ogens in health care settings. Morb. Mortal.
Wkly. Rep. 37:600–602, 607–608.
11.5.4 Serodia Treponema pallidum Particle
Agglutination Test

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE
The Serodia Treponema pallidum particle nontreponemal tests who have signs or mat of particles on the bottom of the mi-
agglutination test (TP-PA) is a treponemal symptoms consistent with late manifesta- crotiter plate well. If no antibodies to T.
test for the serologic detection of antitre- tions of syphilis, or for patients for whom pallidum are present, the particles settle to
ponemal antibodies (3, 4). Partially puri- clinical history indicates that they may the bottom of the well, forming a compact
fied sonicated T. pallidum antigens are have late latent syphilis. As with all trep- button. The unsensitized particle control
used to sensitize gelatin particles. If anti- onemal tests, results of the test remain re- well for each serum sample should also
bodies against syphilis are found in the pa- active for life for most patients even when form a compact button on the bottom of
tient’s serum, an agglutination reaction the patients are adequately treated for the well, indicating an absence of nonspe-
takes place. The TP-PA is intended to be syphilis. cific agglutination.
used as a confirmatory test for reactive re- Serum that contains antibodies to the
sults in a nontreponemal test such as the pathogenic treponemes binds to the anti-
RPR test, for patients with nonreactive gens on the particles, forming a smooth

II. SPECIMEN COLLECTION Serum is the preferred sample, and when handling serum, standard precautions must
be observed (2). It is recommended that persons who handle blood and body fluids
be vaccinated against hepatitis B virus (1). Blood samples should be collected and
processed in such a way as to minimize hemolysis or contamination. Samples that
are too hemolyzed or extremely lipemic or that contain microbial contamination
should not be used since these may interfere with the test.

III. MATERIALS A. Reagents 2. Pipette droppers calibrated to de-


All of the reagents that are required for liver 25 ll for dispensing sample
performance of the test are included in diluent
the kit, which is available commer- 3. 100- and 25-ll automatic safety pi-
cially (Table 11.5.1–1). The user may pette with disposable tips for add-
want to include a minimally reactive ing serum samples and making se-
control that gives 1Ⳮ agglutination for rum dilutions in the microtiter plate
extra QC. Droppers that deliver 25 ll 4. Tray viewer (optional)
for delivery of sensitized and unsen- 5. Automatic vibratory shaker
sitized gelatin particles are included in 6. Disposable latex or nitrile gloves,
the kit. safety glasses, and protective cloth-
B. Additional materials required ing
1. Disposable 96-well microtiter 7. Discard containers and disinfec-
plates with round-bottom wells tants

11.5.4.1
Serodia TP-PA 11.5.4.2

Table 11.5.4–1 Interpretation of agglutination patterns in the TP-PA


Settling pattern of particles Reading Interpretation
Smooth mat of particles covering the entire bottom of the 4Ⳮ Reactive
well; edges may be folded

Smooth mat of particles covering less than the entire bottom 3Ⳮ Reactive
of the well and maybe surrounded by faint ring

Agglutinated particles uniformly covering the bottom of the 2Ⳮ Reactive


well, surrounded by a red circle

Particles forming a large ring with an irregular outer margin 1Ⳮ Reactive


and peripheral agglutination, surrounded by a red circle

Particles concentrated in the shape of a compact ring with Ⳮ/ⳮ Inconclusive


smooth edges and a hole in the center

Particles concentrated in the shape of a button in the center ⳮ Nonreactive


with a smooth round outer margin, with or without a very
small hole in the center

ANALYTICAL CONSIDERATIONS

IV. QUALITY CONTROL A. All new lots of test kits should be tested in parallel with the lot of the kit
currently in use before being placed into use. Results of the two lots should be
comparable. Reactive and nonreactive controls plus 10 serum samples of graded
reactivity (3 reactive, 4 of 1 to 2Ⳮ reactivity, 3 nonreactive) should be used for
the comparison. The reactive and nonreactive controls included with the kit
should be run daily.
B. Automatic pipettors should be calibrated annually.

V. PROCEDURE A. One row of 12 wells is required for the reactive control, four wells are required
for the nonreactive control, and four wells are required for all patient samples.
Place 100 ll of sample diluent in the first well of each control and patient set
of wells. In each additional well per sample or control, add 25 ll of sample
diluent.
B. Make 1:5 dilution of sample by adding 25 ll of sample or control to the first
well and serial twofold dilutions made either through well 4 (1:40 dilution) for
patient samples and the nonreactive control or through well 12 for the reactive
control. Discard 25 ll from the last well.
C. Using the droppers included with the kit, add 25 ll of sensitized particles to
well 4 (final dilution, 1:80) of each patient sample and the nonreactive control
and wells 4 through 12 for the reactive control. Add 25 ll of unsensitized
particles to well 3 of the controls and patient samples.
D. Mix the samples by placing on a vibratory shaker for 30 s. Cover with either
an empty microtiter plate or microplate cover. Let sit for 2 h at room temperature
before reading. The plate may be left overnight to be read the next morning.
E. Read the plate against a white background and read the pattern of agglutination
in each well according to Table 11.5.4–1. Alternatively, a plate viewer can be
used to aid in visualizing the results. Results are read as either reactive or non-
reactive. The unsensitized particle controls should not show any degree of ag-
glutination.
11.5.4.3 Immunology

POSTANALYTICAL CONSIDERATIONS

VI. INTERPRETATION Read the results of agglutination against a white background. Any degree of ag-
glutination (Table 11.5.4–1) in the wells with sensitized particles should be inter-
preted as reactive. Control sera and patient sera must show nonreactive results in
the wells with the unsensitized particles.

A. The titer of the reactive control is read as the last dilution reading 1Ⳮ. The titer
should not vary more than Ⳳ1 doubling dilution from the endpoint titer estab-
lished for the reactive control. The nonreactive control should not have any
agglutination in the 1:80 dilution with sensitized cells. For any sample that
shows agglutination in both the unsensitized and sensitized particles, the man-
ufacturer’s procedure for absorption should be followed. A reactive test result
indicates past or present infection and the test usually remains reactive for life,
even with adequate treatment for syphilis.
B. Any patient sample that shows indeterminate results should be retested or a
second sample should be drawn and tested. Repeated inconclusive results should
be confirmed with another treponemal test, such as the fluorescent treponemal
antibody absorption test.

VII. LIMITATIONS OF THE A. All treponemal tests tend to remain reactive for life for persons who have had
PROCEDURE treponemal infections. Therefore, the TP-PA cannot be used to evaluate re-
sponse to therapy or in determining relapse or reinfection.
B. Tests for syphilis cannot distinguish syphilis from yaws or pinta or endemic
syphilis and may be reactive for persons from areas where these other diseases
have been endemic.
C. False-positive reactions may occur in association with other underlying illnesses
or conditions such as human immunodeficiency virus infection, rheumatic fever,
leprosy, autoimmune diseases, Lyme disease, drug addiction, leprosy, toxo-
plasmosis, or Helicobacter pylori infection.
D. There is a false-positive rate in healthy persons of about 1%. These false-posi-
tive reactions are frequently transient and a second serum sample will be non-
reactive.
E. Nonspecific agglutination, which rarely occurs in the test, may not be resolved
by retesting absorbed serum against the sensitized and unsensitized particles.

REFERENCES 1. Centers for Disease Control. 1987. Update 4. Pope, V., M. B. Fears, W. E. Morrill, A.
on hepatitis B prevention. Morb. Mortal. Wkly. Castro, and S. E. Kikkert. 2000. Comparison
Rep. 36:353–360, 366. of the Serodia Treponema pallidum particle
2. Centers for Disease Control. 1988. Update: agglutination, Captia Syphilis-G, and
universal precautions for prevention of trans- SpiroTek Reagin II tests with standard test
mission of human immunodeficiency virus, techniques for diagnosis of syphilis. J. Clin.
hepatitis B virus, and other blood-borne path- Microbiol. 38:2543–2545.
ogens in health care settings. Morb. Mortal.
Wkly. Rep. 37:377–383, 387–388.
3. Larsen, S. A., V. Pope, R. E. Johnson, and
E. J. Kennedy, Jr. (ed). 1998. A Manual of
Tests for Syphilis, 9th ed. American Public
Health Association, Washington, D.C.
11.6 Detection of Borrelia burgdorferi
Antibodies

Lyme disease is a multisystem disease thema migrans (EM) and arthritis, B. gar- direct means such as antibody detection
caused by Borrelia burgdorferi and trans- inii tends to cause more neurological man- methods.
mitted through the bite of infected Ixodes ifestations and B. afzelii tends to cause Several immunodominant antigens are
scapularis ticks (45). It is currently the chronic skin infections (48). Recent stud- present in B. burgdorferi. They include
most frequent vector-borne infectious dis- ies have also suggested that genetic vari- OspC (23 kDa), flagellin (41 kDa), and
ease in the United States, with the highest ability among B. burgdorferi strains iso- Borrelia membrane protein A (BmpA) (39
incidence in the northeastern and mid- lated from skin lesions of patients in North kDa) in early disease. As the disease pro-
western states (13). Lyme disease is also America may explain the different clinical gresses, several other antigens become im-
prevalent in Europe, where evidence of the manifestations observed in early disease munoreactive (2, 3, 16, 17, 20). Tremen-
disease existed in the early 1900s and (49). dous knowledge has been gathered in
where it is transmitted by Ixodes ricinus Lyme disease usually occurs a few testing for B. burgdorferi antibodies since
complex. days to weeks after the bite of an infected the original assays were developed. First-
B. burgdorferi is a motile spirochete tick and presents in about 80% of individ- generation immunoassays used organisms
with a complex antigenic composition. It uals with a slowly expanding erythema- highly passaged in culture that are cur-
contains a linear chromosome and several tous skin lesion-denominated EM (45) rently known to be devoid of OspC (14,
linear and circular plasmids that encode (Table 11.6–1). 15). Immunoglobulin (IgM) antibodies to
outer surface proteins (OspA through The diagnosis of Lyme disease relies B. burgdorferi appear within 2 weeks of
OspF). Expression of these outer surface on clinical recognition of the EM lesion infection and may persist for prolonged
proteins varies depending on environmen- during early stages of the disease. When periods of time after antimicrobial treat-
tal conditions allowing the organisms to EM is absent, unrecognized, or atypical, ment (3, 19). IgG antibodies also appear
adapt to different conditions. OspA is other diagnostic modalities are needed. within weeks of infection but reach their
preferentially expressed at 25⬚C in the un- Laboratory tests aimed at detecting the peak months after untreated early infec-
fed tick midgut, making it the target of causative agent involve culture, PCR, or tion.
choice for vaccine development (43, 44). microscopy. Unfortunately, these methods Since antigens such as flagellin and
OspC is expressed at 37⬚C after a blood are most sensitive in detecting B. burg- high-molecular-weight antigens, includ-
meal has been obtained by the feeding tick dorferi from EM lesions, for which labo- ing heat shock proteins, are also present in
and in the mammal host after transmission ratory tests are theoretically unnecessary other bacteria, antibody tests using whole-
has occurred (39, 40). This switch in ex- (41). B. burgdorferi can be cultured in organism preparations may detect cross-
pression of outer surface proteins is im- Barbour-Stoenner-Kelly medium, and reactive antibodies (18). The facts that
portant to understand the development of growth detection can take several weeks available antibody assays have not been
antibodies in humans as is described be- of incubation (7). Increased recovery from standardized and that most assays are con-
low. B. burgdorferi is composed of several blood specimens has recently been ob- ducted in populations with low probability
genospecies (genogroups) that cause dif- tained for patients with early disease by for Lyme disease (32) have contributed to
ferent manifestations of the disease. The culturing large volumes of blood (50, 51). misdiagnosis of B. burgdorferi infection
broad group is denominated B. burgdor- PCR has been most useful with synovial and to the belief that serologic assays for
feri sensu lato, which contains the geno- fluid of patients with late disease and can B. burgdorferi are unreliable.
species B. burgdorferi sensu stricto (geno- be used to determine eradication of infec- In 1994 the CDC, in conjunction with
group 1), Borrelia garinii (genogroup 2), tion after treatment (35). The use of a urine the Association of State and Territorial
and Borrelia afzelii (genogroup 3). B. test to detect B. burgdorferi antigens has Public Health Laboratory Directors
burgdorferi sensu stricto most frequently been questioned in a recent evaluation of (ASTPHLD), the Food and Drug Admin-
causes Lyme disease in the United States, its performance (24). Due to the lack of istration (FDA), the NIH, the Council of
while B. garinii and B. afzelii cause dis- sensitivity and the cumbersome nature of State and Territorial Epidemiologists, and
ease in Europe. While B. burgdorferi the aforementioned tests, the laboratory the NCCLS, cosponsored a Conference on
sensu stricto causes most frequently ery- confirmation of Lyme disease relies on in- the Serological Diagnosis of Lyme Dis-

11.6.1
11.6.2 Immunology

ease. Among the recommendations was have included specific antibodies bound to pected to react in first-step serological as-
that a two-step testing approach was immune complexes, reported to be detect- says that use whole borrelial cells as an-
needed for active disease and for a previ- able in patients found to be seronegative tigens, since they contain OspA antigens
ous infection using a sensitive EIA or in- by standard assays (9, 38). Like the func- but could be distinguished by Western im-
direct immunofluorescent-antibody assay tional borreliacidal-antibody assays, the munoblots (4). A few assays using prep-
(IFA) as a first step, followed by Western immune complex antibody assays are still arations of antigens from B. burgdorferi
immunobloting (12). The first step should restricted to a few research laboratories. strains lacking OspA have been developed
detect IgG and IgM antibodies to B. burg- Between 1994 and 1997 two recombi- but are not currently FDA approved (52).
dorferi and when reactive should be fol- nant OspA vaccines to prevent Lyme dis- Since Lyme disease in Europe is
lowed by separate IgG and IgM immu- ease were evaluated in the United States, caused by different B. burgdorferi geno-
noblot tests. and one such preparation was until re- groups and consensus on serology is being
Besides the matrix-based assays, func- cently available for use. Their efficacy in developed (22, 23, 37), the discussion on
tional antibody assays have also been used disease prevention ranges between 76 and methods that follows is restricted to detec-
(1, 11). Although these assays are more 92% after three doses of vaccine (43, 44). tion of B. burgdorferi antibodies in the
cumbersome, they seem to correlate better Sera of vaccinated individuals are ex- United States.
with disease activity. Other types of assays

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE A. First-step testing servatives, and, in some formats, other


More than 40 commercial assays have bacterial antigens to block nonspecific
been cleared by the FDA, and the ma- reactions. Several washing steps re-
jority of them are ELISAs or modifi- move unbound antibodies. Bound an-
cations of EIAs. Since IFAs are less tibodies are detected after the addition
frequently used, as a first-step testing of enzyme-conjugated anti-human im-
the following description is restricted munoglobulins (IgG, IgM, and IgA,
to ELISA or EIA (Table 11.6–2). alone or in combination). After re-
In ELISA, serum is tested for the moval of unbound conjugate by sev-
presence of antibodies reacting with eral washes, a solution containing the
whole-cell sonicates of low-passage B. substrate for the bound enzyme-con-
burgdorferi antigens immobilized to a jugate is added. Color reaction (or
solid surface. Variations to this antigen fluorescence for Vidas) is read with a
preparation are the addition of recom- spectrophotometer (or luminometer)
binant proteins (p39) or purified anti- and absorbance is converted to a nu-
gens and the use of synthetic C6 pep- merical value and translated to cate-
tide, derived from the VlsE protein of gories of reactivity (negative, border-
B. burgdorferi (25). The serum speci- line/equivocal, and positive) based on
men is diluted (1:20 to 1:400) in a dil- a ratio of patient specimen absorbance
uent containing buffer solutions, pre- over a calibrator absorbance.

Table 11.6–1 Lyme disease stages


Early disease: Few days to few weeks after the bite of an infected I. scapularis tick (I.
ricinus in Europe)
Early localized: EM at the site of the tick bite
Early disseminated: EM, accompanied by fever, influenza-like symptoms, headache,
multiple skin lesions
Neurologic manifestations (15%): meningitis, cranial nerve palsies,
motor or sensory radiculoneuritis
Cardiac manifestations (5%): atrioventricular blocks, myopericardi-
tis
Late disease: months after onset of illness
Arthritis: about 60% of untreated patients develop intermittent attacks of arthritis
Chronic neuroborreliosis in up to 5% of untreated patients: chronic axonal neuropathy,
chronic encephalomyelitis (caused by B. garinii in Europe), chronic encephalopathy
(cognitive disturbances)
ACAa chronic skin lesion caused by B. afzelii in Europe
a
ACA, achrodermatitis chronica atrophicans.
B. burgdorferi Serology 11.6.3

Table 11.6–2 B. burgdorferi first-step antibody assaysa


Manufacturer/ Immunoglobulin(s)
Kit name Antigen(s) used Note(s)
distributor detected
bioMérieux Vidas Lyme IgG and IgM Whole borrelial lysate IgG, IgM Automated enzyme-linked
fluorescence assay

Bio-Rad Anti-Borrelia (Lyme) micro- Whole borrelial lysate plus re- IgG, IgA, IgM E. coli proteins used to ab-
plate EIA combinant P39 sorb cross-reactive anti-
bodies

Diamedix Immunosimplicity anti-Bor- Whole borrelial lysate IgM


relia burgdorferi IgM test
kit
Immunosimplicity anti-Bor- IgG, IgM
relia burgdorferi IgG/IgM
test kit

Focus Lyme disease IFA IgG Fixed B. burgdorferi IgG Indirect IFA
Technologies Lyme disease IFA IgM IgM

GenBio Borrelia Dot G test Whole borrelial lysates, re- IgG Antigens applied in a dot
Borrelia Dot M test combinant high-molecular- IgM format on a strip
weight antigen, purified fla-
gellin, recombinant P39, and
recombinant OspC

Immunetics C6 B. burgdorferi (Lyme) C6 synthetic peptide derived IgG, IgM Peptide sequence highly con-
ELISA kit from V1sE protein of B. served among all genospe-
burgdorferi cies of B. burgdorferi

MarDx/Trinity B. burgdorferi EIA (IgG, Whole borrelial lysate IgG, IgM


Biotech IgM) test system
IgM IgM
IgG IgG

Meridian Premier Human Lyme Whole borrelial lysate IgG, IgM

Sigma Borrelia burgdorferi IgG/ Whole borrelial lysate IgG, IgM


IgM

Remel Rapidot Lyme disease test Whole borrelial lysate spotted IgG, IgM Rapid assay on test modules.
onto membranes Amenable for physician
offices.

Immunowell Borrelia Whole borrelial lysate with ad- IgG, IgA, IgM Use of Escherichia coli pro-
dition of recombinant P39 teins to absorb cross-reac-
tive antibodies

Immunodot Borrelia Whole borrelial lysate plus re- IgG, IgA, IgM Antigens applied in dot for-
combinant P39; purified fla- mat on a strip. E. coli is
gellin used as absorbant.

Wampole Borrelia burgdorferi IgG/ Whole borrelial lysate IgG, IgM


IgM ELISA

Wampole PreVue Recombinant OspA, OspB, IgG, IgM Immunochromatographic test


OspC, p93, and flagellin performed in test devices.
Use serum or whole blood.

Zeus Borrelia burgdorferi ELISA Whole borrelial lysate IgG Ⳮ IgM, IgG,
test system IgM
a
List of some of the commercially available tests.
11.6.4 Immunology

I. PRINCIPLE (continued) B. Second-step testing: Western blot- removed by washing steps. Enzyme-
ting conjugated anti-human IgG and IgM
Specimens testing borderline or posi- are reacted with the two separate strips,
tive by the first step are assayed in and unbound conjugate is removed by
Western blots (immunoblots) where B. washing. After the addition of a sub-
burgdorferi antigens have been sepa- strate for the bound enzyme, colored
rated electrophoretically by size and bands representing reactivity to the dif-
transferred (blotted) to membranes. ferent immobilized antigens will de-
Diluted serum specimens are tested in velop. Band recording is visually
separate assays for IgG and IgM anti- determined by comparison to the reac-
bodies to the different antigenic com- tivity of a band locator control and of
ponents bound to the membranes, usu- a weak intensity control.
ally in strips. Unbound antibodies are

II. SPECIMEN COLLECTION The most appropriate specimen for testing is serum. Lipemic or hemolyzed speci-
mens should be avoided. Once serum has been separated from whole blood, it
should be stored refrigerated (4 to 8⬚C) for several days or frozen (ⳮ20 to ⳮ70⬚C)
if testing is delayed beyond a few weeks or for long-term storage.
Since development of antibodies requires a few days to weeks after infection, it
is often necessary to test acute- and convalescent-phase sera. The best results are
accomplished when both specimens are tested in parallel in the same assay under
the same conditions. Testing of other types of specimens, such as synovial fluid or
CSF, has not yet been standardized.

III. MATERIALS As summarized in Table 11.6–2, many netics released an ELISA using the C6
formats are currently available for first- peptide, a synthetic peptide derived
step testing for B. burgdorferi antibodies. from the VlsE antigen of B. burgdor-
feri (27). Recombinant antigens have
A. Antigens been evaluated in Europe as well as by
Most commercial first-step assays in several investigators in the United
the United States use whole-cell soni- States, but few are commercially avail-
cates of low-passage B. burgdorferi able (10, 21, 29, 30, 36).
B31, the first isolate of B. burgdorferi Western blot assays commercially
sensu stricto cultured from an I. sca- available at this time also use B. burg-
pularis tick. Several manufacturers dorferi strain B31, except that of Focus
have added the recombinant p39 anti- Technologies, formerly MRL, which
gen to the whole-cell antigen prepa- uses strain CB (a genogroup 1 B. burg-
ration. Gen-Bio and Alexon have pro- dorferi sensu stricto human isolate)
duced dot EIAs using recombinant and (Table 11.6–3).
purified antigens, and recently Immu-

Table 11.6–3 Commercially available Western immunoblots


B. burgdorferi
Manufacturer Kit name
strain used
Focus Technologies Lyme disease B. burgdorferi Geno- CB
http://www.focusanswers.com group 1 Western Blot IgG
(800) 838-4548 Lyme disease B. burgdorferi Geno-
group 1 Western Blot IgM
Immunetics Qualicode B. burgdorferi IgG Western B31
(800) 227-4765 blot kit
Qualicode B. burgdorferi IgM Western
blot kit
MarDx Diagnostics, Inc. B. burgdorferi (IgG) Marblot strip test B31
(800) 221-3391 system
B. burgdorferi (IgM) Marblot strip test
system
B. burgdorferi Serology 11.6.5

III. MATERIALS (continued) B. Serum diluent C. Other materials required as for any
Due to the presence in B. burgdorferi immunoassay
of antigens common to other bacteria, 1. Calibrated pipettors
several manufacturers have added bac- 2. Rocking platforms or shakers
terial proteins in some first-step assays 3. Timers
in order to absorb cross-reactive anti- 4. Serological pipettes
bodies from patient serum. 5. Multichannel incubating trays for
immunoblots

ANALYTICAL CONSIDERATIONS

IV. PROCEDURE Commercially available FDA-cleared kits provide instructions for proper perfor-
mance of assays.

A. Recommendations for testing


Special attention should be paid to the following factors.
1. Use kits within their expiration dates.
2. Avoid interchanging reagents from different lot numbers.
3. The following recommendations are particularly applicable for first-step tests
such as ELISAs.
a. Positive, low positive, and negative controls as recommended by the man-
ufacturer should be included in every run.
b. It is highly recommended that an in-house low positive well-characterized
control be included in each run to assess run performance.
c. Prior to placing a new kit lot in use, it is advisable to run a panel of well-
characterized frozen sera that encompasses all the ranges of reactivity. In
our experience differences in detection of IgM antibodies exist when dif-
ferent lots from the same manufacturer are used.
4. The following recommendations are applicable to Western immunoblots.
a. Run respective positive, weak positive, and negative controls as recom-
mended by the manufacturer. Some recommend running only the weak
and negative controls in every run and the positive (or band locator) only
once per new kit.
b. Inclusion of in-house controls showing bands of significance not present
in the band locator is also suggested.
5. Run paired sera in the same run when comparing reactivities of different
specimens obtained from the same patient at different time points.
B. Analytical factors
1. Maintain room temperature conditions (20 to 25⬚C). Day-to-day variations
in room temperature affect the reliability and consistency of results. When
room temperature exceeds ranges, the assay should be performed in another
area of the laboratory (if possible) where conditions are met.
2. Incubation times and use of rotators and shakers should be adhered to as
indicated by manufacturer unless in-house studies have proven them not to
affect results.
3. Although Western blot kits are also provided with the appropriate instruc-
tions for performance, there are guidelines published by the NCCLS (doc-
ument M34-A [33]). These guidelines address antigen preparation, electro-
phoresis, transfer of antigens to the matrix, calibration, and interpretation,
among other issues important to this method.
11.6.6 Immunology

POSTANALYTICAL CONSIDERATIONS

V. INTERPRETATION A. First-step tests


Interpretation guidelines are provided by manufacturers. Usually they involve
a comparison (ratio) of patient’s serum reactivity (absorbance for ELISA) to
the reactivity of a calibrator included in the assay (usually a sample testing low
positive). This ratio is translated to an index or units such that those samples
reacting at about the same intensity of the calibrator are labeled as borderline
(equivocal) or positive (reactive). Those with reactivity greater than that of those
calibrators are reported as positive. Some in-house tests generate these ratios
based on a reactivity that exceeds that of negative controls by 3 standard de-
viations.
Expected results:
1. Specimens containing cross-reactive antibodies as well as those from patients
with early Lyme disease usually test borderline or low positive.
2. Sera from patients with early disseminated disease are low positive to posi-
tive (sometimes two to three times above the cutoff).
3. Sera from patients with late disease or those who have received two or three
doses of OspA vaccine are high positive (4), usually greater than three times
the cutoff.
B. Western immunoblots
If blots are produced in-house, guidelines provided by NCCLS document M34-
A are recommended (33). At this writing three commercial sources of Western
immunoblots are available (Table 11.6–3), and they provide sufficient guidelines
for interpretation. Although this method offers greater specificity than first-step
tests, it suffers great limitations due to the subjectivity inherent to visual band
scoring. Some investigators have suggested a densitometric analysis of bands,
but currently this approach is not widely used (17). Manufacturers have tried to
address this issue by including a weak control that usually contains antibodies
to the 41-kDa antigen (flagellin).
1. Band scoring
a. Bands with intensity similar to or greater than that of the 41-kDa antigen
of the weak control are scored. Adherence to this is particularly important
when scoring IgM bands in sera from patients with early disease. IgM
reactivities to the 41- and 23-kDa (OspC) proteins usually exceed the
intensity of the weak control for these patients.
b. Use the band locator provided by the manufacturer to compare the re-
active bands in the patient strip and record the bands.
2. Results
a. A positive IgM blot shows two of these three significant bands: 23 kDa
(OspC), 39, or 41 kDa (17).
b. A positive IgG blot shows 5 of the following 10 significant bands: 18,
23 (OspC), 28, 30, 39, 41, 45, 58, 66, and 93 kDa (16).
c. CDC-ASTPHLD guidelines state that the IgM criteria should be only of
diagnostic use during the first 4 weeks of disease but the IgG criteria can
be used to support a clinical diagnosis at any disease stage.
d. Individuals who have received more than two doses of OspA vaccine
develop high titers of IgG to the recombinant antigen of 31 kDa. IgM
reactivity is of lesser intensity and duration than the IgG antibodies. In
addition, those individuals with high titers of IgG anti-OspA antibodies
may show reactivity to other antigens of low molecular size that most
likely represent breakdown products of OspA during blot preparation (4,
31).
B. burgdorferi Serology 11.6.7

VI. POSTANALYTICAL A. First-step assays


RECOMMENDATIONS 1. Guidelines established by the manufacturer concerning run acceptability
should be followed.
2. Laboratories performing tests for B. burgdorferi antibodies should participate
in an approved proficiency testing program, such as those offered by different
states or the CAP survey.
3. It is important to emphasize that current guidelines recommend that all spec-
imens reactive by first-step tests be referred for second-step IgG and IgM
immunoblots and that the final result be that of the immunoblots. Necessary
arrangements should be made to refer the specimens to a reference laboratory
or establish the Western blot method in the laboratory if the volume of
specimens allows it.
4. Absorbance of specimens may vary slightly from run to run; therefore, the
numerical values are not as precise as values of chemical blood parameters.
This is of particular importance when comparing reactivities of sera obtained
from the same individual at different time points. Paired sera should be run
in parallel using the same lot of reagents under the same run conditions in
order to obtain a meaningful comparison.
5. Since first-step tests are quantitative and the results are derived more objec-
tively than with Western blotting, they better assess the amount of antibodies
present. Therefore, these tests are better utilized to determine decline in an-
tibodies than Western immunoblotting.
6. Sensitivity and specificity
Variable levels of sensitivity are observed with the available first-step assays,
as has been demonstrated in proficiency testing of the CAP (6). Most fre-
quently, lack of sensitivity involves the detection of IgM antibodies during
early disease (2, 3, 8, 16, 26, 46). For a population of patients with culture-
confirmed EM of short duration, IgM antibodies were more frequently de-
tected by immunoblotting than by ELISA. The sensitivity of first-step tests
during the acute phase is about 30 to 40%, with a specificity of approximately
90% (2, 3, 16). About 60% or more of patients presenting with a disease of
more than 1 week’s duration show antibodies by first-step tests (3). Sensi-
tivity increases to about 90% if patients are tested within 2 weeks into con-
valescence.
B. Western immunoblots
1. It is important to assess the blot reactivities of the entire run as a whole,
regardless of whether the run met criteria of acceptability based on the con-
trol materials. Intensity of immunoblot bands correlates with reactivity as
measured by the first-step tests, and usually the specimens included in a blot
run have a spectrum of reactivity by ELISA and blotting. If blot bands are
present across the board and they do not seem to correlate with the ELISA
values, the entire run may need to be questioned. Lack of adherence to
procedural details, room temperature, or other factors may have affected the
run.
2. If great discrepancies exist between ELISA and immunoblot reactivities,
both tests may need to be repeated.
3. Laboratories performing Western immunoblots should participate in an ap-
proved proficiency testing program, such as those available through different
state health departments or the CAP survey. Although there is not a “gold
standard” method to compare results and materials included in proficiency
surveys have been questioned (42), proficiency testing programs have con-
tributed to a better understanding of the performance of available tests (6).
11.6.8 Immunology

VI. POSTANALYTICAL 4. When immunoblot testing is recently introduced into the laboratory, the use
RECOMMENDATIONS of a panel of monoclonal antibodies to various antigens may aid in identi-
(continued) fying the bands of significance. Such panel is available through the CDC
and consists of monoclonal antibodies to p93, p62, p41, p39, p37 (FlaA),
OspB, OspA, OspD, OspC, and p22. Other monoclonal antibodies have also
been produced by a number of investigators.
5. Although the OspA vaccine is no longer commercially available, it is advis-
able to become familiar with the blot reactivities of sera of those individuals
who received such preparations. Since patients who have developed natural
antibodies after an infection with B. burgdorferi do not usually develop anti-
OspA antibodies, the vaccination reactivity observed in blots is quite char-
acteristic. The presence of reactivity to low-molecular-size antigens in vac-
cinated individuals is different and should not be confused with reactivities
to the significant bands used in the IgG blot criteria (23 kDa [OspC] and 18
kDa) (4). Furthermore, vaccination does not seem to affect the interpretation
of blots for individuals who failed vaccination and developed Lyme disease
(4). Such individuals usually developed low titers of antibody to OspA (4,
28), and as mentioned above, vaccination elicits mostly IgG antibodies and
does not affect the IgM blot interpretation (4).
6. Sensitivity and specificity
During early stages of Lyme disease the sensitivity of IgM blots is about
40% in the acute phase and 60 to 80% during convalescence. The sensitivity
increases for patients with disseminated disease; in our experience, for pa-
tients with early disseminated disease with EM and neurological involvement
the sensitivity of IgM blots is close to 100%. In later stages of disease with
encephalopathy and arthritis the IgG blots have sensitivities of 84 and 100%,
respectively (16).
Methods aimed at detecting antibodies to B. burgdorferi, the causative
agent of Lyme disease, have many limitations. Contributing factors are the
complex antigenic composition of B. burgdorferi, the lack of standardization
of methods, and their use in unselected patient populations. The American
College of Physicians has proposed that antibody tests be restricted to those
patients with a pretest probability of Lyme disease of 0.20 to 0.80 (5, 47).
Testing of patients with a low probability (⬍0.20) of Lyme disease will most
likely yield false-positive tests. According to their recommendation, patients
with a high pretest probability should be treated and not tested (34). The
current approach to antibody testing involves the use of two steps: a sensitive
ELISA or IFA that should detect both IgG and IgM antibodies, followed by
separate IgM and IgG immunoblots. The role of these methods is to support
the clinical diagnosis of Lyme disease, and their indiscriminate use should
be discouraged.
Future tests to detect B. burgdorferi antibodies most likely will include
recombinant or synthetic antigens in a quantitative format. Such an approach
seems not so distant since such tests are already being developed and eval-
uated (27).
B. burgdorferi Serology 11.6.9

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11.7 Serodiagnosis of Rickettsial Infections

11.7.1 Introduction

Human rickettsial diseases are divided prevalences as high as 65 cases of HME All rickettsial infections present pre-
into those caused by species in the families per million population in Searcy County, dominantly with fever, headache, myal-
Rickettsiaceae and Anaplasmataceae. Ark., and 522 cases of HGE per million gias, and malaise, and for RMSF and mu-
Members of the genus Rickettsia infect en- population in Jackson County, Wis. (6). E. rine typhus, a maculopapular rash is
dothelial cells and cause vasculitis, ewingii is serologically cross-reactive with present in approximately 85 and 50% of
whereas those in the Anaplasmataceae E. chaffeensis, and a few studies have patients, respectively. The undifferen-
family include Ehrlichia chaffeensis, shown that about 10% of infections diag- tiated clinical presentation makes clinical
which infects monocytes and mononu- nosed as HME may actually be due to E. diagnosis difficult, and laboratory studies
clear phagocytes, and Anaplasma phago- ewingii (2). HME is generally more severe may be helpful if a normal leukocyte count
cytophilum (formerly Ehrlichia phagocy- than HGE, but both have case fatality rates or leukopenia with a left shift are observed
tophila, Ehrlichia equi, and the HGE of up to 2%, and infections are substan- concomitantly with thrombocytopenia and
agent) and Ehrlichia ewingii, which infect tially more severe in the context of im- increases in serum hepatic transaminase
granulocytes, chiefly neutrophils. Mem- munocompromise and human immuno- activities. Since delays in diagnosis and
bers of the genus Rickettsia are further di- deficiency virus. Of lower national therapy are associated with increased se-
vided into the spotted fever and typhus se- prevalence, but of great local importance, verity and fatality, diagnosis and initiation
rologic groups based on the presence is the flea-borne murine typhus caused by of therapy based upon clinical suspicion
(spotted fever group) or absence (typhus Rickettsia typhi and by the recently rec- are mandatory; therapy should not be held
group) of rickettsial outer membrane pro- ognized Rickettsia felis (4). This infection pending laboratory diagnostic tests.
tein A (rOmpA) and lipopolysaccharides. is recognized principally in South Texas RMSF may be diagnosed in the acute
Members of each group are strongly cross- and southern California, although a wide phase by skin biopsy and demonstration of
reactive, and infections cannot be differ- distribution wherever rats and fleas exist R. rickettsii antigens by direct fluorescent
entiated by routine serological testing. Oc- is known. As for RMSF, disease reflects antibody or immunohistochemical stain-
casionally, reactions across serological underlying vasculitis because of infection ing (7); PCR is not widely available and
groups occur, and differentiation is of endothelial cells systemically. Al- has not demonstrated any additional in-
achieved by comparing titers of antibody though generally perceived as a mild ill- crease in sensitivity during the acute phase
to specific rickettsial antigens. Similarly, ness, murine typhus is associated with a (8, 9). HME and HGE are diagnosed
cross-reactions between E. chaffeensis and case fatality rate approaching 2%. Orien- chiefly by specific PCR and less sensi-
A. phagocytophilum are detected in be- tia tsutsugamushi is the cause of scrub ty- tively by examination of Romanowsky-
tween 3 and 30% of infections; differen- phus, a chigger-transmitted rickettsial in- stained peripheral blood or buffy coat
tiation is usually achieved by comparison fection that occurs mostly in eastern parts smears for the presence of intracyto-
of specific antibody titers. of Asia, including Indonesia, and northern plasmic bacterial clusters called morulae
Among vector-borne infections in parts of Australia. Scrub typhus imported in monocytes (HME) or neutrophils
North America, next to Lyme disease, to North America and Europe is not infre- (HGE). Because of the unavailability or
tick-borne Rocky Mountain spotted fever quent and requires specific serological insensitivity of these tests for most clinical
(RMSF) caused by Rickettsia rickettsii is tools for diagnostic confirmation. In ad- laboratories, serologic testing is the
the second most prevalent and is associ- dition, Q fever is caused by Coxiella bur- method most frequently selected for con-
ated with a case fatality rate of up to 8.5% netii, an obligate intracellular bacterium firmation of a clinical diagnosis.
(1, 3). The next most frequently reported not phylogenetically related to either Rick- The most frequent serologic test for
vector-borne infections in North America ettsiaceae or Anaplasmataceae, and rarely confirmation of rickettsial and ehrlichial
are human monocytic (HME) and granu- arthropod transmitted. Diagnoses are often infections is the indirect fluorescent-anti-
locytic (HGE) ehrlichiosis, caused by E. made by referral of sera to public health body test, although additional tests such as
chaffeensis and A. phagocytophilum. Na- laboratories that possess the expertise to solid-phase EIA (dot blot) and the latex
tionally, these each exceed more than 100 conduct and interpret the complicated tests agglutination assay may provide useful di-
reported cases per year, with regional required for this infection. agnostic information and are commer-

11.7.1.1
11.7.1.2 Immunology

cially available. Still available but strongly ploy recombinant rickettsial and ehrlichial sults from non-FDA-cleared tests offered
discouraged owing to a significant lack of antigens hold great promise but have not through commercial reference laborato-
sensitivity and specificity is the Weil-Felix been cleared by the Food and Drug Ad- ries. However, it should be the responsi-
agglutination of Proteus antigens OX-2, ministration (FDA) for in vitro diagnostic bility of the laboratory director to deter-
OX-19, and OX-K, which are part of the use. Most are not currently commercially mine whether the performing referral
“febrile agglutinins” panel (5). An increas- available, although some referral labora- laboratory’s developmental and validation
ing variety of alternative serodiagnostic tories may offer such tests as alternatives data support acceptance of the diagnostic
tests and antigen preparations are reported to established methods. Current standards results.
in peer-reviewed literature. Tests that em- generally do not permit acceptance of re-

REFERENCES 1. Billings, A. N., J. A. Rawlings, and D. H. 6. McQuiston, J. H., C. D. Paddock, R. C. Hol-


Walker. 1998. Tick-borne diseases in Texas: man, and J. E. Childs. 1999. The human ehr-
a 10-year retrospective examination of cases. lichioses in the United States. Emerg. Infect.
Tex. State J. Med. 94:66–76. Dis. 5:635–642.
2. Buller, R. S., M. Arens, S. P. Hmiel, C. D. 7. Procop, G. W., J. L. Burchette, Jr., D. N.
Paddock, J. W. Sumner, Y. Rikhisa, A. Un- Howell, and J. E. Sexton. 1997. Immunope-
ver, M. Gaudreault-Keener, F. A. Manian, roxidase and immunofluorescent staining of
A. M. Liddell, N. Schmulewitz, and G. A. Rickettsia rickettsii in skin biopsies. A com-
Storch. 1999. Ehrlichia ewingii, a newly rec- parative study. Arch. Pathol. Lab. Med.
ognized agent of human ehrlichiosis. N. Engl. 121:894.
J. Med. 341:148–155. 8. Sexton, D. J., S. S. Kanj, K. Wilson, G. R.
3. Centers for Disease Control and Preven- Corey, B. C. Hegarty, M. G. Levy, and E.
tion. 2001. Summary of notifiable diseases, B. Breitschwerdt. 1994. The use of a poly-
United States 1999. Morb. Mortal. Wkly. Rep. merase chain reaction as a diagnostic test for
48:1–104. Rocky Mountain spotted fever. Am. J. Trop.
4. Dumler, J. S., J. P. Taylor, and D. H. Med. Hyg. 50:59–63.
Walker. 1991. Clinical and laboratory fea- 9. Standaert, S. M., T. Yu, M. A. Scott, J. E.
tures of murine typhus in South Texas, 1980 Childs, C. D. Paddock, W. L. Nicholson, J.
through 1987. JAMA 266:1365–1370. Singleton, Jr., and M. J. Blaser. 2000. Pri-
5. Hechemy, K. E., R. W. Stevens, S. Sasowski, mary isolation of Ehrlichia chaffeensis from
F. E. Michaelson, E. A. Casper, and R. N. patients with febrile illnesses: clinical and mo-
Philip. 1979. Discrepancies in Weil-Felix and lecular characteristics. J. Infect. Dis.
microimmunofluorescence test results for 181:1082–1088.
Rocky Mountain spotted fever. J. Clin. Micro-
biol. 9:292–293.
11.7.2 Indirect Fluorescent-Antibody Test

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE
The indirect fluorescent-antibody (IFA) complished by reacting serum from the morphology of the bacteria. For a patient
test, also called indirect immunofluores- patient with rickettsial antigens immobi- with a consistent clinical illness and epi-
cence and microimmunofluorescence, is lized onto glass slides; antibodies, if pres- demiologic history, the detection of a high
the most frequently used and best-char- ent in the serum, are detected with a fluo- titer of antibodies in a single serum sam-
acterized serological test for rickettsial and rescein isothiocyanate (FITC)-labeled ple, a seroconversion to a predetermined
ehrlichial infections (1, 3, 7–9, 11–16). anti-human isotype reporter. Reporter sig- minimal titer, or a fourfold increase in an-
IFA detection of human antibodies to nal is detected by UV microscopy using tibody titer is considered evidence of in-
Rickettsia, Ehrlichia, and Anaplasma spe- filters appropriate to detect FITC (apple fection.
cies antigens (rickettsial antigens) is ac- green) fluorescence that highlights the

II. SPECIMEN COLLECTION Serum samples for rickettsial serology should be obtained by venipuncture either
into standard red-top tubes or into serum separator tubes. Samples should be ob-
tained preferably at the time of acute illness and 2 to 4 weeks later. Some com-
mercial assays may also permit the analysis of antibodies in heparinized plasma,
whole blood, and capillary blood. The use of serum is strongly recommended. For
Rocky Mountain Spotted Fever (RMSF), antibody may be detected as early as 3
days after onset of illness, and nearly all patients have antibodies demonstrated by
21 days (8, 12, 14). For murine typhus, more than 50% of patients are seropositive
within the first 6 days of illness, and 100% are seropositive by 13 to 15 days (10).
For both human monocytic (HME) and human granulocytic (HGE) ehlichiosis, up
to 40% of patients are seropositive at presentation, and nearly all will be seropositive
within 1 month (2, 9). Aliquots of acute-phase samples should be stored frozen for
simultaneous testing with convalescent-phase samples. If serum is unable to be
tested within 24 h, it should be stored at ⳮ20⬚C until testing can be conducted.

III. MATERIALS There are few suppliers of high-quality an- mobilized onto multiwell, Teflon-coated
tigen slides or IFA kits for detection of glass slides. Some antigen slide prepara-
rickettsial and ehrlichial antibodies (Table tions have two or more rickettsial or ehr-
11.7.2–1). In addition, the CDC and some lichial antigens within a single well on the
public health laboratories provide or use slide, allowing simultaneous detection of
dimethyl sulfoxide-stabilized frozen in- antibodies reactive with multiple species.
fected-cell antigen suspensions that may In addition, some manufacturers will sup-
be applied to glass slides as needed. In ply individual components of kits, includ-
general, antigen preparations available ing antigen slides, conjugates, positive
range from whole infected cells to highly control sera, negative control sera, im-
purified cell-free bacterial suspensions im- munoglobulin M (IgM) removal diluent,

11.7.2.1
11.7.2.2 Immunology

Table 11.7.2–1 Manufacturers of rickettsial and ehrlichial serodiagnostic kits and


reagentsa
Antigen (disease) Focus Technologiesb PanBio, Ltd.c
R. rickettsii (RMSF) IFA IFA, DS-EIA, LA
R. typhi (murine typhus) IFA IFA, DS-EIA
E. chaffeensis (HME) IFA IFA
A. phagocytophilum (HGE) IFA IFA
a
IFA, indirect fluorescent-antibody test; DS-EIA, dipstick EIA; LA, latex agglutination test.
b
Focus Technologies, Inc., Diagnostics Customer Service, 10703 Progress Way, Cypress, CA 90630-
4717. Phone: (714) 220-1900, (800) 445-0185. Fax: (714) 220-1182. Website: http://
www.focusanswers.com.
c
PanBio, Ltd. (Australia), 116 Lutwyche Rd., Windsor, Brisbane 4030, Queensland, Australia. Phone:
61 (0) 7 3357 1177. Fax: 61 (0) 7 3357 1222. E-mail: PanBio@PanBio.com.au. Website: http://
www.panbio.com.au. PanBio, Inc. (Maryland), 9075 Guilford Rd., Columbia, MD, 21046. Phone:
(410) 381-8550. Fax: (410) 381-8984. e-mail: Carl_Stubbings@PanBio.com.au.

III. MATERIALS (continued) mounting medium, and buffered diluent. chamber for incubation of slides, and a
The assays will generally require addi- timer. A fluorescent microscope with ap-
tional supplies, such as coverslips for the propriate excitation filters is required for
antigen slides, test tubes and racks for examination of stained slides.
making serum dilutions, a humidified

IV. QUALITY ASSURANCE A positive and a negative control serum should be tested in parallel with all patient
specimens. Most commercially available kits will provide both and will give spe-
cific instructions regarding their preparation and use and interpretation of results.
In general, positive control samples have been obtained from individual patients
recovered or recovering from the rickettsial infection, or pools of samples from
such patients; separate controls may be provided for IgG and IgM analyses. Neg-
ative control samples are generally derived from healthy individuals without any
history of the rickettsial infection and may be supplied as either individual samples
or pools from a number of individuals. Controls should be diluted in parallel and
at the same time as patient samples, and the test should be conducted at the same
time for controls as for test samples. Some manufacturers provide control sera
prediluted. Under these circumstances, it is useful for each laboratory to have ad-
ditional positive and negative control sera of established titer available for testing
to control for problems with diluting or dilution buffers. Each positive control serum
provided by commercial manufacturers will have a target titer that should be con-
firmed at least with each new lot of antigen slides and new lot of conjugate. Man-
ufacturers’ instructions should be followed and generally call for replication of
stated titers within twofold dilutions. Each new lot of conjugate should be tested
for reactivity at the recommended dilution in the absence of any primary serum to
ensure that direct reaction with the antigen slides is not occurring.
Should positive control serum not react or not achieve the stated range of titer,
or if negative control serum should become reactive, patient results should not be
accepted and the tests should be repeated after appropriate troubleshooting. It is a
reasonable practice to periodically review the clinical records of patients with ser-
oconversion, a fourfold increase in antibody titers, or high IgM titers to ascertain
whether the clinical circumstances are consistent with the rickettsial or ehrlichial
infection identified. A repetitive pattern of inconsistent clinical manifestations
should be an indication for the laboratory director to investigate and/or educate
regarding appropriate, sensitive, and specific utilization of rickettsial IFA serology.
Currently, no proficiency testing program is available from the CAP, although
a pilot program to assess clinical laboratories’ serologic tests for tick-borne path-
IFA Test 11.7.2.3

IV. QUALITY ASSURANCE ogens has had at least one distribution as of this writing. A consortium of clinicians,
(continued) clinical laboratorians, researchers, and the CDC, called the Consensus Approach
for Ehrlichiosis (“CAFÉ”) Society, has made recommendations for clinical and
laboratory diagnosis of HME and HGE and has initiated a program of sample
sharing to assess the need for standardization (15). It is intended that as the program
matures and recommendations are made, it may operate as a repository for corrob-
orated samples to be distributed for proficiency testing.

ANALYTICAL CONSIDERATIONS

V. PROCEDURE In general, IFA test procedures for the various species are similar and have several
steps. It is good practice to record into which well each serum or control will be
placed prior to starting the assay.

A. Slides are allowed to warm to room temperature.


B. A small volume (usually 10 to 25 ll) of each appropriate dilution of positive
control, negative control, patient serum, and patient serum depleted of IgG (for
IgM detection) is added to the appropriate well on a multiwell antigen slide.
Some manufacturers supply control sera in a prediluted format. Dilutions are
determined by manufacturers, but most agree that a screening dilution of 1:64
should be used for IgG and polyvalent tests; dilutions for IgM tests may vary.
C. Incubation periods vary by manufacturer but are generally 30 min to 1 h at
ambient temperature or at 37⬚C.
D. Slides are rinsed in buffer and washed by one or more immersions in buffer,
and buffer salts are removed by a brief distilled-water rinse prior to drying.
E. Each well receives an appropriate volume (usually 10 to 25 ll) of isotype-
specific conjugate.
F. Repeat the incubation and wash steps (steps V.C and D). Some protocols include
a step for counterstaining with reagents such as Evan’s blue or Eriochrome black
prior to or during the final wash step.
G. After the slides are dry, each well receives a small volume (10 to 25 ll) of
mounting medium, usually a glycerol–phosphate-buffered saline mixture.
H. Coverslips are placed over the wells on the slides, avoiding trapped air bubbles.
Care should be taken to avoid excess mounting medium as it will seep from
under the coverslip and contaminate the microscope objectives or immersion
oil, if used. Excess mounting medium may be removed by capillary action with
an absorbent blotting paper.
I. Slides are examined at a final magnification of between ⳯200 and ⳯1,000
using a fluorescent microscope with appropriate filters to allow excitation and
emission for FITC.
J. If the slides cannot be viewed immediately, they may be kept at 4⬚C in the dark
for 24 h.

Since a fourfold change in titer offers the most sensitive seroconfirmation, sera
that react at the screening dilution should be titrated. To do so, sera should be
serially diluted (after removal of IgG for IgM titer determinations) in doubling
dilutions starting at the screening dilution (usually 1:64) until an endpoint titer
where no definite fluorescent bacteria are identified is reached.
A reactive sample will allow definite fluorescent visualization of the bacterial
morphology. Rickettsia spp. appear as small (approximately 0.3 by 1.5 lm) bacilli
and occasionally coccobacilli, whereas Ehrlichia chaffeensis and Anaplasma phag-
ocytophilum will appear predominantly as tiny (0.2 by 1.0 lm) coccobacilli and
cocci. Some antigen preparations utilize bacteria free of host cells, prepared by
11.7.2.4 Immunology

V. PROCEDURE (continued) growth in chicken embryo yolk sacs or by density gradient purification from in-
fected tissue culture cells. When bacteria are purified from yolk sacs, a diluent that
includes normal yolk sac as a 10% suspension in diluting buffer is advisable to
absorb any antibodies reactive with yolk sac antigens. Antigen preparations derived
from whole infected cells (Rickettsia rickettsii or Rickettsia typhi grown in Vero or
other cell lines, E. chaffeensis grown in DH82 or Vero cells, or A. phagocytophilum
grown in HL-60 cells) may have different appearances from those in cell-free prep-
arations. In general, it is best to only interpret bacteria that are within cells in these
preparations. The bacteria will be localized within the cytoplasm as short bacilli for
R. rickettsii and R. typhi (occasionally in nuclei for R. rickettsii) and as clusters or
inclusions of coccobacilli and cocci in the cytoplasms of the host cells for E. chaf-
feensis and A. phagocytophilum.
Slides should be examined for the presence of fluorescent bacteria in several
locations, as staining intensity may vary considerably even in a single well. Bacteria
at the periphery of the well should not be evaluated. Various intensities of fluores-
cence may be observed, and many manufacturers suggest recording this in a semi-
quantitative manner (0 to 4Ⳮ). However, the best practice is to assess the mor-
phology of each antigen reacted with each sample for the definition of morphology,
the distribution, and the quantity of intracellular bacteria compared with the positive
and negative control samples. The positive control sample will provide the approx-
imate distribution and an appropriate comparative morphology, whereas the nega-
tive control will provide a meaningful level of background fluorescence. Fluores-
cence of bacterial morphology in samples that does not exceed that identified on
the negative control should be considered negative. Optimally, fluorescent bacteria
should not appear in negative control samples.

POSTANALYTICAL CONSIDERATIONS

VI. INTERPRETATION IFA testing for detection of antibodies to R. rickettsii, R. typhi, E. chaffeensis, and
A. phagocytophilum is the most sensitive method for confirmation of infection (Ta-
ble 11.7.2–2). In general, it is good practice to establish appropriate screening and
cutoff titers for positive samples in each laboratory, a situation that is not possible
for most clinical laboratories owing to the lack of sufficient numbers of well-char-
acterized samples. Thus, most laboratories and laboratory directors will rely upon
the validation performed by the manufacturer. In most cases, the CDC case defi-
nition provides a serologic cutoff that is widely accepted and corroborated by man-
ufacturers of rickettsial and ehrlichial serodiagnostic tests. For the IFA test, the
current CDC seroepidemiologic criterion for diagnostic confirmation of suspected

Table 11.7.2–2 Sensitivities and specificities of serological tests for confirmation of


RMSF, murine typhus, HME, and HGE
Disease Serological assaya Sensitivity (%) Specificity (%)
RMSF IFA IgG 89–100 99–100
IFA IgM 83–85 100
LA 79–100 94–100
Murine typhus IFA IgG ⱖ83 ⱖ93
IFA IgM 53–85 99
Dot blot 88 92
HME IFA IgG 88 Not determined
IFA IgM 86 Not determined
HGE IFA IgG 82–100 82–100
IFA IgM 27–37 83–100
a
IFA, indirect fluorescent-antibody test; LA, latex agglutination.
IFA Test 11.7.2.5

VI. INTERPRETATION RMSF (5) requires a fourfold rise in titer of antibody to R. rickettsii antigen; for a
(continued) serological test that screens samples at a dilution of 1:64, this implies a minimum
titer of 128 if the acute-phase titer is ⬍64. The IFA test serological criterion for
“probable” infection in a patient with suspected RMSF is a single titer of ⱖ64.
Application of these criteria for diagnosis of individual patients generally requires
a “probable” diagnosis with a titer of at least 64. Similar CDC laboratory criteria
exist for confirmation of the diagnosis of HME and HGE in clinically suspect cases,
but no specific cutoff levels are advocated (6). Some suppliers recommend that
initial dilutions of sera begin at 1:8, and a fourfold increase would occur with a
minimum titer of 16 or 32. However, prior studies suggest that dilutions lower than
approximately 1:64 often result in nonspecific fluorescence; thus, a minimum titer
of 64 would be required before any sample is considered specifically reactive with
R. rickettsii. Although not extensively studied, similar results have been observed
in IFA testing for E. chaffeensis and A. phagocytophilum.
As antibodies are rarely detected in the acute phase of RMSF and murine typhus
and infrequently (ⱕ 30% of patients) in HME or HGE, a second or convalescent-
phase sample must be obtained 2 to 4 weeks later for optimal diagnostic sensitivity
(2, 9, 12, 14). More than 99% of patients with HGE and HME are seropositive at
1 month postinfection, and IgG or polyvalent reactions may persist for years. Al-
though IgG antibodies may be frequent in early infection, IgM antibodies are fre-
quently detected only during the first 45 to 60 days postinfection and may be useful
to confirm recent exposure or infection; IgM antibodies may infrequently persist
for a year or more.
Overall, serologic testing for antibodies reactive with R. rickettsii, R. typhi, E.
chaffeensis, and A. phagocytophilum should be conducted only when a reasonable
level of clinical suspicion exists. Although specificity of the reaction is relatively
high (ⱖ92%), the predictive value of a positive test is only moderate unless the
prevalence of the infection or the pretest probability of infection is relatively high.
Likewise, since sensitivity is high in all of these tests (88 to 100%), the predictive
value of a negative test is very high when paired acute- and convalescent-phase
samples are compared. The predictive value of a negative test is low when only an
acute-phase sample is tested, since sensitivity in that phase of infection is low
(generally ⬍40%). It is also particularly important to conform with the principle
of comparing acute- and convalescent-phase sera since a significantly higher pro-
portion of persons have serologic evidence of infection by R. rickettsii, E. chaf-
feensis, and A. phagocytophilum than report manifestations of infection. Whether
these observations reflect the occurrence of subclinical infections or antigenic stimu-
lation with cross-reactive antigens or “nonpathogenic” species after tick bites is
undetermined.
A number of preexisting conditions may dispose patients to false-positive se-
rologic reactions for the various Rickettsia, Ehrlichia, and Anaplasma spp. (Table
11.7.2–3). It must be understood that the R. rickettsii IFA test detects antibodies
broadly reactive with spotted fever group rickettsiae and more weakly reactive with
typhus group rickettsiae; the converse is true for reactions with R. typhi. Thus,
seroconfirmation of RMSF or murine typhus is dependent upon a strong clinical
history consistent with the geographically likely infection or upon more specific
serologic tests that are conducted only in reference and public health laboratories.
A beneficial aspect of this nonspecificity is the ability to broadly apply the RMSF
and murine typhus serologic tests. Thus, these may be used to detect antibodies
reactive with other spotted fever group or typhus group rickettsiae that are domes-
tically rare (Rickettsia akari or R. felis) or may have been acquired during inter-
national travel, including such agents as Rickettsia conorii (Mediterranean spotted
fever), Rickettsia africae (African tick bite fever), or Rickettsia prowazekii (epi-
demic typhus), among many. Occasional cross-reactions with other infectious
11.7.2.6 Immunology

Table 11.7.2–3 Conditions that may lead to false-positive serologic tests for rickettsial
diseasesa
Confounding
R. rickettsii R. typhi E. chaffeensis A. phagocytophilum
condition or agent
Spotted fever Ⳮ Ⳮ Ⳳ
Typhus Ⳮ Ⳮ Ⳳ
HME Ⳮ
HGE Ⳮ
Syphilis Ⳮ
Lyme disease Ⳮ
Leptospirosis Ⳮ
Q fever Ⳮ Ⳮ
Brucella infection Ⳮ
Bartonella sp. Ⳮ
Cholera Ⳮ
Typhoid Ⳮ
Shigellosis Ⳮ Ⳮ
Malaria Ⳮ Ⳮ
Epstein-Barr virus Ⳮ Ⳮ
Dengue Ⳮ
Rheumatoid factor Ⳮ Ⳮ Ⳮ Ⳮ
ANA Ⳮ Ⳮ Ⳮ
ANCA Ⳮ
Antiplatelet antibody Ⳮ
Pregnancy Ⳮ
a
ANA, antinuclear antibody; ANCA, antineutrophil cytoplasmic antibody. Ⳮ, the condition or agent is
documented to cause a false-positive test; Ⳳ, there is potential for serologic cross-reactivity, although
documentation is infrequent.

VI. INTERPRETATION agents are also observed, but under ordinary circumstances, the cross-reactive titers
(continued) are at least fourfold lower than the titer for the homologous rickettsial infectious
agent.
In a similar manner, serologic tests for E. chaffeensis and A. phagocytophilum
may yield cross-reactions with each other and related species (6, 16). For example,
Ehrlichia ewingii, the agent of a newly recognized form of granulocytic ehrlichiosis,
induces antibodies reactive with E. chaffeensis, as do veterinary infections with
other Ehrlichia spp. (E. canis, E. muris, E. ruminantium) and some Anaplasma spp.
(e.g., A. phagocytophilum) (4). In general, the majority of infections result in a
fourfold higher titer of IFA to the homologous infectious agent, but as antigens are
not available for all Ehrlichia and Anaplasma spp., a high degree of suspicion based
upon clinical manifestations and epidemiologic history is required for the highest
degree of predictive value. Approximately 3 to 30% of patients with HGE will
develop antibodies reactive with E. chaffeensis, but only a small proportion (⬍
10%) will not be resolved by comparison of titers.
A disproportionate number of sera from patients with HGE and HME will also
react in some serological tests for Lyme disease (17, 19). The basis for this reaction
is still not clear, and it is speculated to result from such diverse circumstances as
serological cross-reactivity between antigens of bacteria in different phylogenetic
classes or coinfections with multiple tick-transmitted infectious agents. Thus, Bor-
relia burgdorferi and ehrlichial serologies must be carefully interpreted in the con-
text of patient clinical manifestations and epidemiologic history.
Owing to the use of A. phagocytophilum-infected HL-60 cells of human deri-
vation, whole-cell-infected antigen preparations may yield false-positive results for
IFA Test 11.7.2.7

VI. INTERPRETATION patients with autoimmune disorders, including those with antinuclear, antiplatelet,
(continued) and anti-smooth muscle antibodies (18). The false-positive results can be minimized
by adherence to careful morphological criteria for interpretation and examination
at a high magnification (usually ⳯1,000).

REFERENCES 1. Aguero-Rosenfeld, M. E., F. Kalantarpour, 11. Magnarelli, L. A., J. W. Ijdo, J. S. Dumler,


M. Baluch, H. W. Horowitz, D. F. Mc- R. Heimer, and E. Fikrig. 1998. Reactivity
Kenna, J. T. Raffalli, T.-C. Hsieh, J. Wu, J. of human sera to different strains of granulo-
S. Dumler, and G. P. Wormser. 2000. Se- cytic ehrlichiae in immunodiagnostic assays.
rology of culture-confirmed cases of human J. Infect. Dis. 178:1835–1838.
granulocytic ehrlichiosis. J. Clin. Microbiol. 12. Newhouse, V. F., C. C. Shepard, M. D. Re-
38:635–638. dus, T. Tzianabos, and J. E. McDade. 1979.
2. Bakken, J. S, I. Haller, D. Riddell, J. J. A comparison of the complement fixation, in-
Walls, and J. S. Dumler. 2002. The serolog- direct fluorescent antibody, and microagglu-
ical response of patients infected with the tination tests for the serological diagnosis of
agent of human granulocytic ehrlichiosis. rickettsial diseases. Am. J. Trop. Med. Hyg.
Clin. Infect. Dis. 34:22–27. 28:387–395.
3. Brouqui, P., E. Salvo, J. S. Dumler, and D. 13. Philip, R. N., E. A. Casper, J. N. Mac-
Raoult. 2001. Diagnosis of granulocytic ehr- Cormack, D. J. Sexton, L. A. Thomas, R. L.
lichiosis in humans by immunofluorescence Anacker, W. Burgdorfer, and S. Vick. 1977.
assay. Clin. Diagn. Lab. Immunol. 8:199–202. A comparison of serologic methods for diag-
4. Buller, R. S., M. Arens, S. P. Hmiel, C. D. nosis of Rocky Mountain spotted fever. Am. J.
Paddock, J. W. Summer, Y. Rikhisa, A. Un- Epidemiol. 105:56–67.
ver, M. Gaudreault-Keener, F. A. Manian, 14. Philip, R. N., E. A. Casper, R. A. Ormsbee,
A. M. Liddell, N. Schmulewitz, and G. A. M. G. Peacock, and W. Burgdorfer. 1976.
Storch. 1999. Ehrlichia ewingii, a newly rec- Microimmunofluorescence test for the sero-
ognized agent of human ehrlichiosis. N. Engl. logical study of Rocky Mountain spotted fever
J. Med. 341:148–155. and typhus. J. Clin. Microbiol. 3:51–61.
5. Centers for Disease Control and Preven- 15. Walker, D. H. 2000. Diagnosing human ehr-
tion. Rocky Mountain spotted fever—1996 lichiosis: current status and recommendations.
case definition. http://www.cdc.gov/epo/ ASM News 66:287–290.
dphsi/casedef/ 16. Walls, J. J., M. Aguero-Rosenfeld, J. S.
rocky_mountain_spotted_fever_current.htm. Bakken, J. L. Goodman, D. Hossain, R. C.
6. Centers for Disease Control and Preven- Johnson, and J. S. Dumler. 1999. Inter- and
tion. Ehrlichiosis—2000 case definition. http:/ intralaboratory comparison of Ehrlichia equi
/www.cdc.gov/epo/dphsi/casedef/ehrlichio- and human granulocytic ehrlichiosis (HGE)
sis_current.htm. agent strains for serodiagnosis of HGE by the
7. Childs, J. E., J. W. Sumner, W. L. Nichol- immunofluorescent-antibody test. J. Clin. Mi-
son, R. F. Massung, S. M. Standaert, and C. crobiol. 37:2968–2973.
D. Paddock. 1999. Outcome of diagnostic 17. Wong, S. J., G. S. Brady, and J. S. Dumler.
tests using samples from patients with culture- 1997. Serological responses to Ehrlichia equi,
proven human monocytic ehrlichiosis: impli- Ehrlichia chaffeensis, and Borrelia burgdor-
cations for surveillance. J. Clin. Microbiol. feri in patients from New York State. J. Clin.
37:2997–3000. Microbiol. 35:2198–2205.
8. Clements, M. L., J. S. Dumler, P. F. Fiset, 18. Wong, S. J., and J. A. Thomas. 1998. Cy-
C. L. Wisseman, Jr., M. J. Snyder, and M. toplasmic, nuclear, and platelet autoantibodies
M. Levine. 1983. Serodiagnosis of Rocky in human granulocytic ehrlichiosis patients. J.
Mountain spotted fever: comparison of IgM Clin. Microbiol. 36:1959–1963.
and IgG enzyme-linked immunosorbent as- 19. Wormser, G. P., H. W. Horowitz, J. S.
says and indirect fluorescent antibody test. J. Dumler, I. Schwartz, and M. Aguero-Ro-
Infect. Dis. 148:876–880. senfeld. 1996. False-positive Lyme disease se-
9. Dawson, J. E., D. B. Fishbein, T. R. Eng, M. rology in human granulocytic ehrlichiosis.
A. Redus, and N. R. Greene. 1990. Diagnosis Lancet 347:981–982.
of human ehrlichiosis with the indirect fluo-
rescent antibody test: kinetics and specificity.
J. Infect. Dis. 162:91–95.
10. Dumler, J. S., J. P. Taylor, and D. H.
Walker. 1991. Clinical and laboratory fea-
tures of murine typhus in South Texas, 1980
through 1987. JAMA 266:1365–1370.
11.7.3 Solid-Phase (Dot Blot) EIA

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE
An alternative method for detection of strate, such as 5-bromo-4-chloro-3-indo- timate of serum antibody concentration, or
rickettsial antibodies is based upon reac- lylphosphate and p-nitroblue tetrazolium titer, may be determined. Theoretically, it
tions of antibodies with rickettsial anti- chloride, is detected by visual observation is possible to develop isotype-specific se-
gens immobilized to membranes, the of a blue-black reaction on the nitrocel- rological results; however, currently avail-
solid-phase EIA, or dot blot (1, 2). This lulose. Unlike the situation with IFA test- able formats detect only polyvalent re-
method utilizes a principle similar to that ing, where serum titer is determined by se- sponses. The main advantages of this
for indirect fluorescent antibody (IFA) rial dilution of serum samples, the EIA approach are the lack of need for fluores-
testing except that serum antibodies are re- takes advantage of the binding properties cence microscopy, the relative ease of
acted with rickettsial antigens immobi- and kinetics of antibody-antigen interac- operation in a dipstick format, the stability
lized onto nitrocellulose and in turn these tions by reacting a single dilution of serum of antigen preparations, diminution of
bound antibodies are detected with anti- with diminishing quantities of rickettsial subjective fluorescent determinations, and
human antibody isotype reagent coupled antigen. Thus, by coupling several dimin- the development of a permanent record.
to a reporter enzyme. Reporter signal ishing quantities of antigen on a single The format is best used when only a lim-
developed from bound enzyme, such as al- strip of nitrocellulose and reacting with a ited number of tests are conducted and a
kaline phosphatase, acting upon a sub- single dilution of serum, a quantitative es- rapid turnaround time is desirable.

II. SPECIMEN COLLECTION Serum samples obtained as described above for immunofluorescence are recom-
mended for use with solid-phase immunoassays. The manufacturer also provides
for the ability to test anticoagulated whole blood or capillary blood.

III. MATERIALS The dot blot kits are provided with re- Additional supplies that are needed to con-
agents mandatory for the reactions, in- duct these tests include a heat block or wa-
cluding assay antigen strips, reaction cu- ter bath capable of maintaining 50 Ⳳ 1⬚C,
vettes, sample diluent, enhancer reagent, a reaction vessel capable of holding at
conjugate reporter reagent, and developer least 50 ml of water, a timer, distilled or
or substrate solution. While the antigen deionized water, pipettes, absorbent tow-
strips are often stable at room temperature, els or paper, and control sera.
the liquid reagents require refrigeration.

11.7.3.1
Solid-Phase (Dot Blot) EIA 11.7.3.2

ANALYTICAL CONSIDERATIONS

IV. QUALITY ASSURANCE Since the dot blot method has an additional step and reagents not used in IFA
testing, each test strip integrates internal reagent controls that ensure that all assay
reagents, except for antigen, are working properly. A positive and negative control
serum should be tested in parallel with each run to ensure reactivity of human serum
with the rickettsial antigen. Currently, no proficiency testing program is available
from the CAP.

V. PROCEDURE Specific instructions are provided by the manufacturer with each kit and should be
followed precisely.

A. Sufficient numbers of assay strips are labeled for patient identification.


B. Serum is diluted by placing 10 ll of serum or 20 ll of whole blood into a
premeasured quantity of diluent to achieve a 1:200 dilution.
C. Assay strips are prewetted for 4 min and then placed sequentially into each of
the following for 5 min, and each step is followed by washing and blotting dry.
1. Warmed diluted serum
2. Sodium chloride “enhancer” solution
3. Reporter conjugate
4. Substrate
D. Assay strips are allowed to completely dry prior to reading and result interpre-
tation. The manufacturer does not advocate facilitated drying with instruments
such as hair dryers, but drying may be enhanced by ambient airflow or gentle
fanning.
E. Reacted strips are evaluated by visual inspection for the presence of colored end
product in each control and patient sample spot on the nitrocellulose strips.
Reaction with diminishing antigen quantities correlates with antibody titer
determined by IFA testing and is interpreted as nonreactive (no dots), weakly
reactive (one or two dots, equivalent to IFA titers of 16 to 128), or reactive
(three or four dots, IFA titers of ⱖ256). Each strip includes internal positive
(human immunoglobulin) and negative (phosphate-buffered saline) reagent con-
trols to ensure proper function of the reporter conjugate and substrate reagents,
and these must react appropriately prior to interpretation. Because these internal
reagent controls do not adequately control for the antigen, a known positive and
negative control should be tested with each run. Although solid-phase EIA re-
sults correlate well with IFA titers, for demonstration of either seroconversion
or a fourfold change in antibody titer, both acute- and convalescent-phase sera
should be tested by the same method, preferably at the same time.

POSTANALYTICAL CONSIDERATIONS

VI. INTERPRETATION Solid-phase EIA has been developed and is available for Rickettsia rickettsii, Rick-
ettsia conorii, Rickettsia typhi, Coxiella burnetii (Q fever), and Orientia tsutsuga-
mushi (scrub typhus). Additional formats that allow simultaneous screening for
antibodies reactive with several infectious agents, including combined tests for spot-
ted fever, scrub typhus, and murine typhus or dengue, typhoid, leptospirosis, scrub
typhus, and salmonella are also available. Favorable comparisons with IFA results
have been published for the use of R. typhi (2) and the O. tsutsugamushi dot blot
EIA (3), and anecdotal reports suggest that the R. rickettsii assay may also be useful
for confirmation of infections with spotted fever group rickettsiae (1). Although no
specific criteria for interpretation of solid-phase EIA results are currently recom-
11.7.3.3 Immunology

VI. INTERPRETATION mended by the CDC, good qualitative correlation with IFA testing for antibodies
(continued) to R. typhi exists when performed as a single serum test or when considering a
seroconversion as a change from nonreactive to reactive. Thus, a single serum
sample reactive by dot blot EIA provides probable evidence of infection in a patient
with clinical and epidemiologic histories consistent with suspected rickettsial in-
fection.
Dot blot was compared with IFA testing in only one literature report and only
for R. typhi (2). Using convalescent-phase sera, sensitivity and specificity compared
to immunoglobulin G (IgG) IFA testing where the cutoff titer was 128 were 91.8
and 87.7%, and titers were well correlated overall (r ⳱ 0.84) with those obtained
by IgG IFA testing. Thus, for a 50% pretest probability of infection, the positive
predictive value is 88%, and for a 20% pretest probability of infection, the predictive
value of a negative test is 81%. Similar predictive values are obtained even if a titer
cutoff of 64 is used.

REFERENCES 1. Hilton, E., J. DeVoti, J. L. Benach, M.L. 3. Weddle, J. R., T. C. Chan, K. Thompson,
Halluska, D. J. White, H. Paxton, and J. S. H. Paxton, D. J. Kelly, G. Dasch, and D.
Dumler. 1999. Seroprevalence and serocon- Strickman. 1995. Effectiveness of a dot-blot
version for tick-borne diseases in a high-risk immunoassay of anti-Rickettsia tsutsugamushi
population in the northeast United States. Am. antibodies for serologic analysis of scrub ty-
J. Med. 106:404–409. phus. Am. J. Trop. Med. Hyg. 53:43–46.
2. Kelly, D. J., C. T. Chan, H. Paxton, K.
Thompson, R. Howard, and G. A. Dasch.
1995. Comparative evaluation of a commer-
cial enzyme immunoassay for the detection of
human antibody to Rickettsia typhi. Clin.
Diagn. Lab. Immunol. 2:356–360.
11.7.4 Latex Agglutination

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE
Detection of antibodies to Rickettsia rick- present, agglutination of the beads occurs long interval between sample collections
ettsii and Rickettsia typhi has been accom- and may be observed macroscopically. or the convalescent-phase sample is ob-
plished using a rapid and relatively sensi- Because agglutination of coated latex tained after 45 days. Although sera may
tive latex agglutination (LA) method (2, beads follows the principles of serologic be diluted to determine LA titers, as IgM
3). For LA, particles are coated with an agglutination reactions, it detects immu- antibodies diminish in convalescence, so
extract of purified, cell-free rickettsiae noglobulin M (IgM) antibodies better than will the detected LA titer. Thus, LA is
called erythrocyte-sensitizing substance IgG antibodies, is more sensitive as a di- most useful as a screening tool during the
(ESS) that is in part composed of sero- agnostic tool early after infection than as early phases of infection, or when early-
group-reactive lipopolysaccharides (4). a seroepidemiologic tool, and may be acute-phase and early-convalescent-phase
This reagent has been prepared only from prone to prozone reactions. (7 to 21 days postonset) sera are obtained.
Rickettsia spp.; thus, LA is not available Unlike the situation for IFA and dot The latest case definition for laboratory
for either Ehrlichia or Anaplasma spp. blot assays that sensitively detect both IgG confirmation of Rocky Mountain spotted
Currently, LA reagents are commercially and IgM, comparison of acute- and con- fever (RMSF) by LA requires either a
available only for R. rickettsii. The coated valescent-phase sera may not yield an ap- fourfold rise in antibody titer or a single
latex beads are mixed with dilutions of se- propriate result, especially if there is a LA titer of ⱖ128 (1).
rum. If specific rickettsial antibodies are

II. SPECIMEN COLLECTION Serum samples to be tested by LA for R. rickettsii are obtained as outlined for IFA
testing. Samples should be obtained preferably at the time of acute illness and 14
to 21 days after onset. Some may elect to obtain an intermediate sample 7 to 10
days postonset to help establish an earlier confirmation of infection. Samples should
be tested immediately or frozen until used. It is a good practice to test acute- and
convalescent-phase samples simultaneously for the most accurate comparison of
antibody titers.

III. MATERIALS The R. rickettsii LA kit is provided with be refrigerated. Additionally required are
reagents required for the reactions, includ- a rotating platform with a humidified
ing ESS antigen-coated latex beads, dilut- cover, such as the type used for rapid
ing buffer, control sera, black rotation plasma reagin testing, and a 56⬚C heat
slides, and stirring sticks; control sera may block or water bath for complement in-
be provided with the kit or purchased sep- activation.
arately. Antigen-coated latex beads should

11.7.4.1
11.7.4.2 Immunology

ANALYTICAL CONSIDERATIONS

IV. QUALITY ASSURANCE Each LA test should include a positive and negative control, and the titer of the
positive control should be confirmed for each test kit lot. A result Ⳳ1 dilution is
acceptable for positive control titer. The antigen-coated latex beads should not be
used if aggregation exists prior to mixing with test or control sera or if the reagents
are contaminated. Currently, no proficiency testing program is available from the
CAP.

V. PROCEDURE Specific instructions are provided by the manufacturer with each kit and should be
followed precisely.

A. The positive control, negative control serum, and patient sera are diluted 1:16,
and all sera are heat inactivated (56⬚C for 30 min).
B. Forty microliters of diluted serum is placed onto one corner of a well on the
mixing slide and 20 ll of well-mixed antigen-coated latex beads is placed di-
agonally in the same well.
C. The serum and latex beads are mixed with a wooden stick.
D. The slide is covered to prevent evaporation and rotated for 6 min at 100 to 120
rpm.
E. After an additional 5 min without rotation, slides are read under a fluorescent
lamp or other appropriate illumination.
F. Agglutination is examined by gently tilting the slide to ascertain the degree of
clumping or aggregation, which is recorded as follows.
1. Negative (no aggregation of latex beads)
2. Weakly reactive (fine aggregation of latex beads)
3. Reactive (definite aggregation of latex beads)
G. All reactive samples are titrated by serial twofold dilutions and each dilution is
retested.
The reciprocal of the final dilution with definite agglutination establishes the
titer of the sample. A fourfold increase in titer between acute- and convalescent-
phase sera from a patient confirms the diagnosis of RMSF in a patient with
consistent clinical and epidemiologic findings. Likewise, for a patient with a
consistent clinical illness, a single serum with a titer of ⱖ128 provides evidence
for probable RMSF. Although LA results correlate well with IFA titers, for
demonstration of either seroconversion or a fourfold change in antibody titer,
acute- and convalescent-phase sera should be tested by the same method.

POSTANALYTICAL CONSIDERATIONS

VI. INTERPRETATION LA has been developed for R. rickettsii, Rickettsia conorii, and R. typhi but is
available only for R. rickettsii. Favorable comparisons with IFA results have been
published for R. rickettsii LA (2, 3). Sensitivity and specificity compared to IFA
were ⱖ79 and ⱖ96%. In a multicenter evaluation study that examined 2,250 sera
from areas of endemicity, LA had a positive predictive value of 77 to 94% and a
negative predictive value of 96 to 97%.
Latex Agglutination 11.7.4.3

REFERENCES 1. Centers for Disease Control and Preven- 4. Murphy, J. R., P. Fiset, L. B. Snyder, and
tion. Rocky Mountain spotted fever—1996 C. L. Wisseman, Jr. 1979. Antibody to Rick-
case definition. http://www.cdc.gov/epo/ ettsia mooseri erythrocyte-sensitizing sub-
dphsi/casedef/ stance. Infect. Immun. 24:962–964.
rocky_mountain_spotted_fever_current.htm.
2. Hechemy, K. E., R. L. Anacker, R. N.
Philip, K. T. Kleeman, J. N. MacCormack,
S. J. Sasowski, and E. E. Michaelson. 1980.
Detection of Rocky Mountain spotted fever
antibodies by a latex agglutination test. J. Clin.
Microbiol. 12: 144–150.
3. Hechemy, K. E., E. E. Michaelson, R. L. An-
acker, M. Zdeb, S. J. Sasowski, K. T. Klee-
man, J. M. Joseph, J. Patel, J. Kudlac, L.
B. Elliott, J. Rawlings, C. E. Crump, J. D.
Folds, H. Dowda, Jr., J. H. Barrick, J. R.
Hindman, G. E. Killgore, D. Young, and R.
H. Altieri. 1983. Evaluation of latex-Rickett-
sia rickettsii test for Rocky Mountain spotted
fever in 11 laboratories. J. Clin. Microbiol.
18:938–946.
11.8 Immunoassay Detection of Shiga
Toxin-Producing Escherichia coli

Shiga toxin-producing Escherichia coli undercooked hamburger meat (12, 18). serotypes which are sorbitol fermenting.
(STEC) organisms are responsible for a Since then, cases have been more often However, non-O157 STEC organisms that
broad range of gastrointestinal illnesses, sporadic. A variety of other food and wa- cause disease cannot be readily detected
from mild watery diarrhea to severe hem- ter sources have been reported as vehicles. using SMAC (8, 11, 16, 19).
orrhagic colitis associated with hemolytic- Person-to-person spread and contact with There are several methods available to
uremic syndrome (HUS) (2, 12, 18). These animals have emerged as important meth- laboratories for toxin detection. These in-
organisms cause disease primarily through ods of spread (12, 18). Children and the clude cell culture techniques, commercial
elaboration of one or more Shiga toxins elderly are the individuals most at risk for EIAs, passive latex agglutination methods,
(Stx 1, Stx 2, Stx 2c, Stx 2e) encoded by development of hemorrhagic colitis and and amplification technologies (17). Com-
genes carried on lambda bacteriophages HUS (2, 12, 18). merical kits can be used to detect toxins
(17). Research supports the fact that hem- Over 100 non-O157 serotypes of E. directly in the stool samples, in broth-en-
orrhagic colitis and HUS likely result from coli produce one or both Shiga toxins, and hanced stool cultures, and from colony
the action of these toxins on vascular en- over 50 serotypes have been associated sweeps of confirmed E. coli organisms
dothelium (17). Other virulence factors with hemorrhagic colitis or HUS (12). In growing on agar media (1, 4, 10, 16).
important for pathogenesis include an ad- Europe and Australia, non-O157 serotypes The advantage of using a method that
hesin, intimin, encoded by an eae gene, predominate, especially O111:Hⳮ, O26, detects toxins responsible for disease is
and enterohemorrhagic E. coli (EHEC) and O103:H5, among others (12). Small that theoretically all serotypes associated
hemolysin (hly gene), a potent cytolysin outbreaks of non-O157 serotypes have with hemorrhagic colitis and HUS could
(17). been reported in the United States. How- readily be detected. Such methods are also
E. coli O157:H7 is the major serotype ever, non-O157 serotypes are not system- rapid, allowing a prompt distinction be-
associated with illness in North America. atically and routinely looked for in U.S. tween STEC diarrhea and diarrhea attrib-
In the United States, the incidence of E. laboratories. Those laboratories that have utable to other infectious etiologies and
coli O157:H7 is approximately 2.1 to 2.9/ surveyed for them routinely have found noninfectious syndromes. The disadvan-
100,000 population (5). This is likely an that non-O157 STEC may cause disease tage of this procedure when used alone is
underestimate, since many laboratories do equal in severity to illness caused by O157 that an organism is not available for ser-
not routinely include selective media for (8, 11, 19). otyping and additional epidemiologic
this pathogen in routine stool cultures. E. coli O157:H7 can be detected on studies, such as tracing the source of out-
O157:H7 is more prevalent in certain geo- routine stool screening using sorbitol breaks. Another issue for laboratories is
graphic areas, such as the western and MAC (SMAC) or other selective media cost. Commercial kits for the detection of
midwestern United States. In the 1980s, that readily take advantage of the pheno- toxins are severalfold more expensive than
outbreaks of O157:H7 were associated typic differences between O157 (non- culture-based screening.
primarily with fast-food restaurants and sorbitol fermenting) and other E. coli

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE Commercial immunoassays for the detec- capture, the toxin present in stool speci-
tion of Stx 1 and Stx 2 consist of two ma- mens or culture systems. After incubation
jor methods: (i) EIAs and (ii) latex agglu- at room temperature, unbound material is
tination. The classic EIAs are performed washed from the microtiter wells. A sec-
in a standard microplate format. Available ondary enzyme-conjugated antibody is
kits use either monoclonal or affinity-pu- added, followed by incubation at room
rified polyclonal anti-Shiga toxin capture temperature and washing to remove un-
antibodies bound to microwells to bind, or bound conjugate. Substrate for the enzyme

11.8.1
Immunoassays for STEC 11.8.2

I. PRINCIPLE (continued) is added. If toxin is present a colorimeter are mixed with polymyxin B to enhance
reaction ensues as the antibody-enzyme toxin release and then added to the latex
complex converts the substrate to a reac- particles. These particles agglutinate and
tion product. The color development can settle diffusely over the base of a micro-
be detected visually or spectrophotomet- titer plate well when mixed with speci-
rically (1, 15, 17). mens containing toxin. If no toxins are
Reversed passive latex agglutination present, the latex particles will settle to
uses latex particles sensitized with anti- form a tight button (10, 17).
bodies against Stx 1 and 2. Test samples

II. SPECIMEN TYPES, Patients should be instructed to excrete directly into a clean, dry container and not
COLLECTION, AND TRANSPORT to take a specimen from the toilet water. The specimen should be free of urine.
Stool specimens should be transported to the laboratory as quickly as possible at 2
to 8⬚C (refrigerator temperatures). Diarrheal stool specimens are preferable to rectal
swabs. The latter, if collected, should have visible fecal matter and should be placed
immediately into transport media. Specimens that will also be cultured for enteric
pathogens should be placed in Cary-Blair or other enteric transport media if trans-
port time exceeds 2 h.
Testing can be performed directly on stool samples, on broth samples after over-
night incubation, and on culture plates after incubation for 16 to 24 h at 37⬚C. For
EIAs, stool samples may be stored at 2 to 8⬚C for up to 7 days. If testing is not to
be performed within that time frame, samples should be frozen at ⳮ20 to ⳮ70⬚C.
Repeated freeze-thawing of samples should be avoided because this leads to deg-
radation of toxin.

III. MATERIALS Commercial monoclonal and polyclonal Often there are additional supplies re-
reagents used in the immunoassays typi- quired but not provided by the manufac-
cally come packaged as kits. All reagents turers. These include, but are not limited
necessary for test performance are in- to, wooden applicator sticks, pipettes,
cluded. Examples of additional reagents tubes needed for dilutions, deionized wa-
supplied include positive and negative ter, squirt bottle, graduated cylinder, and
control materials, specimen diluent, wash an EIA reader capable of reading absorb-
buffers, color substrates, and stop solution. ance at 450 or 630 nm.

ANALYTICAL CONSIDERATIONS

IV. QUALITY ASSURANCE At the time of testing, all reagents should be examined for leakage, contamination,
and stability. Reagents from different lot numbers should not be mixed or inter-
changed. Each time an immunoassay is performed, a positive and negative control
Include QC information on
reagent container and in
should be included. Commercial kits contain a set of QC reagents, and each man-
QC records. ufacturer defines specific limits for the positive and negative control values. Ideally,
previously positive and negative patient specimens should be included as controls,
if not with every run then certainly when changing to a new lot of reagents and
when training new personnel.
When reading the EIA results visually, it is particularly important to adhere to
the manufacturer’s guidelines. In the case of colorimetric reactions, an intense color
is required for a definitive positive result. A pale color should be considered in-
determinate and the assay should then be repeated or, alternatively, the sample can
be read using a spectrophotometer. Similarly, when using a spectrophotometer a
result should be within the optical density (OD) cutoffs for positivity or negativity.
Any indeterminate assay should be repeated. In general, if a laboratory is experi-
encing a significant number of indeterminates or low positives (⬃5% of specimen
results), the most common reason is insufficient washing of the microwells. As
11.8.3 Immunology

IV. QUALITY ASSURANCE mentioned above, automated plate washers should be avoided, as sufficient washing
(continued) cannot be achieved (personal observations).
For interpretation of the latex agglutination results, the agglutination reaction in
the control latex wells is compared to that in the sensitized latex wells. Specimens
with indeterminate results should be retested, preferably by a different toxin method.
With both EIA and latex agglutination methods, direct stool testing leads to more
frequent observations of nonspecific results possibly related to interfering sub-
stances. Specificity is enhanced when testing is performed on broth cultures or
colonies picked from culture plates.
Since non-E. coli pathogens, such as Enterobacter and Shigella dysenteriae, may
also produce verocytotoxins, it is suggested that attempts be made to confirm the
source of toxin as one of the serotypes of E. coli associated with disease. This may
require simultaneous culture with identification of the enterics as E. coli and sub-
sequent testing of isolates for toxin production. Alternatively, sending positive stool
samples or toxin-positive enrichment broths to a public health or reference labo-
ratory for confirmation is an option.

V. PROCEDURES Instructions on test performance are provided with each Food and Drug Adminis-
tration (FDA)-cleared kit. All commercially available assays have procedures for
direct stool testing, broth culture, and plate culture methods. A brief overview of
It is imperative that these
cultures be handled in a EIA and latex agglutination methods follows (1, 10, 15).
biosafety hood.
A. Specimen preparation for EIA methods
1. Direct stool testing
a. The stool sample should be mixed thoroughly prior to pipetting.
b. For liquid samples, a defined aliquot is added to the manufacturer’s spec-
imen diluent using a scored transfer pipette. The volume of stool and the
amount of specimen diluent are specified by each manufacturer.
c. For solid specimens (nonpipettable) a 3- to 4-mm-diameter portion (pellet
sized) is added to specimen diluent and then emulsified using a wooden
applicator stick. Samples added to the specimen diluent should be mixed
using a vortex.
d. Patient samples transported in Cary-Blair medium can be added directly
to the microplate without further dilution.
2. Broth methods
a. The broth-enhanced method requires fresh stool or stool in Cary-Blair
medium to be added to 5 ml of MacConkey broth, TSB, modified TSB,
GN broth, or E-Z Coli broth (Difco Laboratories, Detroit, Mich.).
b. The sample is incubated overnight at 37⬚C.
c. A defined volume of broth growth is then added to specimen diluent prior
to EIA testing.
3. Culture plate methods
After the stool sample has been plated to MAC or a SMAC plate and incu-
bated for 16 to 24 h at 37⬚C, a colony sweep of a pure subculture or individual
colonies can be removed with a loop and placed in the specimen diluent for
EIA testing.
Observe standard precautions. B. Addition of specimen to microtiter plates
1. All reagents and microtiter wells should be allowed to reach room tempera-
ture (20 to 25⬚C). Reagents should be thoroughly mixed.
2. The required number of microwells, enough for each patient and the positive
and negative control, should be removed from the pouch.
Immunoassays for STEC 11.8.4

V. PROCEDURES (continued) 3. Add negative control to a test well and a positive control to a second well.
4. Using a transfer pipette, add a measured amount of dilute specimen or un-
diluted stool in Cary-Blair medium to each well. Care should be taken not
to splash into adjacent wells. This can be accomplished by inserting the
pipette tip no more than halfway into the well and allowing the sample to
slowly run down into the well.
5. Seal or cover the microwells and incubate at room temperature for 1 h.
Observe standard precautions. C. Washing unbound material
1. After incubation, the contents of the wells should be aspirated or shaken out
into a biohazard container.
2. Each well is washed with a buffered solution using a squirt bottle. After the
wells are filled with buffer, they are aspirated or inverted, and then the plates
are forcefully tapped on clean paper towels. This process is critical and
should be repeated three or four times as suggested by the manufacturer. The
use of an automated plate washer is not recommended (personal observa-
tions).
Observe standard precautions. D. Addition of detection antibody-enzyme conjugate
1. Depending upon which manufacturer’s kit is used, the addition of detection
antibody (usually a few drops) as specified by the manufacturer, followed
by enzyme conjugate or enzyme conjugate alone, occurs at this step.
2. The microwells are incubated at room temperature (22 to 27⬚C) for 30 min
and then washed as in step V.C.2 above.
Observe standard precautions. E. Addition of color substrate
Two free-falling drops of color substrate are added, followed by a short incu-
bation period at room temperature.
Observe standard precautions. F. Addition of stop solution
1. One or two drops of stop solution are added to each well.
2. The microwells should be read for the colorimetric reaction either visually
or spectrophotometrically within 10 to 30 min after the addition of the stop
solution, depending upon the manufacturer.
Observe standard precautions. G. Latex agglutination (10)
1. Specimen preparation
Direct stool testing is currently not recommended by the manufacturer.
a. Colony sweep testing
(1) After overnight culture of the stool sample on SMAC, a volume of
cells equal to a large paper matchstick head is placed into a micro-
centrifuge tube which contains 0.1 ml of the polymyxin B solution.
(2) The sample is incubated at 37⬚C (35 to 39⬚C) for 30 min. without
shaking.
(3) A total of 0.1 ml of 25% kaolin suspension in phosphate-buffered
saline is added, and the solution is mixed again and centrifuged at
12,000 rpm for 10 min. The supernatant is used for testing.
b. BHI agar enrichment method
(1) An aliquot of stool is inoculated onto SMAC or other selective media
and incubated overnight at 37⬚C.
(2) Bacterial cells from a dense area of colonies are picked up with a
sterilized cotton swab and are inoculated onto a BHI agar plate. The
entire plate is inoculated.
(3) The plate is incubated at 37⬚C for 6 h.
(4) A volume of growth equivalent to a paper matchstick head is sus-
pended in 0.1 ml of polymyxin solution.
(5) The suspension is incubated at 37⬚C for 30 min, with intermittent
shaking (every 5 to 10 min). This ensures optimal extraction of ver-
otoxins.
(6) A total of 0.1 ml of kaolin suspension is added.
11.8.5 Immunology

V. PROCEDURES (continued) (7) The suspension is mixed briefly and centrifuged at 12,000 rpm for
10 min.
(8) The supernatant is used for the test specimen.
c. Stationary culture from BHI agar
(1) A test isolate of E. coli is inoculated onto BHI agar and incubated
for 16 to 20 h at 37⬚C.
(2) A volume of cells equivalent to three paper matchstick heads is sus-
pended in 0.5 ml of polymyxin solution using a microbiological loop
or cotton swab.
(3) The suspension is incubated at 37⬚C for 30 min, with intermittent
shaking (every 5 to 10 min). This ensures optimal extraction of ver-
otoxins.
(4) The suspension is centrifuged at 12,000 rpm for 10 min.
(5) The supernatant is used for the test specimen.
d. Shaking broth method
(1) An E. coli test isolate is inoculated into 5 ml of Casamino Acid yeast
extract broth in a test tube.
(2) The broth is incubated aerobically, with shaking (120 to 150 strokes/
min), at 37⬚C for 16 to 20 h.
(3) The broth is then centrifuged at 900 ⳯ g for 15 to 20 min.
(4) The supernatant is used for the test specimen.
2. Reversed passive latex agglutination (10)
a. The microtiter plate is arranged so that each specimen is allotted two
wells, one for testing with the sensitized latex reagent and the other for
testing with the control latex reagent. The positive control and the reagent
control (negative control) are allotted to two and one well each, respec-
tively.
b. A total of 25 ll of the supernatant (test specimen, see above) is added to
each of the two allotted wells.
c. A total of 25 ll of sensitized latex is added to the first well and 25 ll of
the control latex is added to the second well for each specimen to be
tested.
d. For the positive control, 25 ll is added to two wells and 25 ll each of
sensitized and the control latex is added to the respective wells. For the
negative reagent control, 25 ll of diluent and 25 ll of sensitized latex
are added to the allotted well.
e. The plates are mixed well using a plate mixer or by carefully tapping by
hand.
f. The plates are sealed with a lid and then incubated for 16 h at room
temperature.
g. Agglutination patterns are observed by placing the plate over a black
surface.

POSTANALYTICAL CONSIDERATIONS

VI. INTERPRETATION Before reading patient specimen results, the positive and negative control wells
should be examined. Any color in the negative control well with the EIA method
constitutes an invalid result and the entire run should be repeated. For reversed
passive latex agglutinations if agglutination in the negative control well is seen, this
constitutes an invalid result and the kit should not be used.

A. EIA methods (1, 15)


As indicated above, results for an EIA can be read either visually or spectro-
photometrically. It is imperative that the manufacturer’s guidelines be followed
Immunoassays for STEC 11.8.6

VI. INTERPRETATION implicitly. A negative result is colorless and will yield a low OD reading. A
(continued) definitive positive is characterized by a strong (usually yellow) color. A positive
will demonstrate an OD reading usually well above the cutoff. Some manufac-
turers have created an “indeterminate” value or range. Results that fall into this
category typically have a pale or faint color or an OD reading that falls some-
where between the positive and negative cutoffs. Sample with indeterminate
results should be retested (see item IV above). A positive result indicates the
presence of Stx 1 or Stx 2.
Currently there are two FDA-cleared EIA kits available, the Premier EHEC
kit (Meridian Diagnostics, Inc., Cincinnati, Ohio) and the ProSpecT STEC mi-
croplate assay (Alexon-Trend, Inc., Ramsey, Minn.) (Table 11.8–1). Only the
Premier EHEC assay has been extensively reviewed in the literature (11, 13,
14, 16). Published reports indicate sensitivity in the range of 82 to 100% and
specificity in the range of 99 to 100% depending upon the comparative methods
and whether O157:H7 alone or all STEC organisms are considered (11, 13, 14,
16). These reported sensitivities are significantly better than SMAC culture re-
sults reported in the same studies (50 to 60%), even when only O157:H7 de-
tection is considered (9, 11, 13, 14, 16). Both commercial assays have better
sensitivity and specificity when testing is performed on broth-enhanced cultures
as opposed to direct stool samples (1, 15).
Clearly, the major advantage of these assays is improved detection of non-
O157 serotypes. On the other hand, there have been problems with false-positive
results leading to unnecessary public health concerns (6). The Council of State

Table 11.8–1 Commercial EIA reagents


Assay and
Method Notes FDA cleared?
manufacturer
Premier EHEC; Microwell EIA Monclonal capture Yes; approved for
Meridian Diag- antibody; poly- direct stool test-
nostics, Inc., clonal rabbit anti- ing and broth
Cincinnati, Ohio bodies specific for and plate cul-
Stx; goat anti-rab- tures
bit antibody con-
jugated to horse-
radish peroxidase

ProSpecT STEC Microplate EIA Rabbit polyclonal Yes; approved for


microplate assay; anti-Stx 1 and 2 direct stool test-
Alexon Trend, capture antibodies; ing and broth
Inc., Ramsey, monoclonal mouse cultures
Minn. anti-Stx 1 and 2
antibody conju-
gated to horserad-
ish peroxidase for
detection

VTEC-Screen (II) Reversed passive Polymyxin B and ka- No; multicenter tri-
Seiken; Denka latex agglutina- olin are added to als are in pro-
Seiken Co., Ltd., tion stool samples to gress in the
Tokyo, Japan increase toxin re- United States
lease and to en-
hance specificity,
respectively. Latex
particles are sensi-
tized with Stx 1
and 2 rabbit poly-
clonal antibodies.
11.8.7 Immunology

VI. INTERPRETATION and Territorial Epidemiologists adopted a position paper (9) calling for culture
(continued) confirmation of positive results from immunoassays (9).
B. Reversed passive latex agglutination
A commercial non-FDA-cleared kit, the VTEC-Screen (II) Seiken (Denka Sei-
ken Co. Ltd, Tokyo, Japan) detects the presence of Stx 1 and Stx 2. The agglu-
tination reactions in the control latex wells are compared to those in the sensi-
tized latex wells.
Preliminary studies (3, 4, 7) indicate a sensitivity slightly less than that of
the traditional verocytotoxin assay. One study on 239 strains representing 50
human and animal serotypes noted a clinical sensitivity of 100% and a speci-
ficity of 97% compared to the verocytoxin assay and PCR methods to detect
toxin genes (3). This product is currently undergoing clinical trials in the United
States.

REFERENCES 1. Alexon Trend, Inc. 2000. ProSpecT Shiga 10. Denka Seiken Co. Ltd. 2001. VTEC-Screen
toxin E. coli (STEC) microplate assay package (II) Seiken product package insert, p. 1–6.
insert. Alexon Trend, Inc., Ramsey, Minn. Denka Seiken Co. Ltd., Tokyo, Japan.
2. Banatvala, N., P. M. Griffin, K. D. Greene, 11. Fey, P. D., R. S. Wickert, M. E. Rupp, T. J.
T. J. Barrett, W. F. Bibb, J. H. Green, J. G. Safranek, and S. H. Hinrichs. 2000. Preva-
Wells, and the Hemolytic Uremic Syndrome lence of non-O157:H7 Shiga toxin-producing
Study Collaborators. 2001. The United Escherichia coli in diarrheal stool samples
States national prospective hemolytic uremic from Nebraska. Emerg. Infect. Dis. 6:530–
syndrome study: microbiologic, serologic, 533.
clinical and epidemiologic findings. J. Infect. 12. Karch, H., M. Bielaszewska, M. Bitzan, and
Dis. 183:1063–1070. H. Schmidt. 1999. Epidemiology and diag-
3. Bettelheim, K. A. 2001. Development of a nosis of Shiga toxin-producing Escherichia
rapid method for the detection of verocyto- coli infections. Diagn. Microbiol. Infect. Dis.
toxin-producing Escherichia coli (VTEC). 34:229–243.
Lett. Appl. Microbiol. 32:1–5. 13. Kehl, K. S., P. Havens, C. E. Behnke, and
4. Beutin, L., S. Zimmermann, and K. Gleier. D. W. Acheson. 1997. Evaluation of the Pre-
1996. Rapid detection and isolation of Shiga- mier EHEC assay for detection of Shiga toxin-
like toxin (Verocytotoxin)-producing Escher- producing Escherichia coli. J. Clin. Microbiol.
ichia coli by direct testing of individual ente- 35:2051–2054.
rohemolytic colonies from washed sheep 14. Mackenzie, A. M. R., P. Lebel, E. Orrbine,
blood agar plates in the VTEC-RPLA assay. P. C. Rowe, L. Hyde, F. Chan, W. Johnson,
J. Clin. Microbiol. 34:2812–2814. P. McLaine, and The Synsorb Pk Study In-
5. Centers for Disease Control and Preven- vestigators. 1998. Sensitivities and specifici-
tion. 2001. Preliminary FoodNet data on the ties of Premier E. coli O157 and Premier
incidence of foodborne illnesses—selected EHEC enzyme immunoassays for diagnosis of
sites, United States, 2000. Morb. Mortal. infection with verotoxin (Shiga-like toxin)-
Wkly. Rep. 50:241–246. producing Escherichia coli. J. Clin. Microbiol.
6. Centers for Disease Control and Preven- 36:1608–1611.
tion. 2001. University outbreak of calicivirus 15. Meridian Diagnostics. 2001. Premier EHEC
infection mistakenly attributed to Shiga toxin- product package insert, p. 1–7. Meridian Di-
producing Escherichia coli O157:H7—Vir- agnostics, Cincinnati, Ohio.
ginia, 2000. Morb. Mortal. Wkly. Rep. 16. Novicki, T. J., J. S. Daly, S. L. Mottice, and
50:489–491. K. C. Carroll. 2000. Comparison of sorbitol
7. Chart, H., G. A. Willshaw, and T. Cheasty. MacConkey agar and a two-step method
2001. Evaluation of a reversed passive latex which utilizes a chromogenic agar to detect
agglutination test for the detection of verocy- and isolate enterohemorrhagic Escherichia
totoxin (VT) expressed by strains of VT-pro- coli. J. Clin. Microbiol. 38:547–551.
ducing Escherichia coli. Lett. Appl. Microbiol. 17. Paton, J. C., and A. W. Paton. 1998. Patho-
32:370–374. genesis and diagnosis of Shiga toxin-produc-
8. Chiao, E., S. Mottice, G. Dowdle, J. Daly, ing Escherichia coli infections. Clin. Micro-
C. Barton, and K. C. Carroll. 2000. Demo- biol. Rev. 11:450–479.
graphic data, virulence factors and clinical 18. Tarr, P. I. 1995. Escherichia coli O157:H7:
findings of non-O157 Shiga toxin-producing clinical, diagnostic, and epidemiological as-
E. coli (STEC) in Utah, abstr. 904, p. 151. pects of human infection. Clin. Infect. Dis.
Abstr. 40th Intersci. Conf. Antimicrob. Agents 20:1–10.
Chemother. American Society for Microbiol- 19. Tarr, P. I., and M. A. Neill. 1996. Perspec-
ogy, Washington, D.C. tive: the problem of non-O157:H7 Shiga toxin
9. Council of State and Territorial Epidemi- (Verocytotoxin)-producing Escherichia coli.
ologists. 2001. Position statements 2000-ID- J. Infect. Dis. 174:1136–1139.
#1. Available at http://www.cste.org.
Immunoassays for STEC 11.8.8

SUPPLEMENTAL READING Acheson, D. W. K., and J. L. Jaeger. 1999. Shiga Kehl, S. C. 2002. Role of the laboratory in the
toxin-producing Escherichia coli. Clin. Microbiol. diagnosis of enterohemorrhagic Escherichia coli
Newsl. 21:183–188. infections. J. Clin. Microbiol. 40:2711–2715.
Boyce, T. G., A. G. Pemberton, J. G. Wells, and
P. M. Griffin. 1995. Screening for Escherichia
coli O157:H7—a nationwide survey of clinical
laboratories. J. Clin. Microbiol. 33:3275–3277.
Griffin, P. M., and R. V. Tauxe. 1991. The epi-
demiology of infections caused by Escherichia
coli O157:H7, other enterohemorrhagic E. coli,
and the associated hemolytic uremic syndrome.
Epidemiol. Rev. 13:60–98.
11.9 Serodiagnosis of Helicobacter pylori

In 1983, Marshall and Warren proposed tiple gastroduodenal diseases have empha- Commercial ELISAs detecting anti-H.
the possible association of Helicobacter sized the importance of accurate and pylori serum IgG are the serologic tests of
pylori with peptic ulcer disease and gastric prompt diagnosis of symptomatic individ- choice for the primary screening of pa-
cancer (17). In February 1994, the NIH uals. tients with uncomplicated infections. As-
Consensus Development Conference con- Multiple methods for assessment of H. says to detect anti-H. pylori IgA in sera
cluded that H. pylori infection is the major pylori infection are used in the clinical set- have reduced and variable sensitivities (39
cause of peptic ulcer disease and that all ting (Table 11.9–1). Invasive methods re- to 82%) compared to those of serum IgG
patients with documented peptic ulcer dis- quire biopsy by endoscopy and are rec- immunoassays. Serum IgA studies may be
ease associated with H. pylori infection ommended for patients with complicated useful in testing of symptomatic individ-
should receive antimicrobial therapy (20). infections who are suspected of having uals with equivocal or negative IgG find-
The prevalence of peptic ulcer disease peptic ulcer disease. Serologic testing rep- ings. A key advantage of follow-up serum
exceeds 6.5 million cases in the United resents a primary screening method for the IgA testing is that the same serum speci-
States, approximating a rate of 2.5%, or diagnosis of H. pylori infection in individ- men may be used. In one study, greater
2,500 cases per 100,000 individuals (23). uals with uncomplicated infections. Infec- than 7% of patients with negative serum
In developed countries, the overall prev- tion with H. pylori results in a vigorous IgG results were found to have detectable
alence of H. pylori infection ranges from local and systemic humoral response to anti-H. pylori serum IgA and symptoms
25 to 30% (5). Seroprevalence increases multiple antigens (14, 24, 25). In contrast consistent with H. pylori infection (13). As
with age, ranging from 5 to 27% in early to serum immunoglobulin M (IgM), serum determined by using the IgA immunoas-
childhood to levels exceeding 50% in IgA and IgG levels persist for months and say from HYCOR Biomedical (Irvine, Ca-
adults greater than 50 years of age (2, 12, years and correlate with active infection in lif.), five of six IgA-positive, IgG-negative
15, 17, 22). Seroprevalence studies dem- untreated individuals (1, 11, 19, 21). patients had peptic ulcers by endoscopy.
onstrate an acquisition rate in adults of 3 Only a minority of individuals did not Salivary and urinary antibody assays have
to 4% per decade. H. pylori infection is a show evidence of systemic seroconversion shown limited utility. Serum IgA assays
likely contributing factor in the develop- following infection (21). Anti-H. pylori lack the requisite sensitivity to serve as
ment of gastric neoplastic diseases, such serum IgG levels are more consistently el- primary screening tests but may be useful
as gastric adenocarcinoma and mucosa-as- evated than serum IgA levels. Conse- in cases in which infection is strongly sus-
sociated lymphoid tissue lymphoma (5). quently, serum IgG immunoassays yield pected and the serum IgG result is nega-
H. pylori infection is considered to in- superior sensitivities and specificities tive or equivocal.
crease the risk of gastric adenocarcinoma, compared with those of serum IgA assays
with pooled odds ratios of 1.9 to 2.5 (6, (16).
15). H. pylori and its association with mul-

PREANALYTIAL CONSIDERATIONS

I. PRINCIPLE (SERUM IgA OR


IgG ELISA)
ELISAs depend on the adsorption of pro- will bind to specific antigens on the solid peroxidase which then binds to the H. py-
tein or carbohydrate antigens to plastic phase, forming antigen-antibody com- lori antigen-antibody complexes. Excess
surfaces such as polystyrene (solid phase). plexes. Excess antibody is removed by conjugate is removed by washing and fol-
When antigens bound to the solid phase washing. Washing is followed by the ad- lowed by the addition of the chromogenic
make contact with a patient’s serum, H. dition of goat anti-human IgG (or anti-hu- substrate tetramethylbenzidine (TMB). If
pylori-specific IgG (or IgA), if present, man IgA) conjugated with horseradish specific IgG (or IgA) antibodies to H. py-

11.9.1
Serodiagnosis of H. pylori 11.9.2

lori antigens are present in the patient’s color change is indicative of the presence plate reader. ELISA-based detection of se-
serum, TMB is altered and a blue color of anti-H. pylori IgG in the patient’s serum rum anti-H. pylori IgG is the preferred ap-
develops. When the enzymatic reaction is and can be detected by absorbance spec- proach for antibody screening in assess-
stopped with 1 N sulfuric acid (H2SO4), trophotometry in an ELISA microwell ment of chronic H. pylori infection.
the contents of the wells turn yellow. The

II. SPECIMEN COLLECTION Optimal detection of anti-H. pylori IgG antibodies depends upon the presence of
fresh serum samples. Although point-of-care testing (POCT) kits for detection of
anti-H. pylori IgG and total antibodies in whole blood are available, assay sensitiv-
ities are reduced compared to those of serum-based ELISAs (8). Recommended
laboratory-based ELISAs include HM-CAP (Enteric Products, Westbury, N.Y.), H.
pylori IgG ELISA (Wampole Laboratories, Cranbury, N.J.), and Premier IgG
Table 11.9–1 Diagnostic and follow-up ELISA (Meridian Biosciences, Cincinnati, Ohio). A minimum volume of 50 ll is
testing approaches for assessment of H. recommended for collection in case repeat testing is required to clarify equivocal
pylori infection findings. Ten microliters of serum is used for testing. Serum specimens should be
Testing approach stored at 2 to 8⬚C if testing will occur within 48 h of collection. If prolonged storage
is required for batch testing, storage at ⳮ20⬚C or lower is recommended to mini-
Diagnostic testing
Noninvasive mize proteolysis of antibodies. Caution must be exercised to avoid hemolyzed,
IgG serology lipemic, or bacterially contaminated sera, as these samples may yield spurious re-
IgA serology sults (e.g., false positive). Sera should not be heat inactivated prior to testing.
Fecal antigen detection In order to facilitate in-office POCT for H. pylori infection, rapid serum and
Urea breath testing whole-blood IgG immunoassays have recently been developed by several manu-
Invasivea facturers (e.g., Quickvue H. pylori test by Quidel, San Diego, Calif., and Flexsure
Histopathology (special stains) HP by Beckman Coulter, Palo Alto, Calif.). Whole-blood assays may use small
Rapid urease testing volumes (drops) of capillary blood for POCT. A rapid latex agglutination test format
(Pyloriset, Orion Diagnostics, Espoo, Finland) is also available to detect serum anti-
H. pylori IgG. Such immunoassays produce a qualitative result in 4 to 10 min using
Follow-up testing
Noninvasive heparinized whole-blood or capillary blood specimens. Compared to those of serum
Fecal antigen detection EIAs, whole-blood serologic assays have reduced sensitivities (80 to 90% versus
Urea breath testing ⬎90%), with comparable specificities (70 to 80%) and lower overall accuracy (5).
a
Requires biopsy sampling by esophagogastro- Therefore, negative results must be confirmed by a laboratory-based serum IgG or
duodenal endoscopy. IgA ELISA, fecal antigen testing, or urea breath testing.

III. MATERIALS H. pylori antigens (derived from strain greater than 20 different immunogenic
ATCC 43504 and others) are commonly proteins identified in H. pylori (14).
used as reagents bound to solid phase for Goat anti-human IgG is commonly
detection of H. pylori-specific antibodies. used for detection of H. pylori-specific an-
Pooled H. pylori antigens of high-molec- tibodies in human serum. Goat IgG is con-
ular-weight surface-associated proteins, jugated with enzymes such as horseradish
acid extracts, or whole-cell lysates (soni- peroxidase to facilitate chromogen detec-
cates) are used in most serologic assays. tion. Chromogenic substrates such as
Various cytosolic and cell surface-associ- TMB are included for colorimetric detec-
ated proteins (18) represent immunodom- tion by absorbance spectrophotometry. A
inant antigens recognized by serum anti- single- or dual-wavelength microplate
bodies of infected individuals (14). reader capable of absorbance spectropho-
Immunodominant antigens include house- tometry at the appropriate wavelength
keeping enzymes such as ATP synthase (e.g., 450 nm for TMB) is necessary for
F1, RecA, and heat shock proteins HspA quantitative optical density determina-
and HspB. Essential colonization factors tions. If dual-wavelength spectrophotom-
such as urease subunit UreB and flagellar etry is performed, the reference filter is
protein FlaA also comprised a group of fixed at 600 to 650 nm.
11.9.3 Immunology

ANALYTICAL CONSIDERATIONS

IV. QUALITY ASSURANCE AND Blank reagent, positive, and negative controls must be included with each assay.
QUALITY CONTROL Commercial kits also provide calibrators and controls that should be included with
each assay. All of the kits described in this procedure provide serum controls.
Calibrators for verifying cutoff values should be provided in commercial serologic
kits and are useful for maintaining reproducible assay performance. The reagent
blank (usually the serum diluent provided in the kit) should be included with each
assay to set the baseline for optical density readings on an ELISA plate reader.
When read against an air blank, the absorbance should remain below 0.200 unit at
the recommended wavelength. Negative controls (provided in kit) and patient serum
negative controls should be included with each assay and yield readings below
0.200 absorbance unit. Positive controls should include low and high positive con-
trols and may include prior reactive patient sera. Dilutions of known positive sera
may provide useful low positive controls. Popular H. pylori serologic kits, such as
the Wampole Laboratories H. pylori IgG ELISA kit, recommend verification of
immune status ratios and stated ranges for high positive, low positive, and negative
controls. Proficiency testing is available by subscribing to the VR3 (serology) sur-
vey of the CAP.

V. PROCEDURE (SERUM IgG Serologic testing for assessment of H. pylori infection depends upon the detection
ELISA) of serum antibodies (IgA, IgG, or total antibodies) by EIAs. The recommended
approach for primary screening in the clinical laboratory is serum IgG detection by
ELISA (16). Samples for which the results are negative or equivocal may be retested
with serum IgA ELISA.

A. Serum preparation
Dilute the test sera from each individual patient in serum diluent provided by
the manufacturer. Typically, 10 ll of patient or control sera will be diluted in
200 ll of diluent (1:21 dilution) prior to testing.
B. Primary H. pylori antigen-serum antibody binding
1. Add calibrators, controls, and patient sera (dilutions) to individual wells.
Serum diluent must be added to reagent blank well for optical density cali-
bration.
2. Incubate each well at room temperature for 20 to 30 min.
3. Wash strips of wells by light agitation or shaking liquid from wells. Alter-
natively, wells may be aspirated by vacuum-assisted suction using a Pasteur
pipette tip at an angle. Repeat procedure a minimum of three times for suf-
ficient removal of nonspecific antigen-antibody interactions.
C. Detection of anti-H. pylori antibodies
1. Add conjugate to each well. Conjugate or conjugated antibody usually is
goat anti-human IgG linked covalently with horseradish peroxidase as re-
porter enzyme.
2. Incubate secondary antibodies in each well for 20 to 30 min.
3. Wash strips of wells by light agitation and aspiration as described above.
Repeat procedure a minimum of three times for sufficient removal of non-
specific antigen-antibody interactions.
4. Add chromogenic substrate (e.g., TMB) solution to each well and incubate
at room temperature for 10 to 15 min.
5. Stop reaction with 1 N H2SO4.
Serodiagnosis of H. pylori 11.9.4

V. PROCEDURE (SERUM IgG 6. Chromogenic reactions should be read on an ELISA plate reader equipped
ELISA) (continued) with an appropriate filter (e.g., for horseradish peroxidase-TMB, 450 nm).
If dual-wavelength absorbance spectrophotometry will be performed, the ref-
erence filter should be used at 600 to 650 nm.

POSTANALYTICAL CONSIDERATIONS

VI. INTERPRETATION Commercial EIAs detecting anti-H. pylori serum IgG are the tests of choice (16)
for primary screening of patients with uncomplicated infections. Patients with mild
dyspeptic symptoms or asymptomatic patients at risk may be screened by serologic
testing. A positive serology should be confirmed with direct detection of H. pylori
by fecal antigen testing or urea breath testing to verify the presence of active in-
fection. Patients with severe symptoms or “alarm” features such as weight loss or
bleeding should be diagnosed by endoscopy. Serologic results should be interpreted
qualitatively as the presence or absence of antibodies. Overall, the medians of the
sensitivity and specificity for H. pylori serology kits were 92 and 83%, respectively
(16). Performance varied significantly among commercial serologic kits, with top
performers exceeding 90% in sensitivity and specificity and bottom performers
failing to reach 90% in sensitivity or 80% in specificity (16). Anti-H. pylori sero-
logic assays from various commercial sources have variable sensitivities (57 to
100%) and specificities (31 to 100%) (16). Importantly, positive (95 to 100%) and
negative (84 to 89%) predictive values for serology were comparable to those of
histology, rapid urease testing, and urea breath testing (4). ELISA serologic testing
had the lowest cost per correct diagnosis, but overall accuracy was reduced com-
pared to that of stool antigen or urea breath testing (25). Patients infected with
“Helicobacter heilmannii” were usually negative by anti-H. pylori IgG assays (9).
Serum IgG immunoassays have markedly reduced sensitivities (50 to 60% versus
⬎90%) with samples from human immunodeficiency virus type 1-infected patients
(7). Therefore, a negative result in this setting must be confirmed by urea breath
testing or gastric biopsy. In addition to qualitative serologic approaches, notable
decreases in titer may be obtained within 6 to 12 months of completion of anti-
microbial therapy with quantitative serologies (3). However, at least 50% of patients
remain seropositive 6 months or longer following successful treatment (3, 10, 21).

REFERENCES 1. Blecker, U., S. Lanciers, B. Hauser, D. I. 6. Eslick, G. D., L. L. Lim, J. E. Byles, H. H.


Mehta, and Y. Vandenplas. 1995. Serology Xia, and N. J. Talley. 1999. Association of
as a valid screening test for Helicobacter py- Helicobacter pylori infection with gastric car-
lori infection in asymptomatic subjects. Arch. cinoma: a meta-analysis. Am. J. Gastroenterol.
Pathol. Lab. Med. 119:30–32. 94:2373–2379.
2. Cullen, D. J., B. J. Collins, K. J. Christian- 7. Fabris, P., L. Bozzola, P. Benedetti, M.
sen, J. Epis, J. R. Warren, I. Surveyor, and Scagnelli, R. Nicolin, V. Manfrin, C. Scar-
K. J. Cullen. 1993. When is Helicobacter py- paro, and F. De Lalla. 1997. H. pylori infec-
lori infection acquired? Gut 34:1681–1682. tion in HIV-positive patients. A serohistolog-
3. Cutler, A., A. Schubert, and T. Schubert. ical study. Dig. Dis. Sci. 42:289–292.
1993. Role of Helicobacter pylori serology in 8. Faigel, D. O., N. Magaret, C. Corless, D. A.
evaluating treatment success. Dig. Dis. Sci. Lieberman, and M. B. Fennerty. 2000. Eval-
38:2262–2266. uation of rapid antibody tests for the diagnosis
4. Cutler, A. F., S. Havstad, C. K. Ma, M. J. of Helicobacter pylori infection. Am. J. Gas-
Blaser, G. I. Perez-Perez, and T. T. Schu- troenterol. 95:72–77.
bert. 1995. Accuracy of invasive and nonin- 9. Hilzenrat, N., E. Lamoureux, I. Weintrub,
vasive tests to diagnose Helicobacter pylori E. Alpert, M. Lichter, and L. Alpert. 1995.
infection. Gastroenterology 109:136–141. Helicobacter heilmannii-like spiral bacteria in
5. Dunn, B. E., H. Cohen, and M. J. Blaser. gastric mucosal biopsies. Prevalence and clini-
1997. Helicobacter pylori. Clin. Microbiol. cal significance. Arch. Pathol. Lab. Med.
Rev. 10:720–741. 119:1149–1153.
11.9.5 Immunology

REFERENCES (continued) 10. Hirschl, A. M., and M. L. Rotter. 1996. Se- 18. Megraud, F. 1996. Helicobacter pylori: bac-
rological tests for monitoring Helicobacter py- terial factors and interaction with the epithelial
lori eradication treatment. J. Gastroenterol. cells. Yale J. Biol. Med. 69:35–37.
31(Suppl. 9):33–36. 19. Morris, A. J., M. R. Ali, G. I. Nicholson, G.
11. Hunt, R. H. 1997. Peptic ulcer disease: defin- I. Perez-Perez, and M. J. Blaser. 1991.
ing the treatment strategies in the era of Hel- Long-term follow-up of voluntary ingestion of
icobacter pylori. Am. J. Gastroenterol. Helicobacter pylori. Ann. Intern. Med.
92:36S–40S. 114:662–663.
12. Isaacson, P. G. 1994. Gastric lymphoma and 20. NIH Consensus Conference. 1994. Helico-
Helicobacter pylori. N. Engl. J. Med. bacter pylori in peptic ulcer disease. NIH Con-
330:1310–1311. sensus Development Panel on Helicobacter
13. Jaskowski, T. D., T. B. Martins, H. R. Hill, pylori in Peptic Ulcer Disease. JAMA 272:65–
and C. M. Litwin. 1997. Immunoglobulin A 69.
antibodies to Helicobacter pylori. J. Clin. Mi- 21. Perez-Perez, G. I., B. M. Dworkin, J. E.
crobiol. 35:2999–3000. Chodos, and M. J. Blaser. 1988. Campylo-
14. Kimmel, B., A. Bosserhoff, R. Frank, R. bacter pylori antibodies in humans. Ann. In-
Gross, W. Goebel, and D. Beier. 2000. Iden- tern. Med. 109:11–17.
tification of immunodominant antigens from 22. Sipponen, P., T. U. Kosunen, I. M. Samloff,
Helicobacter pylori and evaluation of their O. P. Heinonen, and M. Siurala. 1996. Rate
reactivities with sera from patients with dif- of Helicobacter pylori acquisition among
ferent gastroduodenal pathologies. Infect. Im- Finnish adults: a fifteen year follow-up. Scand.
mun. 68:915–920. J. Gastroenterol. 31:229–232.
15. Kosunen, T. U., A. Aromaa, P. Knekt, A. 23. Sonnenberg, A. 1996. Cost-benefit analysis of
Salomaa, H. Rautelin, P. Lohi, and O. P. testing for Helicobacter pylori in dyspeptic
Heinonen. 1997. Helicobacter antibodies in subjects. Am. J. Gastroenterol. 91:1773–1777.
1973 and 1994 in the adult population of Vam- 24. Tinnert, A., A. Mattsson, I. Bolin, J. Dal-
mala, Finland. Epidemiol. Infect. 119:29–34. enback, A. Hamlet, and A. M. Svenner-
16. Laheij, R. J., H. Straatman, J. B. Jansen, holm. 1997. Local and systemic immune re-
and A. L. Verbeek. 1998. Evaluation of com- sponses in humans against Helicobacter pylori
mercially available Helicobacter pylori serol- antigens from homologous and heterologous
ogy kits: a review. J. Clin. Microbiol. strains. Microb. Pathog. 23:285–296.
36:2803–2809. 25. Vakil, N., D. Rhew, A. Soll, and J. J. Ofman.
17. Marshall, B. J., and J. R. Warren. 1984. Un- 2000. The cost-effectiveness of diagnostic
identified curved bacilli in the stomach of pa- testing strategies for Helicobacter pylori. Am.
tients with gastritis and peptic ulceration. Lan- J. Gastroenterol. 95:1691–1698.
cet 1:1311–1315.
11.10 Total Viable Cell Counting
Procedure

I. PRINCIPLE Trypan blue allows rapid observation of


nonviable cells present in a peripheral
blood mononuclear cell suspension
(PBMCS). A count of viable cells present
in a PBMCS allows the suspension to be
adjusted accordingly for further use in as-
says.

II. SPECIMEN A PBMCS in a known volume of cell culture medium

III. MATERIALS A. Trypan blue (0.4%; Sigma catalog


no. T81540, 100-ml bottle)
1. Filter as needed.
2. Store at room temperature.
B. Hemacytometer with appropriate
coverslip
C. Pipettes

IV. QUALITY CONTROL A. Perform daily.


B. A drop of trypan blue is mixed with a drop of distilled water. The preparation
is screened for contamination by bacteria.

V. PROCEDURE A. In a 5-ml test tube, add 90 ll of the 0.4% trypan blue solution to 10 ll of the
cell suspension.
B. Mix cells and trypan blue and let stand for 1 to 3 min.
C. Make sure hemacytometer is clean; place appropriate clean coverslip on empty
unit.
D. Load one chamber of the double-sided hemacytometer with 10 to 12 ll of cell-
trypan blue mixture. Avoid air bubbles.
E. Allow cells to settle; let stand for at least 1 min.

11.10.1
11.10.2 Immunology

VI. READING AND REPORTING A. Screen chamber under low power.


RESULTS B. Use 40⳯ high-power magnification to count cells.
C. The blue-tinged cells are counted as dead, nonviable cells. The translucent cells
are counted as live, viable cells.
D. Count all the cells present inside the large four corner squares of the hemacy-
tometer. Record as viable or nonviable.
E. Calculate percentages of viable and nonviable cells.
F. Report total viable cell count.

VII. CALCULATIONS A. Total viable cell count

冢 冣
Total no. of cells in 4 large squares
⳯ 104 ⳯ volume of cell suspension
4 [number of squares counted]
⳯ dilution factor

B. Dilution factor

90 ll of trypan blue Ⳮ 10 ll of cells


⳱ 10 (dilution factor)
10 ll of cells

SUPPLEMENTAL READING Kruse, P. F., and M. K. Patterson. 1973. Tissue


Culture—Methods and Applications, p. 406. Ac-
ademic Press, New York, N.Y.
11.11 Peripheral Blood Mononuclear Cell
Cryopreservation Method

I. PRINCIPLE To be able to cryopreserve peripheral


blood mononuclear cells (PBMCs) for
utilization in future testing

II. SPECIMEN Blood collected in heparin or acid citrate dextrose

III. MATERIALS AND REAGENTS A. Fetal calf serum (heat inactivated at F. Nalgene “Mr. Frosty” (catalog no.
56⬚C for 30 min) 288-383; Curtis Matheson Scientific,
B. RPMI 1640 medium Houston, Tex.)
C. Ficoll-Hypaque G. Optional: cryotube holder that sta-
D. Dimethyl sulfoxide (DMSO) (catalog bilizes tubes on ice (Corning work-
no. 27,043-1; Aldrich, Milwaukee, station from Fisher Scientific; cata-
Wis.) log no. 03-374-37A)
DMSO must be fresh. Once the bottle H. Plasticware, pipettes
is opened, the shelf life of DMSO I. Sterile phosphate-buffered saline,
should not exceed 6 months. Ca2ⴐ and Mg2ⴐ free
E. Cryogenic vials (cryovials): 2-ml Use for washing cells.
polyethylene vial with screw cap
(catalog no. 03-341-20; Fisher Sci-
entific)
䊓 NOTE: Polypropylene cryovials
are not recommended for storage in
liquid nitrogen unless sealed in Cry-
oflex tubing (catalog no. 12-565-177;
Fisher) using a heat sealer (catalog no.
01-812-15; Fisher).

IV. PROCEDURE A. Label cryovials and chill at ⳮ20⬚C for 30 min.


B. Prepare freezing solution (90% fetal calf serum plus 10% DMSO); chill on ice.
C. Prepare PBMCs by Ficoll-Hypaque density centrifugation; wash twice at 4⬚C.
Adjust cells, and wash again at 4⬚C. (After Ficoll separation, keep cells chilled
or on ice at all times.)
D. Resuspend cells in freezing solution at 107 cells/ml; aliquot 0.5 to 1.0 ml of the
cell suspension per cryovial. Be sure cryovial caps are securely tightened.
E. Immediately place cryovials in “Mr. Frosty” and transfer to a ⳮ70⬚C freezer.
Alternatively, place cryovials in a Cryomed freezing chamber, lowering the
temperature at ⳮ1⬚C per minute to ⳮ70⬚C.
F. Transfer cryovials to liquid nitrogen within 24 h.

11.11.1
11.11.2 Immunology

V. THAWING PROCEDURE A. Transfer cryovial from liquid nitrogen to a 37⬚C water bath. If liquid nitrogen
has seeped into the cryovial, loosen the cap slightly to allow the nitrogen to
escape during thawing.
䊏 Some cryovials have been reported to explode during the thawing process.
To minimize this risk, use only unbreakable polyethylene vials for storage in
liquid nitrogen (see item III above). If polypropylene tubes are used, they should
be sealed in Cryoflex tubing.
B. Hold the cryovial in the surface of the water bath with an occasional gentle flick
during thawing. Do not leave cryovial unattended during the thawing process.
(It is important for cell viability that the cells are thawed and processed quickly.)
When a small bit of ice remains in the cryovial, transfer the cryovial to the
biosafety hood. Dry off the outside of the cryovials before opening to prevent
contamination.
C. Add RPMI 1640 plus 10% fetal calf serum dropwise into the cryovial containing
the cell suspension, slowly (over 2 min), up to a volume which doubles the
original volume (e.g., add 1 ml of medium to a vial containing 1 ml of cell
suspension).
D. Transfer the cell suspension to a 15-cc conical-bottom centrifuge tube contain-
ing 8 ml of RPMI 1640 plus 10% fetal calf serum; wash twice by centrifugation,
gently resuspending the cells between washes.
E. Determine cell number and viability.

REFERENCE 1. Laboratory Technologists Committee of the


Adult AIDS Clinical Trials Group. 2002.
Consensus protocol for PBMC cryopreserva-
tion and thawing, version 3.0. http://aactg.s.-
3.com/pub/download/vir/freezingproto-
col.doc.
11.12 Lymphocyte Proliferation Assay
[Updated March 2007]

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE
Measuring the proliferative capability of such as phytohemagglutinin or pokeweed each well. The isotope is incorporated into
lymphocytes is important in the evaluation mitogen, recall antigens such as Candida the newly synthesized DNA of the prolif-
of an individual’s immune system. This albicans and tetanus toxoid, and other an- erating cells, and after a 6-h incubation the
can be achieved in an assay which cultures tigens which may be of specific interest cells are harvested. A scintillation counter
lymphocytes with either a mitogen or an- (such as human immunodeficiency virus is used to measure the activity of the tri-
tigen that can stimulate a proliferative proteins gp120 or p24). Separated periph- tiated thymidine, expressed as counts per
response and then labels the cells with a eral blood mononuclear cells (PBMCs) are minute. This measurement is directly pro-
radioactive marker to allow for measure- incubated at 37⬚C for 6 days in a 96-well portional to the level of lymphocyte pro-
ment of response (1). plate with a mitogen or antigen in tripli- liferation.
The lymphocyte proliferation assay cate wells. [3H]thymidine is then added to
(LPA) exposes lymphocytes to mitogens

II. SPECIMEN Whole blood collected in an acid-citrate-dextrose (ACD) or heparin tube. These
samples must be processed within 30 h of draw time.

III. REAGENTS AND SUPPLIES A. Reagents 5. 1-ml sterile polystyrene aspirating


1. LPA medium (see Appendix pipettes
11.12–1 for preparation) 6. 20-, 100-, and 200-ll pipettors
2. Ficoll-Paque PLUS 7. Multichannel pipettor
3. 1⳯ phosphate buffer solution 8. Pipette tips to deliver 5 to 200 ll
(PBS) 9. 15-ml sterile polystyrene centri-
4. Crystal violet (see Appendix fuge tubes with conical bottoms
11.12–1 for preparation) 10. 50-ml sterile polypropylene cen-
5. Trypan blue trifuge tubes with conical bottoms
6. [3H]thymidine, 6.7 Ci/mmol, 1 11. 12- by 75-mm sterile polystyrene
mCi/ml culture tubes with closures (snap
7. Pooled heat-inactivated human cap)
AB serum (Cambrex) 12. Glass microfiber filter paper
8. Mitogens (assay dependent) 13. 6-ml glass scintillation vials with
9. Recall antigens (assay dependent) cap
10. Specific antigens of interest (assay 14. Scintillation fluid—Betafluor. If
dependent) you are using a TopCount ma-
B. Supplies chine, then you will need Micro-
1. Pipet-Aid scint 0.
2. 10-ml sterile polystyrene pipettes 15. Unifilter plate, top seal, backing
3. 5-ml sterile polystyrene pipettes tape (if using a TopCount ma-
4. 2-ml sterile polystyrene aspirating chine)
pipettes

11.12.1
11.12.2 Immunology

III. REAGENTS AND SUPPLIES 16. 3H waste containers (check with C. Equipment
(continued) your institution regarding what 1. Variable-speed centrifuge
type of waste container for 3H you 2. Cell harvester
are to use) 3. Liquid scintillation counter or
17. Hemacytometer with coverslip TopCount machine
18. Ziploc bags 4. Biosafety cabinet with vacuum trap
19. 96-well round-bottom plates system for aspiration
20. Steriflips (0.2-lm pore size) 5. Incubator, 37⬚C, 5% CO2, 96%
21. PES filter unit, 150 ml (0.2-lm relative humidity
pore size; 50-mm-diameter mem- 6. Microscope
brane)

ANALYTICAL CONSIDERATIONS

IV. QUALITY CONTROL Calibration

A. Scintillation counter
Include QC information on 1. Normalization of scintillation counter must be done once a week.
reagent container and in 2. Background and 3H standards must be assayed with each run.
QC records.
3. Preventive maintenance should be performed twice per year.
B. TopCount machine
1. Normalization of TopCount machine must be done once a week.
2. Preventive maintenance should be performed twice per year.
C. Pipettors
Pipettors should be calibrated twice per year.
D. Equipment
All laboratory equipment should have preventive maintenance performed ac-
cording to the manufacturers’ instructions.

V. PROCEDURE 䊓 NOTE: All of the pipetting and filtering should take place in a biosafety cabinet
using sterile technique, and all reagents should be at room temperature when used.
All radioactive materials must be used in designated areas and must be disposed of
It is imperative that these
cultures be handled in a properly per the institution’s radiation safety guidelines.
biosafety hood.
A. Plate preparation
LPA plates can be made in advance and stored at ⳮ70⬚C in Ziploc polystyrene
Observe standard precautions. bags until they are needed. LPA plates consist of triplicate wells containing 100
ll of either medium (background), antigen, or mitogen prepared at 2⳯ in me-
dium. The medium used is LPA medium plus 20% pooled heat-inactivated
human AB serum to yield a final concentration of 10% pooled human AB serum
after the PBMCs are added. Some commonly used final concentrations are as
follows.
1. Tetanus toxoid: 2.5 and 1.25 lg/ml
2. C. albicans: 50 and 10 lg/ml
3. Pokeweed mitogen: 10 lg/ml
A titration is recommended to determine the optimal concentration for all stim-
ulants.
B. PBMC separation
1. Centrifuge ACD or heparin tube for 10 min at 200 ⳯ g at room temperature.
2. Gently invert the Ficoll-Paque PLUS bottle twice.
3. Using a pipette, remove the plasma from the ACD or heparin tube and place
into a snap-cap tube. This can be aliquoted and frozen, or discarded if
desired.
4. Dilute the blood twofold using 1⳯ PBS. Mix gently by pipetting up and
down.
Lymphocyte Proliferation Assay 11.12.3

V. PROCEDURE (continued) 5. Pipette 5 ml of Ficoll-Paque PLUS into a 15-ml conical tube.


6. Carefully layer the blood onto the 5 ml of Ficoll-Paque PLUS. Be sure not
to let the diluted blood penetrate the Ficoll-Paque PLUS.
7. Centrifuge the conical tube containing the diluted blood on Ficoll-Paque
PLUS at 300 to 400 ⳯ g for 30 min at room temperature.
8. After centrifugation, four layers will be visible. Gently aspirate the top clear
layer, which contains PBS and any remaining plasma. Be careful not to
disturb the cloudy layer underneath, which contains the mononuclear cells.
If you do not have an in-house vacuum system, then use a 5-ml pipette to
remove the top clear layer. Eject fluid into a waste container, such as an
empty 15-ml conical tube, and treat as biohazardous waste.
9. The cloudy mononuclear cell layer is removed next by placing the tip of a
pipette over the layer and gently moving the pipette in a circular motion
while applying suction with the pipette. The entire layer should be removed
and transferred to a 15-ml conical tube containing 5 ml of 1⳯ PBS. Care
should be taken to remove as little as possible of the Ficoll-Paque PLUS
layer below the mononuclear cells. Centrifuge the conical tube at 200 ⳯ g
for 10 min at room temperature.
10. After centrifugation, aspirate the supernatant, making sure not to disturb
the cell pellet.
11. Pipette 10 ml of 1⳯ PBS into the conical tube containing the pellet. Using
the pipette, thoroughly mix to resuspend the pellet.
12. Centrifuge the conical tube at 200 ⳯ g for 10 min at room temperature.
13. After centrifugation, aspirate the supernatant without disturbing the pellet.
14. Pipette 1 ml of LPA medium into the conical tube containing the pellet.
Using the pipette, thoroughly mix to resuspend the pellet.
15. Count the PBMCs using the total viable cell counting procedure (see pro-
cedure 11.9). This procedure uses trypan blue dye to count the viability and
number of PBMCs. To better distinguish mononuclear cells, it is recom-
mended that the cell count be performed using crystal violet.
16. Adjust the concentration of the cells to 106/ml with LPA medium. The total
number of cells needed for a plate can be found by multiplying the number
of wells by 100,000. A minimum of 600,000 is usually required, which
allows you to do one control triplicate and one mitogen triplicate.
17. Retrieve the appropriate premade LPA plate from the freezer. After remov-
ing the polystyrene bag, put the plate in the incubator to defrost.
18. Once the plate is fully defrosted, label it appropriately with patient infor-
mation, study information, and date.
19. Mix PBMCs well and add 100 ll of the PBMC suspension to each well of
the plate that contains a stimulant, changing tips every triplicate. Also add
100 ll in triplicate to the medium control wells.
20. Tap plate gently to mix.
21. Place the plate in the incubator for 6 days.
22. On the sixth day, add 50 ll of [3H]thymidine working solution per well to
yield 1 lCi/well. The working solution should be made fresh monthly (or-
der new stock vial of [3H]thymidine every 6 months).
23. Tap plate gently to mix.
24. Return the plate to the incubator for 6 h.
25. After 6 h, harvest the plate using a cell harvester.
11.12.4 Immunology

V. PROCEDURE (continued) 26. Leave the filters to dry overnight. The following day, put the filters in 6-
ml glass scintillation vials and add 2 ml of a scintillation fluid such as
Betafluor to each of these vials. Alternatively, you can use a Filtermate
harvester to harvest the entire plate onto a Unifilter plate. Leave the Unifilter
plate to dry overnight. The following day, put backing tape on the plate.
Add 30 ll of Microscint 0 to each well, and put a top seal on the plate.
27. Count each vial or well for 1 min using a liquid scintillation counter or
TopCount machine.

POSTANALYTICAL CONSIDERATIONS

VI. CALCULATIONS A. To determine net counts


Net counts ⳱ (experimental counts per minute ⳮ background unstimulated
counts per minute)
B. To determine stimulation index (SI)
SI ⳱ (experimental counts per minute/background unstimulated counts per min-
ute)

VII. INTERPRETATION An SI greater than 3 (antigens) to 5 (mitogens) is generally considered to be a


significant response to the specific antigenic or mitogenic stimulus.

REFERENCES 1. Fletcher, M. A., N. Klimas, R. Morgan, and


G. Gjerset. 1992. Lymphocyte proliferation,
p. 213–219. In N. R. Rose, E. Conway de Ma-
cario, J. L. Fahey, H. Friedman, and G. M.
Penn (ed.), Manual of Clinical Laboratory Im-
munology, 4th ed. American Society for Mi-
crobiology, Washington, D.C.

SUPPLEMENTAL READING Froebel, K. S., N. G. Pakker, F. Aiuti, M. Bofill,


H. Choremi-Papadopoulou, J. Economidou, C.
Rabian, M. T. L. Roos, L. P. Ryder, and F. Mie-
dema. 1999. Standardization and quality assur-
ance of lymphocyte proliferation assays for use in
the assessment of immune function. J. Immunol.
Methods 227:85–97.

APPENDIX 11.12–1 Reagent Information and Preparation


A. LPA medium
1. 48.5 ml of RPMI 1640
2. 0.5 ml of 200 mM L-glutamine for a final concentration of 2 mM L-glutamine
3. 0.5 ml of 10,000-U/ml penicillin and 100-lg/ml streptomycin solution for a final
concentration of 100 U of penicillin per ml and 100 lg of streptomycin per ml in
solution
4. 0.5 ml of 1 M HEPES buffer for a final of 10 mM HEPES buffer
B. Crystal violet
1. 98 ml of deionized H2O
2. 2 ml of glacial acetic acid
3. 0.05 g of crystal violet dye (powder form)
C. [3H]thymidine working solution (20 lCi/ml)
1. 24.5 ml of 1⳯ PBS
2. 0.5 ml of [3H]thymidine at 1 mCi/ml
D. Reagent and special supply information
See table on next page.
Lymphocyte Proliferation Assay 11.12.5

Reagent Manufacturer Catalog no. Address and website Phone no.


[ H]thymidine (1 mCi/ml)
3
Perkin-Elmer NET-027 549 Albany St. (800) 551-2121
Boston, MA 02118

Ficoll-Paque PLUS Amersham Pharmacia 17-1440-03 800 Centennial Ave. (800) 526-3593
P.O. Box 1327
Piscataway, NJ 08855-1327
http://www.apbiotech.com

HEPES buffer Invitrogen 15630-080 1600 Faraday Ave. (800) 955-6288


P.O. Box 6482
Carlsbad, CA 92008
http://www.invitrogen.com

L-Glutamine Bio-Whittaker 17-605E 8830 Biggs Ford Rd. (800) 638-8174


Walkersville, MD 21793
http://www.cambrex.com

Penicillin-streptomycin Invitrogen 15140-148 1600 Faraday Ave. (800) 955-6288


P.O. Box 6482
Carlsbad, CA 92008
http://www.invitrogen.com

RPMI 1640 Bio-Whittaker 12-167F (500 ml) 8830 Biggs Ford Rd. (800) 638-8174
12-167Q (1,000 ml) Walkersville, MD 21793
http://www.cambrex.com

Glacial acetic acid Sigma A-6283 P.O. Box 14508 (800) 325-3010
St. Louis, MO 63178
http://www.sigma-aldrich.com/order

1⳯ PBS HyClone SH30256.01 1725 South HyClone Rd. (800) hyclone


Logan, UT 84321
http://www.hyclone.com

Crystal violet dye (powder) Sigma A-6158 P.O. Box 14508 (800) 325-3010
St. Louis, MO 63178
http://www.sigma-aldrich.com/order

Human AB serum Cambrex 14-490E One Meadowlands Plaza (201) 804-3000


(heat inactivated) East Rutherford, NJ 07073

Trypan blue dye Invitrogen 15250-061 1600 Faraday Ave. (800) 828-6686
P.O. Box 6482
Carlsbad, CA 92008
http://www.invitrogen.com
11.13 Natural Killer Cell Assays
[Updated March 2007]

11.13.1 Introduction
[Procedure added March 2007]

Natural killer (NK) cells are another group NK activity and/or depressed absolute The role of NK cells in viral disease has
of cytolytic lymphocytes, distinct from B numbers of circulating NK cells, has been been known for a long time. The correla-
lymphocytes and T lymphocytes, that par- linked to the development and progression tion between low NK activity and serious
ticipate in both innate immunity and adap- of cancer, chronic and acute viral infec- viral infections in immunocompromised
tive immunity. NK cells are lymphocytes tions (including AIDS), various immuno- hosts after transplantation and in certain
that lack B-cell receptors and T-cell recep- deficiencies, and certain autoimmune dis- congenital immunodeficiencies has been
tors. They are designed to kill certain mu- eases. well documented.
tant cells and virus-infected cells. Nor- These cells characteristically lack CD3 The Practice Parameter for the Diag-
mally present as a small subpopulation of but express CD16, CD56, and CD57. NK nosis and management of Primary Immu-
circulating lymphocytes, NK cells mor- cell proliferation occurs in a number of re- nodeficiency recommends testing NK
phologically appear as so-called “large active and autoimmune conditions. The cells, quantitative and qualitative function,
granular lymphocytes.” Like cytotoxic T transient, reactive proliferations include in patients with severe disease caused by
cells, they attack and kill tumor cells and associations with viral infections, hepati- herpesviruses or papillomaviruses who do
protect against a wide variety of infectious tis, nephrotic syndrome, and lymphoma. not have another defined immunodefi-
microbes. They are “natural” killers be- Chronic, reactive proliferations can be ciency. The assays described in proce-
cause they do not need additional stimu- seen in a number of autoimmune diseases. dures 11.13.2 and 11.13.3 quantify NK
lation or to recognize a specific antigen in In addition, a chronic, clonal proliferative cells by flow cytometry as well as evaluate
order to attack and kill. NK cells appear disorder of NK cells (large granular lym- NK cell function.
to play a role in a variety of human dis- phoproliferative disorder) has been de-
eases. Compromised or absent natural im- scribed.
munity, as measured in vitro by decreased

11.13.1.1
11.13.2 Natural Killer Cell Flow Cytometry
Assay
[Procedure added March 2007]

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE
The Practice Parameter for the Diagnosis countered in patients with decreased NK and Shapiro et al. (4) in 1977. These stud-
and Management of Primary Immunode- cell function in the absence of other de- ies developed the principles of using for-
ficiency (3) states that patients with severe fined immunodeficiency, NK cells should ward and angular light scatter measure-
disease caused by herpesviruses or papil- be specifically assessed by flow cytometry ments from individual cells passing by
lomaviruses who do not have another de- using anti-CD16 clone B73.1. However, it fluid laminar flow through various light
fined immunodeficiency should have phe- is important to note that many clinical im- sources to characterize subpopulations of
notypic and functional assessments of munology laboratories use anti-CD16 cells from heterogeneous populations.
natural killer (NK) cells performed. These clone B73.1 and anti-CD56 conjugated Shapiro et al. expanded upon these studies
patients need to be assessed for complete with the same fluorophore in the same tube to incorporate histochemical fluoro-
absence of all NK cells (all CD56Ⳮ cells), (identifies cells CD16Ⳮ or CD56Ⳮ). In this chromes to identify subpopulations of pe-
classical NK cells (absence of CD56Ⳮ type of assay, patients with the L48H sub- ripheral blood leukocytes. These studies
CD3ⳮ cells but presence of CD56Ⳮ CD3Ⳮ stitution will not be identified. Therefore, established the principles to be used by
cells), and defective NK cell function it is important to use anti-CD16 clone second- and third-generation instruments,
(presence of CD56Ⳮ CD3ⳮ cells but ab- B73.1 alone and not in conjunction with which, with improved data acquisition and
sence of NK cell cytotoxicity). In addition, anti-CD56 in the same tube. In our labo- analysis capability, have produced ex-
the Practice Parameter for Immunology ratory, we use two different clones of anti- traordinarily sophisticated fluorescence-
(3) states that a mutation in the gene CD16: anti-CD16(B73.1) and anti- activated flow cytometers for both bio-
(FCGR3A) coding for CD16 that causes CD16(3G8). If the patient is homozygous medical research and routine clinical
an amino acid substitution results in im- for the FCGR3A mutation, the NK cells diagnostics. Further, the development of
paired NK cell cytotoxicity and suscepti- will be positive for expression of anti- monoclonal antibody technology provided
bility to recurrent or severe herpesvirus in- CD16(3G8) but not with anti- the necessary reagents for the immuno-
fections. Patients with isolated defects of CD16(B73.1). Below is an example of the phenotyping of a broad range of hemato-
cellular immunity who do not have defects assay showing normal NK cells (Fig. poietic cells, which has led to important
of gamma interferon/interleukin 12 axis 11.13.2–1). The cells are gated on the diagnostic procedures such as the more
should be screened for this particular mu- CD3ⳮ population and analyzed for ex- detailed classification of leukemias and
tation in FCGR3A by flow cytometry us- pression of anti-CD16(3G8) and anti- lymphomas.
ing anti-CD16 clone B73.1. Patients ho- CD16(B73.1) and CD56. The percent Whole blood can be stained with anti-
mozygous for this mutation have NK cells gated and absolute values are reported, in bodies against markers found on the cell
that fail to react with a commonly used addition to an interpretation. surface. The antibodies used are directly
anti-CD16 monoclonal antibody (clone Flow cytometric, multiparametric anal- conjugated with a fluorescent dye. The
B73.1) and thus will appear to be CD56Ⳮ ysis of subpopulations of cells from vari- stained cells are read on a flow cytometer
CD16ⳮ CD3ⳮ. Thus, when severe or re- ous biological tissues had its genesis in the and the percentage of fluorescent cells is
current infection due to herpesvirus is en- early studies of Loken et al. (2) in 1976 determined for each antibody.

II. SPECIMEN A. Type


Peripheral blood; EDTA, acid-citrate-dextrose (ACD), or heparin can be used
as an anticoagulant. Depending on the number of antibodies to be run, 1 to 3
ml of peripheral blood should be collected. One hundred microliters of whole
blood is needed for each antibody.

11.13.2.1
11.13.2.2 Immunology

Figure 11.13.2–1 NK cell panel interpretation. As per the 2005 Practice Parameter for the
Diagnosis and Management of Primary Immunodeficiency, a mutation in the Fc gamma
R3III gene affecting NK cell function can be screened for by demonstrating decreased
detection of CD16 using the B73.1 clone. If the CD16-positive population is detectable
using both the anti-CD16 clones (B73.1 and 3G8) it is unlikely that the individual is
homozygous for this mutation.
Natural Killer Cell Flow Cytometry Assay 11.13.2.3

II. SPECIMEN (continued) B. Handling


1. Specimens should be collected and brought immediately to the lab at room
temperature. Specimens can be held for no more than 24 h at room tem-
perature. This must also be kept in mind for specimens sent overnight.
2. Severely hemolyzed specimens or those with large clots should be rejected.

III. REAGENTS AND SUPPLIES A. Adjustable-volume pipettor and G. Phosphate-buffered saline (PBS)
tips H. Transfer pipettes
B. 12- by 75-mm polypropylene tubes I. Antibodies against cell surface
C. Beckman-Coulter Immunoprep re- markers—stored according to man-
agent kits and Q-Prep instrumen- ufacturer’s instructions
tation J. Healthy-donor control peripheral
D. Vortex blood
E. Centrifuge or serofuge K. Fetal calf serum
F. Vacuum source with pipette at- L. Plain red-top Vacutainer tubes
tached M. Flow cytometer

IV. CALIBRATION A. Standards


The flow cytometer must be optically aligned and standardized for fluorescence
intensity.
B. Procedure
1. Refer to flow cytometer instrument manual and startup, QC, and shutdown
procedures.
2. New antibody lots (with the exception of isotype controls and CD45) must
be tested in parallel with the current lot to ensure the sensitivity and speci-
ficity of the new lot.

ANALYTICAL CONSIDERATIONS

V. QUALITY CONTROL Controls


Whole blood from a healthy donor

VI. PROCEDURE A. Label one set of 12- by 75-mm polypropylene tubes for each patient and control.
Label the tubes based on the antibodies or panel ordered. Refer to Table
11.13.2–1.
B. Pipette the appropriate volume of antibodies into all other corresponding tubes.
Change pipette tips between each antibody vial to prevent contamination.
C. Add 100 ll of the control or patient blood to each of the appropriate tubes and
vortex to mix.
D. Vortex all tubes and incubate for at least 15 min at 2 to 8⬚C.
E. Process all tubes containing peripheral blood after incubation. Select the nec-
essary protocols/panels on the flow cytometer and load the sample tubes into a
carousel in the appropriate order.
F. Gate on the lymphocyte population based on bright CD45 expression and low
side scatter. Adjustments to this region should be made as needed. Refer to Fig.
11.13.2–2 for an example of a normal CD45/side scatter histogram.
G. If populations span quadrants, the positive and negative populations can be used
as a guide to set cursors.
H. Percent positive results are obtained from the flow cytometer printouts.
11.13.2.4 Immunology

Table 11.13.2–1 Example of tube labeling for NK cell flow cytometry assay
Tube Panela
1 MsIgG1-FITC MsIgG1-PE CD3-ECD MsIgG1-PC5 CD45-PC7

2 CD16-FITC CD16-PE CD3-ECD CD56-PC5 CD45-PC7


(3G8 clone) (B73.1 clone)
a
MsIgG1, mouse immunoglobulin G1; FITC, fluorescein isothiocyanate; PE, phycoerythrin; ECD, phy-
coerythrin with Texas Red.

Figure 11.13.2–2 Example of a normal CD45/side scatter histogram.

VI. PROCEDURE (continued) I. Surface marker-specific absolute counts are automatically calculated where ap-
propriate.
1. The absolute counts are calculated using the percent positive result along
with the WBC and percent lymphocytes from a tandem complete blood
count.
2. If it is necessary to manually calculate a marker-specific absolute count, refer
to item VII below.

POSTANALYTICAL CONSIDERATIONS

VII. CALCULATIONS To manually calculate a marker-specific absolute count:


Marker-specific absolute count ⳱

(WBC ⳮ cells/ll) ⳯ (% lymphocytes Ⳮ % atypical lymphocytes) ⳯ (% flow marker positive)


10,000

Example: Absolute CD3Ⳮ CD4Ⳮ ⳱

(6,800 cells/ll) ⳯ (37 Ⳮ 0) ⳯ (45.2)


⳱ 1,137 CD3Ⳮ CD4Ⳮ cells/ll
10,000
Natural Killer Cell Flow Cytometry Assay 11.13.2.5

VIII. INTERPRETATION Results format

A. Results are reported as percent positive for each antibody. Histograms for NK
panel specimens must be given to the medical director for an interpretation of
the results (see previous example).
B. Normal range: age-matched reference ranges for NK lymphocyte subsets CD3ⳮ
CD16Ⳮ CD56Ⳮ are from reference 1. All other reference ranges are derived
from healthy-adult peripheral blood samples with a region set on lymphocytes.

REFERENCES 1. Comans-Bitter, W. M., et al. 1997. Immu- 4. Shapiro, H. M., E. R. Schildkraut, R. Cur-
nophenotyping of blood lymphocytes in child- belo, R. B. Turner, et al. 1977. Cytomat-R:
hood. Reference values for lymphocyte sub- a computer-controlled multiple laser source
populations. J. Pediatr. 130:388–393. multiparameter flow cytophotometer system.
2. Loken, M. R., R. G. Sweet, and L. A. Her- J. Histochem. Cytochem. 25:836–844.
zenberg. 1976. Cell discrimination by mul-
tiangle light scattering. J. Histochem. Cyto-
chem. 24:284.
3. Parks, D. R., L. A. Herzenberg, and L. Her-
zenberg. 1989. Flow cytometry and fluores-
cence-activated cell sorting, p. 781–802. In
W. E. Paul (ed.), Fundamental Immunology,
2nd ed. Raven Press, New York, N.Y.
11.13.3 Natural Killer Cell Assay
[Procedure added March 2007]

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE
Natural killer (NK) cells mediate the spon- is known to be NK sensitive. K-562 target incubation, the plate is centrifuged and the
taneous lysis of virus-infected or tumor cells are incubated for 1 h with 51Cr. The supernatants are collected. The amount of
cells. The functional capability of NK targets are then incubated with several dif- 51
Cr released into the supernatants, which
cells can be determined by exposing them ferent serial dilutions of peripheral blood is measured with a gamma counter or
to a known NK-specific target that is ra- mononuclear cells (PBMCs) (effectors) TopCount, can be used to determine the
dioactively labeled. NK cell activity is for 4 h at 37⬚C in a 96-well plate. The lytic capability of the NK cells at each
quantified in this assay by using the K-562 effectors lyse the target cells, releasing effector/target (E/T) ratio.
cell line (see Appendix 11.13.3–1), which 51
Cr into the medium. At the end of the

II. SPECIMEN Whole blood drawn into a heparin (green top) tube; 5 to 10 ml is usually sufficient
to perform this assay. These samples must be processed within 30 h of draw time.

III. REAGENTS AND SUPPLIES A. Reagents (see Appendix 11.13.3–1 9. 12- by 75-mm sterile polystyrene
for preparation) culture tubes without closures
1. 51Cr 10. Hemacytometer with coverslip
2. K-562 cell line 11. PES filter unit, 150 ml (0.2-lm
3. RPMI 1640 (stored at 4⬚C) pore size; 50-mm-diameter mem-
4. Filtered RPMI 1640 complete with brane)
10% fetal bovine serum (FBS) 12. 96-well round-bottom plate
(R10 FBS) (stored at 4⬚C) 13. LumaPlate and top seal (if using
5. Ficoll-Paque PLUS (stored at 4⬚C) TopCount machine)
6. 1% Triton X-100 C. Equipment
7. Crystal violet 1. 20-, 100-, and 200-ll pipettors
8. Trypan blue 2. Pipet-Aid
B. Supplies 3. Vacuum setup in biosafety cabinet
1. Sterile pipette tips to deliver 5 to 4. Centrifuge
200 ll 5. Serofuge
2. 10-ml sterile polystyrene pipettes 6. 51Cr radiation safety waste con-
3. 5-ml sterile polystyrene pipettes tainers
4. 1-ml sterile polystyrene pipettes 7. Gamma counter (or TopCount
5. 2-ml sterile polystyrene pipettes machine)
6. 2-ml sterile polystyrene aspirating 8. 37⬚C, 5% CO2, 96% relative hu-
pipettes midity incubator
7. 15-ml sterile polystyrene conical 9. Microscope
tubes 10. Water bath
8. 12- by 75-mm sterile polystyrene
culture tubes with closures (snap
caps)

11.13.3.1
Natural Killer Cell Assay 11.13.3.2

IV. CALIBRATION A. Gamma counter


1. Cesium standard should be included with each run.
2. Preventive maintenance should be performed according to the manufac-
turer’s recommendations.
B. TopCount machine
1. Normalization of TopCount machine must be done once a week.
2. Preventive maintenance should be performed twice per year.
C. Pipettors
Pipettors should be calibrated twice per year.
D. All laboratory equipment should have preventive maintenance performed ac-
cording to the manufacturers’ instructions.

ANALYTICAL CONSIDERATIONS

V. QUALITY CONTROL A. Medium


When R10 FBS is first made, it should be tested for the presence of contami-
nation by pipetting 2 ml into 5 ml of BHI broth and placing in a 37⬚C, 5% CO2,
Include QC information on 96% relative humidity incubator for 5 days.
reagent container and in
QC records. B. Assay validity
In order for the assay to be considered valid, the spontaneous release must be
ⱕ15% of the maximum release.

VI. PROCEDURE 䊓 NOTE: All of the pipetting and filtering should take place in a biosafety cabinet
using sterile technique, and all reagents should be at room temperature when used.
All radioactive materials must be used in designated areas and must be disposed of
It is imperative that these
cultures be handled in a properly per the institution’s radiation safety guidelines.
biosafety hood.
A. PBMC preparation
1. Centrifuge the heparin tube for 10 min at 200 ⳯ g at room temperature.
Observe standard precautions. 2. Gently invert the Ficoll-Paque PLUS bottle twice.
3. Pipette 5 ml of Ficoll-Paque PLUS into a sterile 15-ml conical tube.
4. Using a 5-ml pipette, remove the plasma from the heparin tube and place
into a sterile snap-cap tube. This can be aliquoted and frozen, or discarded
if desired.
5. Dilute the blood twofold using RPMI 1640. Mix gently by pipetting up and
down.
6. Carefully layer the blood onto the 5 ml of Ficoll-Paque PLUS. Be sure not
to let the diluted blood penetrate the Ficoll-Paque PLUS.
7. Centrifuge the conical tube containing the diluted blood on Ficoll-Paque
PLUS at 300 to 400 ⳯ g for 30 min at room temperature.
8. After centrifugation, four layers will be visible. Using a sterile aspirating
pipette connected to a vacuum system, gently aspirate the top clear layer
that contains RPMI 1640 and any remaining plasma. Be careful not to
disturb the cloudy layer underneath, which contains the mononuclear cells.
If you do not have a vacuum system, then use a 5-ml pipette to remove the
top clear layer. Eject fluid into a waste container, such as an empty 15-ml
conical tube, and treat as biohazardous waste.
11.13.3.3 Immunology

VI. PROCEDURE (continued) 9. The cloudy mononuclear cell layer is removed next by placing the tip of a
pipette over the layer and gently moving the pipette in a circular motion.
The entire layer should be removed and transferred to a sterile 15-ml conical
tube containing 5 ml of RPMI 1640. Care should be taken to remove as
little as possible of the Ficoll-Paque PLUS layer below the mononuclear
cells. Centrifuge the conical tube at 200 ⳯ g for 10 min at room tempera-
ture.
10. After centrifugation, aspirate the supernatant, making sure not to disturb
the pellet.
11. Pipette 10 ml of RPMI 1640 into the conical tube. Thoroughly mix to
resuspend the pellet.
12. Centrifuge the conical tube at 200 ⳯ g for 10 min at room temperature.
13. After centrifugation, aspirate the supernatant, making sure not to disturb
the pellet.
14. Pipette 1 ml of R10 FBS into the conical tube. Using the pipette, thoroughly
mix to resuspend the pellet, being careful not to make bubbles.
15. Count the PBMCs using the total viable cell counting procedure (see pro-
cedure 11.9). This procedure uses trypan blue dye to count the viability and
number of PBMCs. To better distinguish mononuclear cells, it is recom-
mended to perform the cell count using crystal violet.
16. If lytic units are to be determined, the NK cells should be enumerated by
flow cytometry.
17. Determine the amount of R10 FBS and cell suspension required to have
1 ml of the first E/T ratio. The E/T ratio is how many PBMCs there are for
each K-562 cell. Typically, 2.5 ⳯ 106 PBMCs/ml are used for the 50:1
E/T ratio, so adjust the number you need for the first desired E/T ratio
accordingly. If you have 12.5 ⳯ 106 PBMCs, the calculation is 2.5/12.5
⳱ 0.20 ml of cell suspension (from step 14) Ⳮ 0.80 ml of R10 FBS.
18. Label four sterile snap caps for the serial dilutions of PBMCs (effector
cells). Commonly used E/T ratios are 50:1, 25:1, 12.5:1, and 6.25:1.
19. Pipette the appropriate amount of R10 FBS (calculated in step 17) into the
starting E/T ratio snap cap.
20. Pipette 0.5 ml of R10 FBS into the next three snap caps.
21. Mix the cell suspension thoroughly and pipette the appropriate amount
(calculated in step 17) into the starting E/T ratio snap cap.
22. With a new pipette, mix well, and pipette 0.5 ml from the first snap cap
into the second one. Discard pipette.
23. Repeat this procedure until you come to the last snap cap, which will have
a final volume of 1 ml after the addition of cells.
24. Using a 96-well plate, label the first 6 wells in the first row as “S.” These
wells are used to identify any spontaneous lysis of the target cells. (This
creates two sets of triplicate wells.)
25. Label the next 6 wells as “M.” These wells are used to demonstrate the
maximum chromium released by the target cells. (This creates two sets of
triplicate wells.)
26. Skip one row and mark off the patient row(s) in triplicate.
27. Pipette 100 ll of R10 FBS into each of the “S” wells.
28. Pipette 100 ll of 1% Triton X-100 into each of the “M” wells.
29. Pipette 100 ll of the first E/T ratio into each of the first 3 wells for that
patient.
30. Changing tips between triplicates, pipette the remaining E/T ratios into the
appropriate triplicate wells.
Natural Killer Cell Assay 11.13.3.4

VI. PROCEDURE (continued) B. K-562 preparation


1. Thoroughly mix the contents of the K-562 flask and pipette 2 ml into a
sterile snap cap.
2. Count the K-562 cells using the total viable cell counting procedure (see
procedure 11.9). Live K-562 cells appear translucent, while dead K-562
cells stain blue with trypan blue dye.
3. Add or remove K-562 suspension from the original snap cap as needed to
obtain 2 ⳯ 106 to 3 ⳯ 106 K-562 cells to label. This is the recommended
number of cells to label with 100 ll of 1-mCi/ml 51Cr.
4. Serofuge the snap cap at 3,500 rpm for 3 min to pellet.
5. Decant the supernatant, making sure not to disturb the pellet.
6. Resuspend the K-562 pellet in 100 ll of R10 FBS.
7. Add 100 ll of 51Cr to the resuspended K-562 cells. It is recommended that
this be timed so that the K-562 cells will be ready close to when the PBMCs
will be ready, due to the highly cytotoxic nature of 51Cr.
8. Put the snap cap into the bottom of a lead container containing warm water
and place in a 37⬚C CO2 incubator for 1 h. The 51Cr will arrive in a lead
container from the manufacturer. After you have either consumed or dis-
posed of the contents, you will have an empty lead container that you can
use for this incubation.
9. Gently shake the snap cap after 30 min to resuspend the K-562 cells.
10. After the incubation, add 2 ml of R10 FBS to the snap cap containing the
K-562 cells. Mix well.
11. Spin in a serofuge for 3 min at 3,500 rpm.
12. Decant the supernatant into an appropriate radioactive waste container,
making sure not to disturb the pellet.
13. Add 2 ml of R10 FBS to the snap cap. Mix well.
14. Spin in the serofuge for 3 min at 3,500 rpm.
15. Decant the supernatant into an appropriate radioactive waste container.
16. Repeat steps 13 through 15 two more times.
17. After the suspension is decanted for the fourth time, pipette 1 ml of R10
FBS into the snap cap. Mix thoroughly.
18. Count the K-562 cells using the total viable cell counting procedure (see
procedure 11.9).
19. An NK assay utilizing one plate requires 10 ml of 5 ⳯ 104/ml K-562 cells.
Remove from the snap cap the necessary amount of K-562 cells and dilute
to 10 ml with R10 FBS in a sterile 15-ml conical tube. Sample calculation:
if you have 2.3 ⳯ 106 K-562 cells, then the calculations would be as fol-
lows: 5 ⳯ 105 K-562 cells/2.3 ⳯ 106 K-562 cells ⳱ 0.22 ml of resuspended
K-562 (from step 17) Ⳮ 9.78 ml of R10 FBS.
20. Invert the 15-ml conical tube several times to mix it.
21. Pipette 100 ll of K-562 final suspension to each of the “S” wells, changing
tips between triplicates.
22. Pipette 100 ll of K-562 final suspension to each of the “M” wells, changing
tips between triplicates.
23. Pipette 100 ll of K-562 final suspension to each of the patient E/T ratio
wells, changing tips between triplicates.
24. Spin the plate in the centrifuge for 5 min at 80 ⳯ g.
25. After centrifugation, incubate the plate at 37⬚C for 4 h.
26. After the incubation, spin the plate in the centrifuge for 10 min at 300 ⳯ g.
27. Label one nonsterile 12- by 75-mm plastic culture tube without closure for
each well of the plate containing PBMCs and K-562 cells and the sponta-
neous and maximum wells (if using a gamma counter).
11.13.3.5 Immunology

VI. PROCEDURE (continued) 28. Tilt the plate slightly and pipette 100 ll of supernatant from each well and
eject into the appropriate tube. Be careful not to disturb the pellet. Repeat
this procedure for all wells, changing tips every triplicate. Alternatively, if
using a TopCount machine, you can pipette 50 ll of supernatant from each
well to the corresponding well on a LumaPlate. The scintillant is already
in this plate, so you just need to let it dry overnight, and then put the top
seal on the plate in the morning.
29. Load tubes into gamma counter (or plate into TopCount machine) to count
for 1 min per tube or well.
30. Discard waste into a 51Cr radioactive waste container after counting.

VII. CALCULATIONS The mean of the triplicate counts per minute (cpm) is determined for each E/T ratio
and for the spontaneous- and maximum-release wells. Percent specific target cell
lysis is calculated for each E/T ratio as follows.

% Specific lysis ⳱
(mean cpm of experimental release ⳮ mean cpm of spontaneous release)
(mean cpm of maximum release ⳮ mean cpm of spontaneous release)
⳯ 100

POSTANALYTICAL CONSIDERATIONS

VIII. INTERPRETATION The NK cell percent lysis at each E/T ratio can be used to determine the effective-
ness of the subject’s NK cells via comparison with the results for a healthy control.
It should be noted, however, that variation in 51Cr absorption and K-562 cell percent
spontaneous release is to be expected, as the health of the K-562 cell line does have
some minor variability.

SUPPLEMENTAL READING Hay, R., J. Caputo, T. R. Chen, M. Macy, P. Whiteside, T., C. R. Rindalo, and R. B. Her-
McClintock, and Y. Reid (ed.). 1994. American berman. 1992. Cytolytic cell functions, p. 220–
Type Culture Collection (ATCC) Catalogue of 230. In N. R. Rose, E. Conway de Macario, J. L.
Cell Lines and Hybridomas, 8th ed. 1994 refer- Fahey, H. Friedman, and G. M. Penn (ed.), Man-
ence guide. American Type Culture Collection, ual of Clinical Laboratory Immunology, 4th ed.
Manassas, Va. American Society for Microbiology, Washington,
Lozzio, C. B., and B. B. Lozzio. 1975. Human D.C.
chronic myelogenous leukemia cell-line with
positive Philadelphia chromosome. Blood
45:321–334.

APPENDIX 11.13.3–1 Assay Materials


A. K-562 cell line
1. According to the ATCC Catalogue of Cell Lines and Hybridomas (1), the K-562
cell line was established by Lozzio and Lozzio (2) from the pleural effusion of a
53-year-old female with chronic myelogenous leukemia in terminal blast crises.
2. The K-562 cell line can be purchased from the ATCC Catalogue of Cell Lines and
Hybridomas, as CCL 243.
3. The culture medium is RPMI 1640, 90%; FBS, 10%; antibiotic free.
4. The cell line is maintained in T25 flasks. When a concentration of 750,000 to
106/ml is reached, the flask is split 1:10 with culture medium.
Natural Killer Cell Assay 11.13.3.6

APPENDIX 11.13.3–1 (continued) B. Reagent information and preparation


1. R10 FBS
a. 435 ml of RPMI 1640
b. 5 ml of 200 mM L-glutamine for a final concentration of 2 mM L-glutamine
c. 5 ml of 10,000-U/ml penicillin and 10,000-lg/ml streptomycin solution for a
final concentration of 100 U of penicillin and 100 lg of streptomycin per ml
in solution.
d. 5 ml of 1 M HEPES buffer for a final of 10 mM HEPES buffer
e. 50 ml of heat-inactivated FBS
2. 1% Triton X
a. 0.1 ml of Triton X-100
b. 9.9 ml of 1⳯ phosphate-buffered saline
3. Crystal violet
a. 98 ml of deionized H2O
b. 2 ml of glacial acetic acid
c. 0.05 g of crystal violet dye (powder form)
4. Reagent information
See table on next page.

REFERENCES 1. Hay, R., J. Caputo, T. R. Chen, M. Macy, 2. Lozzio, C. B., and B. B. Lozzio. 1975. Human
P. McClintock, and Y. Reid. 1994. American chronic myelogenous leukemia cell-line with
Type Culture Collection (ATCC) Catalogue of positive Philadelphia chromosome. Blood
Cell Lines and Hybridomas, 8th ed. 1994 ref- 45:321–334.
erence guide. American Type Culture Collec-
tion, Manassas, Va.
11.13.3.7 Immunology

Reagent Manufacturer Catalog no. Address and website Phone no.


51
Chromium Perkin-Elmer NEZ-030S 549 Albany St. (800) 762-4000
Boston, MA 02118

Fetal bovine serum Atlanta Biologicals S11550H 2150 Cedars Road, Suite 200 (800) 780-7788
(heat inactivated) Lawrenceville, GA 30043
http://www.atlantabio.com

Ficoll-Paque PLUS Amersham Pharmacia 17-1440-03 800 Centennial Ave. (800) 526-3593
P.O. Box 1327
Piscataway, NJ 08855-1327
http://www.apbiotech.com

HEPES buffer Invitrogen 15630-080 1600 Faraday Ave. (800) 955-6288


P.O. Box 6482
Carlsbad, CA 92008
http://www.invitrogen.com

L-Glutamine Bio-Whittaker 17-605E 8830 Biggs Ford Rd. (800) 638-8174


Walkersville, MD 21793
http://www.cambrex.com

Penicillin-streptomycin Invitrogen 15140-148 1600 Faraday Ave. (800) 955-6288


P.O. Box 6482
Carlsbad, CA 92008
http://www.invitrogen.com

RPMI 1640 Bio-Whittaker 12-167F (500 ml) 8830 Biggs Ford Rd. (800) 638-8174
12-167Q (1000 ml) Walkersville, MD 21793
http://www.cambrex.com

Trypan blue dye Invitrogen 15250-061 1600 Faraday Ave. (800) 955-6288
P.O. Box 6482
Carlsbad, CA 92008
http://www.invitrogen.com

Triton X-100 Sigma X-100 P.O. Box 14508 (800) 325-3010


St. Louis, MO 63178
http://www.sigma-aldrich.com/order

1⳯ phosphate-buffered saline Bio-Whittaker 17-516F 8830 Biggs Ford Rd. (800) 638-8174
Walkersville, MD 21793
http://www.cambrex.com

Crystal violet dye (powder) Sigma A-6158 P.O. Box 14508 (800) 325-3010
St. Louis, MO 63178
http://www.sigma-aldrich.com/order

Glacial acetic acid Sigma A-6283 P.O. Box 14508 (800) 325-3010
St. Louis, MO 63178
http://www.sigma-aldrich.com/order

LumaPlate-96 Perkin-Elmer 6006633 549 Albany St. (800) 762-4000


Boston, MA 02118
11.14 Quantitation of Human Interleukin
4, Interleukin 6, and Gamma
Interferon

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE
Interleukin 4 (IL-4), IL-6, and gamma in- quantify cytokines by ELISA. This pro- oxidase (HRP) is added. After another in-
terferon (IFN-c) are cytokines with many cedure is written for use with kits manu- cubation, tetramethylbenzidine (TMB)
cellular effects, including mediation of the factured by Pierce Endogen. If IL-4, IL-6, substrate is added to the plate. The en-
immune response to infection. Among or IFN-c is present in the clinical speci- zyme-substrate reaction produces a color
other biological activities, IL-4 and IL-6 men, it will bind to an ELISA plate that is change that can be measured spectropho-
stimulate B cells and IFN-c has antiviral precoated with antibody specific for that tometrically. The unknown values can be
activities (6). There is an increasing inter- cytokine. After the patient specimen is determined from a standard curve in
est in using cytokine levels to monitor dis- incubated with a biotinylated detection which the color intensity is directly pro-
ease progression (2). There are several antibody, the plate is washed and then portional to the standard concentration.
commercially prepared kits available to streptavidin conjugated to horseradish per-

II. SPECIMEN Serum, EDTA plasma, heparin plasma, sodium citrate plasma, and urine are ac-
ceptable specimens for cytokine measurement. In addition, in vitro cytokine pro-
duction can be measured in peripheral blood mononuclear cell culture supernatants
or whole blood culture supernatants (1, 7). Specimens should be stored at ⳮ70⬚C.
If more than one ELISA is to be performed, the specimen or supernatant should be
split into an appropriate number of 150-ll aliquots prior to freezing.

III. REAGENTS AND SUPPLIES A. Reagents 6. 12- by 75-mm nonsterile polypro-


1. Human IL-4, IL-6, or IFN-c kit pylene tubes
(Pierce Endogen, Rockford, Ill.) 7. Disposable reagent reservoirs
2. Distilled water 8. 15-ml nonsterile plastic centrifuge
B. Supplies tubes with conical bottoms
1. 5- and 10-ml nonsterile plastic pi- 9. Squirt bottle
pettes 10. Empty box to fit a 96-well plate
2. 20-, 100-, 200-, and 1,000-ll pi- C. Equipment
pettors 1. Vortex
3. Multichannel pipettor 2. ELx800 microplate reader (Bio-
4. 2-liter nonsterile flask or bottle to Tek Instruments, Inc., Winooski,
dilute wash buffer Vt.)
5. Nonsterile pipette tips to deliver 5
to 1,000 ll

11.14.1
11.14.2 Immunology

ANALYTICAL CONSIDERATIONS

IV. QUALITY CONTROL A. All reagents and samples should be brought to room temperature before use.
Do not place in a water bath.
B. Reagents
1. Do not use any reagent that appears cloudy. This may indicate microbial
contamination.
2. Do not mix reagents from different kits.
3. Use a new reagent reservoir for each reagent.
4. When using the multichannel pipettor, use new tips for each row to avoid
cross contamination and bubble formation.
5. TMB substrate solution should be colorless. If it appears blue, discard it.
6. The standard diluent and biotinylated antibody reagent contain sodium azide
as a preservative. If leftover reagent is poured down the drain, it must be
flushed with water for several minutes.
C. Specimens
If any specimen is cloudy, lipemic, hemolyzed, or otherwise unusual, a note
should be made on the plate plan or worksheet and the result should be reported
with this notation.
D. Calibration
1. Standard curve
Each ELISA plate is specific for only one cytokine. This is identified by the
kit being used. A curve using the enclosed standard must be run on each
plate.
2. Medium validation
a. Medium validation must be performed in order to run culture supernatants
and patient specimens (serum, etc.) on the same plate.
b. A standard curve diluted in culture medium is run in parallel with a
standard curve diluted in the Pierce Endogen standard diluent. If the op-
tical density (OD) values of the two curves are within 10% of the mean
for each standard point of both curves then the culture medium is vali-
dated. For example, the average OD values for a particular point of a
standard curve are 0.605 for the medium-diluted curve and 0.685 for the
curve diluted in standard diluent. The mean for these OD values would
be 0.645. The difference between the mean and the OD values would be
0.040, or 5.8%. This means that culture supernatants can be run on a plate
with a curve diluted in standard diluent. If the difference is greater than
10%, culture supernatants must be run on a separate plate with a curve
diluted in the culture medium.
3. Plate reader
The plate reader must be calibrated in accordance with the specific manu-
facturer’s instructions.

V. PROCEDURE Plate setup


A plate plan or worksheet should be filled out completely for each plate to be run.
This can be a diagram of a 96-well plate or a listing of each well. The standards
It is imperative that these
cultures be handled in a and specimens to be run should be written in the places where they will be on the
biosafety hood. plate. The standards should always be run at the beginning of the plate (Fig. 11.14–
1). All standards and samples are to be run in duplicate.

A. Remove the plate from the foil envelope. If a partial plate is being run, the
unused strips should be resealed in the envelope with the desiccant and refrig-
erated as soon as possible.
Quantitation of Human Interleukin 4, Interleukin 6, and Gamma Interferon 11.14.3

Figure 11.14–1 Plate plan for Endogen IL-6 ELISA with 10 samples
1 2 3 4 5 6 7 8 9 10 11 12
A Standard 1 Standard 5 Sample 3 Sample 7
400 pg/ml 10.24 pg/ml
B Standard 1 Standard 5 Sample 3 Sample 7
400 pg/ml 10.24 pg/ml
C Standard 2 Standard 6 Sample 4 Sample 8
160 pg/ml 0 pg/ml
D Standard 2 Standard 6 Sample 4 Sample 8
160 pg/ml 0 pg/ml
E Standard 3 Sample 1 Sample 5 Sample 9
64 pg/ml
F Standard 3 Sample 1 Sample 5 Sample 9
64 pg/ml
G Standard 4 Sample 2 Sample 6 Sample 10
25.6 pg/ml
H Standard 4 Sample 2 Sample 6 Sample 10
25.6 pg/ml

V. PROCEDURE (continued) B. Add 50 ll of the biotinylated antibody reagent to each well using a multichannel
pipettor. This reagent is slightly foamy, so it will be necessary to use new tips
frequently to avoid air bubbles.
C. Mix all samples by gently inverting the tubes.
D. Make 1:2.5 serial dilutions of the appropriate standard. See Appendix 11.14–
1 for details.
E. Add 50 ll of standards or samples to the appropriate wells in duplicate. Check
that no air bubbles are in the pipette tip.
F. Cover the plate with a new adhesive plate sealer and incubate at room tem-
perature (20 to 25⬚C) for 2 h.
G. At the end of 2 h, wash the plate with wash buffer. See Appendix 11.14–1 for
instructions to dilute wash buffer.
1. Empty the plate into the sink and use a squirt bottle to add wash buffer
vigorously.
2. Empty the plate and repeat two more times.
3. After the third wash, pat the plate on paper towels to absorb all excess liquid.
4. An automated plate washer can be used for this process.
H. Add 100 ll of streptavidin-HRP solution to each well. See Appendix 11.14–1
for preparation instructions.
I. Cover the plate with a new adhesive plate sealer and incubate at room tem-
perature for 30 min.
J. After 30 min, wash the plate (see steps V.G.1 to V.G.4).
K. After washing the plate, add 100 ll of TMB substrate solution to each well.
䊓 This reagent is extremely sensitive to light. Do not pour into reagent res-
ervoir until immediately before use. Do not pour out more reagent than nec-
essary.
L. Allow the plate to develop in the dark at room temperature for 30 min (e.g.,
place plate in empty box on lab bench). Do not use a plate sealer for this step.
M. After 30 min, add 100 ll of stop reagent to each well.
N. Read the plate using an automated plate reader at 450 nm. The plate must be
read within 30 min after the addition of the stop reagent.
11.14.4 Immunology

POSTANALYTICAL CONSIDERATIONS

VI. CALCULATIONS A. The standard curve can be created manually or with curve-fitting software. The
cytokine concentration is plotted on the x axis, and the average OD of the
standards is plotted along the y axis. The average OD of each specimen is used
to interpolate its concentration from the standard curve. If curve-fitting software
is used, a linear or point-to-point curve can be chosen. All OD values must
agree within 10% of the duplicate value for that patient, control, or standard.
Example: A patient whose samples have been set up in duplicate has OD
values of 0.123 and 0.167. Average these numbers.

0.123 Ⳮ 0.167 ⳱ 0.29 0.29/2 ⳱ 0.145

Calculate the difference between the mean and one of the values.

0.145 ⳮ 0.123 ⳱ 0.022

Divide the difference by the mean and multiply by 100 to calculate


the percent difference.

0.022/0.145 ⳱ 0.152 ⳯ 100 ⳱ 15.2%

These OD values are greater than 10% from the mean, and this
sample would have to be retested on another run.
B. The results for any diluted specimens must be multiplied by the dilution factor.

VII. INTERPRETATION A. In the absence of disease, cytokines are nearly undetectable in body fluids (6).
See Table 11.14–1.
B. Sensitivity
1. The IL-4 and IFN-c assays are sensitive to ⬍2 pg/ml.
2. The IL-6 assay is sensitive to ⬍1 pg/ml.

VIII. PROCEDURE NOTES A. If the reader does not give an OD value for a specimen or if the cytokine
concentration is greater than the highest standard, that specimen must be diluted
and retested on another run.
B. Culture supernatants are diluted in the culture medium. Serum, plasma, or urine
is diluted in the provided sample diluent.

Table 11.14–1 Pierce Endogen observed cytokine values in body fluidsa


Concn (pg/ml) of cytokine
Body fluid
IL-4 IL-6 IFN-c
Serum Not detected (8) 0–149 (14) 0–1.5 (35)
Plasma Not detected (8) 0–5 (14) 0–2.6 (45)
Urine Not detected (5) 0–0.6 (5) 0.5–1.2 (5)
a
The number of subjects evaluated is in parentheses (3–5).
Quantitation of Human Interleukin 4, Interleukin 6, and Gamma Interferon 11.14.5

REFERENCES 1. Katial, R. K., D. Sachanandani, C. Pinney, 5. Pierce Endogen. 2000. Package insert for hu-
and M. M. Lieberman. 1998. Cytokine pro- man interferon gamma ELISA. Pierce Endo-
duction in cell culture by peripheral blood gen, Rockford, Ill.
mononuclear cells from immunocompetent 6. Remick, D. G. 2002. Protein analysis and bi-
hosts. Clin. Diagn. Lab. Immunol. 5:78–81. oassays of cytokines and cytokine receptors,
2. Lee, B. N., J. G. Lu, M. W. Kline, M. Paul, p. 320–337. In N. R. Rose, R. G. Hamilton,
M. Doyle, C. Kozinetz, W. T. Shearer, and and B. Detrick (ed.), Manual of Clinical Lab-
J. M. Reuben. 1996. Type 1 and type 2 cy- oratory Immunology, 6th ed. ASM Press,
tokine profiles in children exposed to or in- Washington, D.C.
fected with vertically transmitted human im- 7. Wallis, R. S., H. M. Lederman, J. Spritzler,
munodeficiency virus. Clin. Diagn. Lab. J. L. Devers, D. Georges, A. Weinberg, S.
Immunol. 3:493–499. Stehn, M. M. Lederman, and the ACTG In-
3. Pierce Endogen. 2000. Package insert for hu- ducible Cytokines Focus Group. 1998. Mea-
man interleukin 4 ELISA. Pierce Endogen, surement of induced cytokines in AIDS clini-
Rockford, Ill. cal trials using whole blood: a preliminary
4. Pierce Endogen. 2000. Package insert for hu- report. Clin. Diagn. Lab. Immunol. 5:556–
man interleukin 6 ELISA. Pierce Endogen, 560.
Rockford, Ill.

SUPPLEMENTAL READING Aziz, N., P. Nishanian, and J. L. Fahey. 1998.


Levels of cytokines and immune activation mark-
ers in plasma in human immunodeficiency virus
infection: quality control procedures. Clin. Diagn.
Lab. Immunol. 5:755–761.

APPENDIX 11.14–1 Reagent Preparation


Instructions for reagent preparation and standard dilutions from Pierce Endogen (1, 2, 3)
Include QC information on
reagent container and in A. Wash buffer
QC records. In a clean flask or bottle, dilute 50 ml of the 30⳯ wash buffer concentrate with 1.45
liters of distilled water. Label the buffer with the lot number, date made, expiration date,
and initials of the person who made it. Store unused wash buffer in a bottle at 4⬚C.
B. Streptavidin-HRP solution
1. Do not prepare more than 15 min before it will be used. Do not prepare more than
necessary. There is no extra reagent in the kit.
2. Gently vortex the streptavidin-HRP concentrate.
3. In a plastic 15-ml tube, dilute the streptavidin-HRP concentrate 1:40 with streptavidin-
HRP dilution buffer.
4. If an entire plate is being assayed, add 30 ll of concentrate to 12 ml of diluent. If less
than a whole plate is being assayed, add 2.5 ll of concentrate to 1 ml of diluent for
each strip used.
C. Standards
1. Reconstitute one vial of standard with the volume of distilled water listed on the
standard vial. The standard will take about 1 min to dissolve. Use standard within 1
h of reconstitution. Do not store reconstituted standard.
2. Gently invert standard to mix.
3. Make a 1:2.5 serial dilution of the standard. Label six polypropylene tubes for the
standard dilutions as follows.
a. For IL-4 and IL-6, label the tubes 400 pg, 160 pg, 64 pg, 25.6 pg, 10.24 pg, and
0 pg.
b. For IFN-c, label the tubes 1,000 pg, 400 pg, 160 pg, 64 pg, 25.6 pg, and 0 pg.
4. If the specimens to be run are plasma, serum, or urine, the standard is diluted with
the provided standard diluent. If the specimens are culture supernatants, the standard
is diluted with the culture medium unless validation has been performed (see item
IV.D.2 in procedure 11.14). If validation has been done, all specimen types can be
analyzed on the same plate.
a. Add 240 ll of the appropriate diluent to each of the six tubes.
b. Pipette 160 ll of the reconstituted standard to the first tube and gently vortex to
mix.
c. Using the same pipette tip, transfer 160 ll to the second tube and gently vortex to
mix.
d. Continue this for the next three tubes, being careful that there are no air bubbles
during the transfers.
11.14.6 Immunology

APPENDIX 11.14–1 (continued) e. Do not transfer anything into the sixth tube. This is the 0 standard tube and will
contain only diluent or culture medium.
f. If more than one plate is being run, the standard dilutions can be made by increasing
all volumes two- or threefold.

References
1. Pierce Endogen. 2000. Package insert for hu-
man interleukin 4 ELISA. Pierce Endogen,
Rockford, Ill.
2. Pierce Endogen. 2000. Package insert for hu-
man interleukin 6 ELISA. Pierce Endogen,
Rockford, Ill.
3. Pierce Endogen. 2000. Package insert for hu-
man interferon gamma ELISA. Pierce Endo-
gen, Rockford, Ill.

APPENDIX 11.14–2 Suppliers Fisher Scientific


2000 Park Lane Dr.
Pierce Endogen
Pittsburgh, PA 15275
3747 North Meridian Rd.
http://www.fishersci.com
P.O. Box 117
Rockford, IL 61105

Bio-Tek Instruments, Inc.


Highland Park, Box 998
Winooski, VT 05404
11.15 Flow Cytometry Whole-Blood
Intracellular-Cytokine Assay Using
Phorbol Myristate Acetate,
Ionomycin, and Brefeldin A

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE
Cytokines are soluble proteins produced cytokine products. The flow cytometry sicular transport from the rough endo-
by T and B lymphocytes, natural killer method described here measures produc- plasmic reticulum to the Golgi complex)
cells, monocytes, macrophages, and gran- tion of cytokines by T-cell subsets using for 4 h at 37⬚C and 5% CO2. Activated
ulocytes. Specifically, cytokines regulate whole blood. Heparinized blood is stimu- cells are stained with monoclonal antibod-
growth, differentiation, and function of a lated with the polyclonal activator phorbol ies for lymphocyte surface markers, fol-
wide variety of cells and mediate normal myristate acetate plus ionomycin (induces lowed by lysing of RBCs. WBCs are fixed
and pathological responses. Most cytokine intracelluar signal cascades for polyclonal and permeabilized simultaneously and
bioassays examine cytokines at the cell leukocyte activation) and the protein stained with monoclonal antibodies to in-
population level, but they cannot provide transport inhibitor brefeldin A (BFA) (a tracellular cytokines. Stained cells are an-
information concerning the phenotype of fungal metabolite that interferes with ve- alyzed by flow cytometry.
cytokine-producing cells or mechanism of

II. SPECIMEN COLLECTION, A. Specimen collection


TRANSPORT, AND HANDLING 1. Peripheral blood in sodium heparin anticoagulant (2 ml)
2. Vacutainers or other collection tubes should be filled properly to account for
Observe standard precautions. the amount of anticoagulant.
3. Since all specimens should be regarded as potentially infectious, standard
precautions for blood collection and handling should be properly followed.
B. Specimen transport
1. Specimens should be placed in well-stoppered tubes and should be placed
with adequately absorbent material, preferably in a closed leakproof con-
tainer, for delivery.
2. If delivery to reference laboratories is possible within the first 24 h, follow
federal guidelines (International Air Transport Association Dangerous Goods
Regulations, January 2001 [see procedure 15.5]).
3. Specimens should be maintained at room temperature (18 to 22⬚C) during
transportation and storage.
4. Testing must be performed within 24 h from the time of collection.
C. Specimen labeling and request submission
1. Each patient sample must be properly labeled.
2. Each sample should be accompanied by some type of procurement form
which should contain appropriate identifiers, e.g., patient’s name, date of
birth, and gender; date and time of collection, clinician; and other relevant
patient information.

11.15.1
11.15.2 Immunology

II. SPECIMEN COLLECTION, D. Rejection criteria


TRANSPORT, AND HANDLING 1. Inadequate sample volume
(continued) 2. Clotted specimen
3. Hemolyzed blood specimen
4. Specimen more than 24 h old
5. Specimen exposed to cold temperatures

III. MATERIALS A. Reagents (see Appendix 11.15–1 for b. Hanks balanced salt solution
reagent concentrations and prepara- without calcium, magnesium,
tion) or phenol red (HBSS) (Invitro-
Include QC information on 1. Dulbecco’s phosphate-buffered gen)
reagent container and in saline without calcium, magne- c. Saponin (Sigma)
QC records. sium, or phenol red (dPBS) (In- d. HEPES buffer (Invitrogen)
vitrogen, Grand Island, N.Y.) 13. Flow cytometer calibration beads
2. Dimethyl sulfoxide (DMSO) or other calibration reagents (fol-
(Sigma Chemical Co., St. Louis, low manufacturer’s directions)
Mo.) B. Supplies
3. RPMI 1640 with glutamine (In- 1. Adjustable-volume pipette and tips
vitrogen) 2. 12- by 75-mm polypropylene tubes
4. Heat-inactivated fetal bovine se- (BD Biosciences, Franklin Lakes,
rum (FBS) (Invitrogen) N.J.)
5. Monoclonal antibodies including 3. 12- by 75-mm polystyrene tubes
isotype markers against cell sur- (BD Biosciences)
face and cytokine markers— 4. Liquid-waste container with bleach
stored according to manufac- 5. Disposable gloves
turer’s instructions 6. Disposable lab coat
6. Wash buffer (0.5% FBS, 0.1% so- 7. Protective eyewear
dium azide) C. Equipment
7. Sodium azide (J.T. Baker, Phil- 1. Centrifuge with swinging bucket
lipsburg, N.J.) rotor, capable of centrifugation at
8. Phorbol 12 myristate 13 acetate 300 ⳯ g and equipped with aerosol
(PMA) (Sigma) containment canisters
9. Ionomycin (Sigma) 2. Vacuum source with pipette at-
10. BFA (Sigma) tached
11. Lysing solution 3. Flow cytometer
a. NH4Cl (Sigma) 4. Class II biosafety cabinet
b. NaHCO3 (Fisher Scientific, 5. Timer/stop watch
Fair Lawn, N.J.) 6. CO2 incubator (5% CO2)
c. EDTA (Fisher Scientific) 7. Vortex
d. Sterile distilled water
12. Permeabilization buffer
a. 10% Methanol-free formalde-
hyde (Polysciences, Inc., War-
rington, Pa.)

ANALYTICAL CONSIDERATIONS

IV. QUALITY CONTROL Controls

A. Surface and intracellular markers (e.g., CD4 and interleukin-2 [IL-2]) from a
healthy volunteer donor. These cells should be stained in the same manner and
with the same monoclonal antibodies as the patient specimens. These cells
should be run with every cytokine assay.
B. Various blocking reagents may be used to ensure positivity.
C. Unstimulated control sample containing BFA.
D. Commercially available QC substance that is positive for particular cytokine.
Flow Cytometry Whole-Blood Intracellular-Cytokine Assay 11.15.3

V. CALIBRATION A. Optical alignment


The flow cytometer must be optically aligned. Follow manufacturer’s directions.
(Some cytometers require a company engineer.)
B. Calibration
Depending on the flow cytometer manufacturer, either standardized fluorescent
beads or chicken RBCs may be used for calibration and to assess instrument
function. Follow manufacturer’s procedures.
C. Verification with whole-blood preparation
Electrical fluorescent compensation on single stained cells. Follow manufac-
turer’s procedures.

VI. CYTOKINE ASSAY A. Procedure


1. Label each of two 12- by 75-mm polypropylene tubes “Unstimulated” and
“Activated.”
It is imperative that these
cultures be handled in a 2. Add 500 ll of RPMI 1640 to each tube.
biosafety hood. 3. Add 10 ll of BFA to the unstimulated tube.
4. Add 20 ll of PMA, 10 ll of ionomycin, and 10 ll of BFA to the activated
tube.
Observe standard precautions. 5. Add 500 ll of heparinized (sodium heparin) whole blood. Vortex to mix.
6. Incubate for 4 h at 37⬚C in the 5% CO2 incubator. (Note: One milliliter of
activated blood provides enough cells for staining 10 samples, based on
100 ll/test.)
7. Aliquot 100 ll of activated blood into 12- by 75-mm polystyrene tubes
preloaded with monoclonal antibody for surface antigens. (Follow manu-
facturer’s instructions for appropriate volume. Antibody may have to be
titered.)
8. Incubate for 15 min at room temperature in the dark.
9. Add 2 ml of prewarmed (37⬚C) lysing solution per tube. Vortex and incu-
bate for 5 min at room temperature.
10. Centrifuge at 300 ⳯ g for 5 min, and aspirate supernatant. Vortex lightly
to loosen cell pellet. Wash twice with 2 ml of wash buffer and centrifuge
at 300 ⳯ g for 5 min.
11. Aspirate supernatant. Vortex lightly to loosen pellet. Add 500 ll of per-
meabilization buffer. Vortex lightly to mix. Incubate for 15 min at room
temperature.
12. Wash twice with 2 ml of wash buffer. Spin at 300 ⳯ g for 5 min. Aspirate
supernatant. Vortex lightly to loosen pellet.
13. Add cytokine monoclonal antibody for intracellular staining. (Follow man-
ufacturer’s instructions for appropriate volume. Antibody may have to be
titered.) Vortex lightly.
14. Incubate for 25 to 30 min at room temperature in the dark.
15. Wash once with wash buffer. Spin at 300 ⳯ g for 5 min.
16. Aspirate supernatant to loosen pellet, and fix with 300 ll of 1% formal-
dehyde.
B. Flow cytometry analysis
1. Collect 5,000 events in the gate (small nongranular cells) on the flow cytom-
eter. Cells can be stored at 4⬚C and collected the following day.
2. Gate lymphocytes using forward versus side light scatter parameters. Ana-
lyze fluorescent parameters for appropriate surface and cytokine markers.
Negative markers will be set on unstimulated stained cells for the same
antibodies.
11.15.4 Immunology

POSTANALYTICAL CONSIDERATIONS

VII. REPORTING RESULTS Report percentage of surface marker that expresses a particular cytokine (e.g., if
the total percentage of CD4 is 25% and the CD4Ⳮ IL-2Ⳮ is 10%, then 10% divided
by 25% would equal 40%, which is the percentage of CD4 that expresses IL-2).

VIII. INTERPRETATION Normal values

A. Values for at least 50 healthy donors should be ascertained by each laboratory


performing this test.
B. Compare percentage to means and standard deviations of healthy donors.

IX. LIMITATIONS OF TESTING Stimulation with PMA and ionomycin does not occur in vivo.

SUPPLEMENTAL READING Centers for Disease Control and Prevention. Picker, L. J., M. K. Singh, Z. Zdraveski, J. R.
1997. Revised guidelines for performing CD4Ⳮ T- Treer, S. L. Waldrop, P. R. Bergstresser, and
cell determinations in persons infected with hu- V. C. Maino. 1995. Direct demonstration of cy-
man immunodeficiency virus (HIV). Morb. Mor- tokine heterogeneity among human memory/ef-
tal. Wkly. Rep. 46 (RR-2):1–29. fector T cells by flow cytometry. Blood 86:1408–
Jung, T., U. Schauer, C. Heusser, C. Nermann, 1419.
and C. Rieger. 1993. Detection of intracellular
cytokines by flow cytometry. J. Immunol. Meth-
ods 159:197–207.
Maino, V., M. A. Suni, and J. J. Ruitenberg.
1995. Rapid flow cytometric method for measur-
ing lymphocyte subset activation. Cytometry
20:127–133.

APPENDIX 11.15–1 Reagent Preparation


A. PMA
Include QC information on 1. Stock solution of 1 mg/ml is prepared in DMSO, with aliquots frozen at 70⬚C.
reagent container and in 2. Working solution is prepared from stock solution diluted in dPBS for a 1-lg/ml con-
QC records. centration. Final concentration in assay is 20 ng/ml.
B. Lysing solution
1. Stock solution is prepared using 8.02 g of NH4Cl (Sigma), 0.84 g of NaHCO3 (Fisher),
and 0.37 g of EDTA (Fisher) and bringing to a volume of 100 ml with sterile distilled
water. Store at 4⬚C. Keeps for 6 months.
2. Working solution is prepared by adding 10 ml of stock solution to 90 ml of sterile
distilled water. Keeps for a week.
C. Ionomycin
1. Stock solution of 1 mg/ll is prepared in DMSO, with aliquots frozen at ⳮ70⬚C.
2. Working solution is diluted in PBS for a 100-lg/ml concentration. Final concentration
in assay is 1 lg/ml.
D. BFA
1. Stock solution of 5-mg/ml is prepared in DMSO, with aliquots frozen at ⳮ70⬚ C.
2. Working solution is diluted in PBS for a 1-lg/ml concentration. Final concentration
of assay is 10 lg/ml.
E. Permeabilization buffer
1. Dilute 10% methanol-free formaldehyde in HBSS to make a 4% solution.
2. Dissolve 0.1 g of saponin in 100 ml of 4% formaldehyde.
3. Filter and add 1 ml of 1 M HEPES buffer.
4. Cover in foil and store at 4⬚C. Keeps for 2 weeks.
F. Buffer
Add 2.5 ml of heat-inactivated FBS and 0.5 g of sodium azide to 500 ml of PBS for a
0.5% FBS–0.1% sodium azide solution.
11.16 Whole-Blood Lymphocyte
Immunophenotyping Using Cell
Surface Markers by Flow Cytometry

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE
Whole blood can be stained with fluoro- tive, multiparameter analysis of heteroge- cells’ size, internal complexity, and rela-
chrome-labeled monoclonal antibodies neous cell populations on a cell-by-cell tive fluorescence intensity. The percentage
against antigen markers found on the sur- basis (single-cell analysis). During acqui- of fluorescent cells is determined for each
faces of lymphocytes. The stained samples sition, the cells travel past the laser beam antibody. In addition, the use of the light
are treated with a lysing solution to de- and scatter the laser light. The stained cells scatter measurements from the individual
stroy erythrocytes. The flow cytometer is fluoresce. These scatter, and fluorescence cells along with fluorescence allows iden-
an instrument capable of rapid, quantita- signals provide information about the tification of the lymphocyte population.

II. SPECIMEN COLLECTION, A. Specimen collection


TRANSPORT, AND HANDLING 1. Peripheral blood in EDTA (acid citrate dextrose or heparin may also be used)
anticoagulant
2. Vacutainers or other collection tubes should be filled properly to account for
the amount of anticoagulant.
3. Since all specimens should be regarded as potentially infectious, standard
precautions for blood collection and handling should be properly followed.
B. Specimen transport
1. Specimens should be placed in well-stoppered tubes and should be placed
with adequately absorbent material, preferably in a closed leakproof con-
tainer, for delivery.
2. Specimens should be maintained at room temperature (18 to 22⬚C) during
transportation and storage.
C. Specimen labeling and request submission
1. Each patient sample must be properly labeled.
2. Each sample should be accompanied by some type of procurement form
which should contain appropriate identifiers, e.g., patient’s name, date of
birth, and gender; date and time of collection; clinician; and other relevant
patient information.
D. Rejection criteria
1. Inadequate sample volume
2. Clotted specimen
3. Hemolyzed blood specimen
4. Specimen more than 24 h old
5. Specimen exposed to cold temperatures

11.16.1
11.16.2 Immunology

III. MATERIALS A. Reagents (see Appendix 11.16–1 for B. Supplies


reagent concentrations and prepara- 1. Adjustable-volume pipette and tips
tion) 2. 12- by 75-mm polystyrene tubes
1. Lysing reagent (depends on manu- (BD Biosciences, Franklin Lakes,
facturer of flow cytometer) N.J.)
2. Dulbecco’s phosphate-buffered sa- 3. Liquid-waste container with bleach
line (dPBS) without calcium, mag- 4. Disposable gloves
nesium, or phenol red (Invitrogen, 5. Disposable lab coat
Grand Island, N.Y.) 6. Protective eyewear
3. Monoclonal antibodies against cell C. Equipment
surface markers—stored according 1. Vortex
to manufacturer’s instructions 2. Centrifuge with swinging bucket
4. 2% Working solution of 10% For- rotor, capable of centrifugation at
malin neutral buffered solution 300 ⳯ g and equipped with aerosol
(Sigma, St. Louis, Mo.) or 2% containment canisters
working solution of 10% methanol- 3. Vacuum source with pipette at-
free Formaldehyde (PolySciences, tached
Inc., Warrington, Pa.) 4. Flow cytometer
5. Flow cytometer calibration beads 5. Class II biosafety cabinet
or other calibration reagents (fol- 6. Timer/stop watch
low manufacturer’s directions)

IV. CALIBRATION A. Optical alignment


Flow cytometer must be optically aligned. Follow manufacturer’s directions.
(Some cytometers require a company engineer.)
B. Calibration
Depending on the flow cytometer manufacturer, either standardized fluorescent
beads or chicken RBCs may be used for calibration and to assess instrument
function. Follow manufacturer’s procedures.
C. Verification with whole-blood preparation
Electrical fluorescent compensation on single stained cells (unless not required
due to calibration software specifications in which beads mimic the actual spec-
imens). Adjust FL1-FL2 and FL2-FL1, FL2-FL3 and FL3-FL2, and FL3-FL4
and FL4-FL3. Parameters depend on the number of lasers present and which
panel is being used. Follow manufacturer’s procedure.

ANALYTICAL CONSIDERATIONS

V. QUALITY CONTROL Controls

A. Surface-stained cells (e.g., CD3ⴐ CD4ⴐ) from a healthy donor


These cells should be stained in the same manner and with the same monoclonal
antibodies as the patient specimens.
B. Commercially available QC substance (usually stabilized blood cells) that
mimics actual specimens
These cells are stained with the same monoclonal antibodies as patient speci-
mens. Results can be compared to expected values.
C. Frequency
The healthy donor blood should be drawn and run daily. If there are several
working shifts, the blood should be stained and run for each shift. The QC cells
should be stained and run daily.
Lymphocyte Immunophenotyping by Flow Cytometry 11.16.3

V. QUALITY CONTROL D. Expected results


(continued) 1. Healthy donor results should be compared to normal values ascertained by
each laboratory.
2. QC substances should be compared to the individual company’s published
ranges.
E. Corrective action
1. All control results falling outside of the expected ranges should be considered
suspect and should be brought to the attention of the person in charge.
2. Results of cell surface markers performed on blood from apparently healthy
donors may be dependent on the current health of the individual or due to
individual variation. They do not necessarily signify a failure of the test
system. These results should be reviewed to determine if patient results are
reportable. There must be documentation of these reviews.
3. In the event that a serious problem is found in the test system, corrective
action should be performed and documented. If patient’s values are suspect,
they should not be released and a request should be made for submission of
a new specimen.

VI. FLOW CYTOMETRY TWO- A. Panel


COLOR STAINING Isotype, CD45/CD14, CD3/CD4, CD3/CD8, CD3/CD19, CD3/CD(16Ⳮ56), or
CD3/CD16
It is imperative that these B. Procedure (done in a laminar hood with the lights out)
cultures be handled in a 1. Label a set of six tubes for each patient and one set for the normal control.
biosafety hood. 2. Pipette 100 ll (or amount specified by the monoclonal antibody manufac-
turer’s directions) of the control’s or patient’s blood into each tube.
3. Pipette the appropriate volume of antibodies into all the tubes, changing
Observe standard precautions. pipette tips between each antibody vial to prevent contamination. Gently
vortex to mix.
4. Incubate at room temperature in the dark for the appropriate time (check
manufacturer’s recommendations).
5. During the incubation time, remove any blood adhering to the walls of the
tubes with a cotton swab moistened with PBS.
6. After incubation, vortex each sample tube and add appropriate amount of
lysing reagent (per manufacturer’s instructions).
7. Incubate for appropriate time (per manufacturer’s instructions).
8. After the incubation period, centrifuge tubes at 300 ⳯ g for 5 min at room
temperature.
9. Using an aspirating unit, remove the supernatant from the tubes to within
1/8 in. of the bottom. There should be about 200 ll of liquid left in the
tube.
10. Gently vortex to resuspend the pellet.
11. Add 3 ml of PBS to all tubes. Centrifuge tubes at 300 ⳯ g for 5 min at
room temperature.
12. Using the aspirating unit, remove the supernatant from the tube to within
1/8 in. of the bottom. There should be about 200 ll of liquid left in the
tube.
13. Gently vortex each tube to resuspend the cell pellet.
14. Add at least 0.5 ml of a 2% formaldehyde fixative solution to each sample
tube and vortex gently. Cap tubes, place in rack, and cover with aluminum
foil to protect from light.
15. Store at 2 to 8⬚C until tubes can be acquired. This should be no longer than
48 h after they are completed.
11.16.4 Immunology

VII. FLOW CYTOMETY TWO- A. Purity and recovery


COLOR ANALYSIS 1. Specimen must be properly mixed before collection.
2. Draw an electronic gate using forward versus side light scatter around the
Observe standard precautions. small nongranular population (lymphocytes). Care should be taken to include
large granular lymphocytes and at the same time to exclude monocytes and
granulocytes.
3. Draw a second, relatively larger light scatter gate to also include debris and
monocytes.
4. Collect 20,000 total events or sufficient events so that there are at least 2,500
events collected in the lymphocyte population for each tube.
5. Using the first gate (lymphocyte gate), analyze the CD45 CD14 tube on a
dual-parameter histogram plot. The CD45 should be on the x axis, and the
CD14 should be on the y axis. Set quadrants so that they separate the bright
CD45-positive cells from CD45-dim and -negative cells and the CD14-posi-
tive cells from the CD14-negative cells on the y axis (Fig. 11.16–1A and B).
The purity of the lymphocyte gate is the percentage of cells within the gate
that express bright CD45Ⳮ and CD14ⳮ cells. The lymphocyte purity should
optimally be at least 90% but may be as low as 85%. If it is less than 85%,
the initial gate should be checked and if possible redrawn.
6. Using a dual dot plot for the larger gate, set the same quadrants as used for
the smaller lymphocyte gate.
7. Determine the number of cells that meet both criteria for both gates (bright
CD45Ⳮ CD14ⳮ ⳱ total number of lymphocytes).
8. To determine the lymphocyte recovery (Fig. 11.16–1C and D), divide the
number of lymphocytes determined from the analysis regions of the smaller
gate by the total number of lymphocytes in the analysis regions of the larger
gate and multiply by 100.
9. The lymphocyte recovery should optimally be at least 95% but may be as
low as 90%. Optimal gates include as many lymphocytes and as few con-
taminants as possible.
B. Isotype tube
Using the smaller gate, analyze the isotype on a two-parameter dot plot. Set the
quadrant markers to read between no more than 1 to 2% positive in quadrants
1, 2, and 4. If the background is above 2% and found to be present throughout
the rest of the samples, it should be subtracted.
C. Remaining tubes
1. Read the remaining tubes for the same specimen using the isotype quadrant
markers. If it is necessary, the quadrant markers may be optimized.
2. Correct all percentages of markers by dividing the percent positive of gated
cells by the percent lymphocyte purity.
D. QA
1. Check lymphosum (total T cells plus B cells plus natural killer [NK] cells).
Results should be between 95 and 100%.
2. Helper (CD3Ⳮ CD4Ⳮ) plus suppressor (CD3Ⳮ CD8Ⳮ) cells should equal total
T (CD3Ⳮ) cells within Ⳳ 10%. (The presence of gamma delta cells could
affect these results. In that case, the values could be less than ⳮ10%.)
3. The difference between CD3Ⳮ cells in all four tubes should be within 0 to
3%.
4. If any of these values are out of range, try regating or restaining.
Lymphocyte Immunophenotyping by Flow Cytometry 11.16.5

Figure 11.16–1 (A) Forward versus side light scatter histogram showing the smaller light
scatter gate (lymphocytes) which is used for analyzing the remaining tubes from that
specimen. (B) CD45/CD14 histogram from gated R1. The lymphocyte purity is the
percentage of positive cells within the gate that express bright CD45-positive cells that are
negative for CD14 (quadrant LR). The purity in this sample is 97%. The number of cells
in quadrant LR which are bright CD45 positive and negative for CD14 is the number of
gated lymphocytes (3,812 cells). (C) The same light scatter gate as in panel A except that
it has a rather large light scatter region drawn around the lymphocytes (R2). (D) The two-
parameter histogram of CD45/CD14 fluorescence is gated on R2. The number of cells in
quadrant LR which are bright CD45 positive and negative for CD14 is the total number of
lymphocytes (3,847 cells). The lymphocyte recovery is 99%, obtained as follows: (3,812/
3,847) ⳯ 100. FITC, fluorescein isothiocyanate; PE, phycoerythrin.
11.16.6 Immunology

VIII. CD45 GATING AND It is easier to distinguish lymphocytes based on CD45 fluorescence and 90⬚ (side)
THREE-COLOR ANALYSIS scatter. Many nonlymphocyte contaminants of a light scatter gate (e.g., unlysed
RBCs) can be easily eliminated from a CD45 side scatter gate. The presence of
nonlymphocyte elements within the boundaries of the lymphocyte gate is assumed
to be negligible. Using a low side scatter and bright CD45 fluorescence for iden-
tification of lymphocytes, an assumption is made that the only cells meeting these
criteria are lymphocytes and that therefore the lymphocyte purity of the gate is close
to 100%. Therefore, it is not necessary to stain cells for determination of purity.
An isotype control is not necessary, as the negatively labeled populations can serve
this function.

A. Panel
CD45/CD3/CD4, CD45/CD3/CD8, CD45/CD3/CD19, CD45/CD3/
CD(16Ⳮ56), or CD16
B. Staining
Follow two-color procedure using four tubes instead of six.
C. Flow cytometry analysis
1. Set a lymphocyte gate using linear 90⬚ side scatter and log of CD45 fluo-
rescence. Lymphocytes are defined as CD45bright with low side scatter.
2. Set threshold to CD45 fluorescence and adjust so populations of interest are
visible.
3. Collect a minimum of 2,500 events in the CD45bright low-scatter gate.
4. Analyze each tube using the negative and positive populations to determine
cursor settings.
5. Ensure lymphocyte recovery by determining the lymphosum (total T cells
plus B cells plus NK cells). Results should fall within 95 to 100%. If not,
try regating.
6. The presence of nonlymphocyte elements within the boundaries of the lym-
phocyte gate is assumed to be negligible. Therefore, it is not necessary to
report purity (percentage of lymphocytes in analysis gate).
D. QA
Follow two-color procedure.

IX. FLOW CYTOMETRY FOUR- A. Panel


COLOR ANALYSIS (Fig. 11.16–2) CD45/CD3/CD4/CD8, CD45/CD3/CD19/CD(16Ⳮ56), or CD16
B. Staining
Follow three-color procedure.
C. Flow cytometry analysis
Follow three-color procedure.
D. QA
Follow three-color procedure, except that the difference between the total T
cells (CD3) in the two tubes should be 0 to 2%.

X. FLOW CYTOMETRY NO- By using fluorescence triggering on CD45 (setting a threshold on fluorescence in-
WASH METHOD stead of forward and side scatter), which stains all WBCs, it is possible to reduce
contamination of unlysed or nucleated RBCs in the gate and analyze the lymphocyte
population.

A. Staining using CD45 gating three- or four-color panel


1. Label a set of two or four tubes, depending on which panel you are using.
2. Pipette 100 ll (or monoclonal antibody manufacturer’s directions) of the
control’s or patient’s blood into each tube.
Lymphocyte Immunophenotyping by Flow Cytometry 11.16.7

Figure 11.16–2 Four-color immunophenotyping using CD45 and side scatter gating. (A)
Gate (R1) on the bright CD45 nongranular population (lymphocytes). This is the region
used for analysis of both tubes 1 and 2. (B) CD3Ⳮ CD4Ⳮ (quadrant 2) from tube 1. (C)
CD3Ⳮ CD8Ⳮ (quadrant 2) from tube 1. (D) CDⳮ CD4Ⳮ (quadrant 1) from tube 2. (E)
CD3ⳮ CD(16Ⳮ56Ⳮ) (quadrant 1) from tube 2. FITC, fluorescein isothiocyanate; APC,
allophycocyanin; PE, phycoerythrin.

X. FLOW CYTOMETRY NO- 3. Pipette the appropriate volume of antibodies into all the tubes as in the three-
WASH METHOD (continued) and four-color procedures.
4. Incubate at room temperature in the dark for the appropriate time as in the
three- and four-color procedures.
5. After incubation, add lysing reagent and incubate as in the three- and four-
color procedures. Do not wash.
B. Flow cytometry analysis
Follow procedure for three- and four-color acquisition.
C. QA
Follow three- or four-color procedure.
11.16.8 Immunology

POSTANALYTICAL CONSIDERATIONS

XI. REPORTING RESULTS Reference range

A. Reference range values, including percent positive and absolute numbers,


should be calculated from at least 50 healthy adult donors. Pediatric reference
ranges should also be established.
B. Patient values should be compared to healthy donor ranges and reported as
increased or decreased.

XII. INTERPRETATION A. Patient values should be compared to healthy donor values. Increases or de-
creases in values may be related to a variety of diseases. A physician should be
able to interpret these results based on patient symptoms and other clinical data.
B. New sample required if
1. Lymphosum (total T cells plus B cells plus NK cells) is out of range. Results
should be between 95 and 100%.
2. The differences in CD3 values in tubes are not within the matching range (0
to 3% for two- or three-color staining and 0 to 2% for four-color staining).
3. Helper (CD3Ⳮ CD4Ⳮ) plus suppressor (CD3Ⳮ CD8Ⳮ) cells do not equal total
T (CD3Ⳮ) cells within Ⳳ 10%. (The presence of gamma delta cells could
affect these results. In that case the values could be less than ⳮ10%.)

SUPPLEMENTAL READING Centers for Disease Centrol and Prevention. Nicholson, J., P. Kidd, F. Mandy, D. Livnat,
1997. Revised guidelines for performing CD4Ⳮ T- and J. Kagan. 1996. Three-color supplement to
cell determinations in persons infected with hu- the NIAID DAIDS guideline for flow cytometric
man immunodeficiency virus (HIV). Morb. Mor- immunophenotyping. Cytometry 26:227–230.
tal. Wkly. Rep. 46 (RR-2):1–29.
NCCLS. 1998. Clinical Applications of Flow Cy-
tometry: Quality Assurance and Immunopheno-
typing of Lymphocytes. Document H42-A, vol. 18,
no. 21. NCCLS, Wayne, Pa.

APPENDIX 11.16–1 Reagent Preparation


Working solution of methanol-free 2% formaldehyde—stock solution from PolySciences or
Sigma

A. 10% Formaldehyde solution (PolySciences no. 0418), which must be diluted 1:5 with
dPBS to obtain a working concentration of 2% formaldehyde
B. 10% Formalin neutral buffered solution from Sigma (no. HT50-1-1) that is diluted 1:2
with dPBS to obtain a working concentration of 2% formaldehyde
C. Store diluted 2% formaldehyde in an amber glass container or wrap container in alumi-
num foil to avoid exposure to light. May be stored for up to 1 week at 2 to 8⬚C.
11.17 Neutrophil Function Whole-Blood
Flow Cytometric Test for Leukocyte
Adhesion Deficiency

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE
Normal neutrophil function is of great im- endothelium and opsonized microorgan- middle ear, and oropharyngeal mucosa. In
portance in the host defense against bac- isms due to the absence or reduced ex- the assay described here, neutrophils are
terial and fungal infections. As neutrophils pression of a group of cell surface leuko- stimulated with phorbol 12 myristate 13
recognize, adhere to, and phagocytose an cyte adhesion markers. This group of acetate (PMA) and stained with CD11b
invading microbe they generate an oxida- surface glycoproteins includes LFA-1 plus corresponding isotypic monoclonal
tive burst, which kills the pathogen. In the (CD11b/CD18), Cr3 (CD11b/CD18), and antibody. Cellular fluorescence is mea-
leukocyte adhesion deficiency (LAD) P150,95 (CD11c/CD18). Patients with this sured on resting and stimulated neutro-
states, there are defects in the ability of deficiency manifest recurrent infections phils using flow cytometry.
neutrophils to adhere to both the vascular involving the skin, subcutaneous tissues,

II. SPECIMEN COLLECTION, A. Specimen collection


TRANSPORT, AND HANDLING 1. Peripheral blood in EDTA anticoagulant (2 ml)
2. Vacutainers or other collection tubes should be filled properly to account for
Observe standard precautions. the amount of anticoagulant.
3. Since all specimens should be regarded as potentially infectious, standard
precautions for blood collection and handling should be properly followed.
B. Specimen transport
1. Specimens should be placed in well-stoppered tubes and should be placed
with adequately absorbent material, preferably in a closed leakproof con-
tainer, for delivery.
2. Specimens should be maintained at room temperature (18 to 22⬚C) during
transportation and storage.
3. Testing must be performed within 4 h from the time of collection.
C. Specimen labeling and request submission
1. Each patient sample must be properly labeled.
2. Each sample should be accompanied by some type of procurement form
which should contain appropriate identifiers, e.g., patient’s name, date of
birth, and gender; date and time of collection; clinician; and other relevant
patient information.
D. Rejection criteria
1. Inadequate sample volume
2. Clotted specimen
3. Hemolyzed blood specimen
4. Specimen more than 4 h old
5. Specimen exposed to cold temperatures

11.17.1
11.17.2 Immunology

III. MATERIALS A. Reagents (see Appendix 11.17–1 for 7. Flow cytometer calibration beads
reagent concentrations and prepara- or other calibration reagents (fol-
tion) low manufacturer’s directions)
Include QC information on 1. BD FACS Lysing Solution—work- B. Supplies
reagent container and in ing solution diluted 1:10 with dis- 1. Adjustable-volume pipette and tips
QC records. tilled water (BD Biosciences, San 2. 12- by 75-mm polystyrene tubes
Jose, Calif.). (If using different lys- (BD Biosciences, Franklin Lakes,
ing reagent, follow manufacturer’s N.J.)
instructions.) 3. Liquid-waste container with bleach
2. Dulbecco’s phosphate-buffered so- 4. Disposable gloves
lution (dPBS) without calcium or 5. Disposable lab coat
magnesium (Invitrogen, Grand Is- 6. Protective eyewear
land, N.Y.) C. Equipment
3. Hanks balanced salt solution 1. Vortex
(HBSS) without calcium, magne- 2. Centrifuge
sium, or phenol red (Invitrogen) 3. CO2 incubator (5% CO2)
4. CD11b and isotype immunoglobu- 4. Vacuum source with pipette at-
lin G2a (IgG2a) phycoerythrin tached
(PE)-labeled monoclonal antibod- 5. Centrifuge with swinging bucket
ies, stored according to manufac- rotor, capable of centrifugation at
turer’s instructions 300 ⳯ g and equipped with aerosol
5. PMA (Sigma Chemical Co., St. containment canisters
Louis, Mo.) 6. Class II biosafety cabinet
6. 2% Working solution of 10% meth- 7. Timer/stop watch
anol-free formaldehyde 8. Flow cytometer

IV. CALIBRATION A. Optical alignment


Flow cytometer must be optically aligned. Follow manufacturer’s directions.
(Some cytometers require a company engineer.)
B. Calibration
Depending on the flow cytometer manufacturer, either standardized fluorescent
beads or chicken RBCs may be used for calibration to assess instrument func-
tion. Follow manufacturer’s procedures.
C. Verification with whole-blood preparation
Electrical fluorescent compensation on single stained cells. Follow manufac-
turer’s procedures.

ANALYTICAL CONSIDERATIONS

V. QUALITY CONTROL Controls

A. Surface-stained cells (e.g., CD11b) from a healthy donor. These cells should be
stained and stimulated in the same manner and with the same monoclonal an-
tibodies as the patient specimens. Values should fall within established normal
ranges.
B. If for some reason the results for the healthy donor are abnormal (such as re-
sponse to stimulants), then the patient’s results should not be reported. Instru-
ments and reagent integrity should be checked, and if necessary, service on the
flow cytometer should be requested. A new patient sample should be submitted.
Whole-Blood Flow Cytometric Test for LAD 11.17.3

VI. LAD ASSAY A. Procedure


1. Set four tubes per run for both a healthy donor and the patient, labeled as
follows.
It is imperative that these
cultures be handled in a a. IgG2a PE
biosafety hood. b. CD11b PE
c. IgG2a PE Ⳮ PMA
d. CD11b PE Ⳮ PMA
Observe standard precautions. 2. Add 100 ll of whole blood to each tube.
3. Add 10 ll of HBSS to tubes a and b. Add 10 ll of PMA to tubes c and d.
Vortex lightly.
4. Incubate for 15 min at 37⬚C in a CO2 incubator.
5. Add 20 ll of monoclonal antibody (or follow amount suggested in mono-
clonal antibody manufacturer’s directions) to appropriate tubes. Vortex
lightly.
6. Incubate for 15 min in the dark at room temperature.
7. Add 2 ml of BD FACS Lysing Solution. Incubate for 10 min (follow ap-
propriate amount and manufacturer’s instructions if using a different lysing
agent).
8. Centrifuge at 300 ⳯ g for 5 min. Aspirate supernatant.
9. Wash one time with 2 ml of HBSS. Aspirate supernatant.
10. Fix with 0.5 ml of 2% methanol-free formaldehyde.
11. Acquire as soon as possible on the flow cytometer.
B. Flow cytometry analysis
1. Using forward versus side light scatter parameters, set a region around the
large granular cells (neutrophils).
2. Set the mean channel of the histogram from tube a (IgG2a) at around 2.0 on
the log scale and collect specimen.
3. Collect 10,000 events for all four tubes.
4. Using the region around the neutrophils, print the histograms from all four
tubes.
5. Record the mean fluorescence intensities (MFI) for CD11b, CD11b plus
PMA, IgG2a, and IgG2a plus PMA and determine the upregulation index
(UI).

UI ⳱
MFI for CD11b stimulated with PMA/MFI for IgG2a stimulated with PMA
MFI for unstimulated CD11b/MFI for unstimulated IgG2a

POSTANALYTICAL CONSIDERATIONS

VII. REPORTING RESULTS Report the MFI for CD11b stimulated with PMA divided by the MFI for IgG2a
stimulated with PMA, the MFI for unstimulated CD11b divided by the MFI for
unstimulated IgG2A, and the UI.

VIII. INTERPRETATION Normal values


A. Values for at least 50 healthy donors should be ascertained by each laboratory
performing this test.
B. Compare the following values.
1. The MFI for stimulated CD11b divided by the value for stimulated IgG2a
2. The MFI for unstimulated CD11b divided by the value for unstimulated
IgG2a
3. UI
11.17.4 Immunology

VIII. INTERPRETATION C. Values for patients should fall within ranges for healthy donors. However, some-
(continued) times values may be slightly off for various reasons, e.g., the patient’s MFI for
unstimulated CD11b is increased. In cases such as these, it is important to
ascertain that there was a sufficient increase in the MFI for stimulated CD11b.
Patients with LAD will have very little increase in the MFI for CD11b when
the cells are stimulated.
D. Increased values compared to the healthy donor ranges have no significance.

IX. LIMITATION OF TESTING Stimulation with PMA does not occur in vivo.

SUPPLEMENTAL READING Centers for Disease Control and Prevention. O’Gorman, M. R. G., and A. C. Mcnally. 1993.
1997. Revised guidelines for performing CD4Ⳮ T- A rapid whole blood lysis technique for diagnosis
cell determinations in persons infected with hu- of moderate or severe leukocyte adhesion defi-
man immunodeficiency virus (HIV). Morb. Mor- ciency. Ann. N. Y. Acad. Sci. 677:427–430.
tal. Wkly. Rep. 46(RR-2):1–29. Todd, R. F., and D. R. Freyer. 1988. The CD11/
Hassan, N. F., D. E. Campbell, and S. D. Doug- CD18 leukocyte glycoprotein deficiency. Hema-
las. 1988. Phorbol myristate acetate oxidation of tol. Oncol. Clin. N. Am. 2:13–31.
2⬘7⬘ dichlorofluorescein by neutrophils from pa-
tients with chronic granulatomatous disease. J.
Leukoc. Biol. 43:317–322.
Malech, H. L., and J. I. Gallin. 1987. Neutro-
phils in human diseases. N. Engl. J. Med.
317:687–694.

APPENDIX 11.17–1 Reagent Preparation


A. PMA
Include QC information on Stock solution of 1 mg/ml is prepared in dimethyl sulfoxide, with aliquots frozen at
reagent container and in ⳮ70⬚C. Working solution is diluted in HBSS for a 1-lg/ml concentration. Final con-
QC records. centration in assay is 90 ng/ml.
B. Working solution of methanol-free 2% formaldehyde—stock solution from PolySciences
or Sigma
1. 10% Formaldehyde solution (PolySciences no. 0418), which must be diluted 1:5 with
dPBS to obtain a working concentration of 2% formaldehyde
2. 10% Formalin neutral buffered solution from Sigma (no. HT50-1-1) that is diluted
1:2 with dPBS to obtain a working concentration of 2% formaldehyde
3. Store diluted 2% formaldehyde in an amber glass container or wrap container in
aluminum foil to avoid exposure to light. May be stored for up to 1 week at 2 to 8⬚C.
11.18 Flow Cytometric Test for Chronic
Granulomatous Disease

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE
Normal neutrophil function is of great im- to hydrogen peroxide (H2O2, which kills oxidative burst activity, which is easily
portance in the host defense against bac- the pathogen). In chronic granulomatous converted to hydrogen peroxide. DHR, a
terial and fungal infections. As neutrophils disease (CGD), microbial killing is defec- nonfluorescent compound, reacts with hy-
recognize, adhere to, and phagocytose an tive because neutrophils from patients drogen peroxide and is oxidized to rho-
invading organism, they generate an oxi- with CGD lack a respiratory burst. The as- damine-123, a green fluorescent com-
dative burst which results in the reduction say described here uses dihydrorhoda- pound. Cellular fluorescence is measured
of molecular oxygen to superoxide. The mine-123 (DHR-123) and phorbol 12 using flow cytometry.
superoxide produced is rapidly converted myristate 13 acetate (PMA) to measure

II. SPECIMEN COLLECTION, A. Specimen collection


TRANSPORT, AND HANDLING 1. Peripheral blood in EDTA anticoagulant (2 ml)
2. Vacutainers or other collection tubes should be filled properly to account for
Observe standard precautions. the amount of anticoagulant.
3. Since all specimens should be regarded as potentially infectious, standard
precautions for blood collection and handling should be properly followed.
B. Specimen transport
1. Specimens should be placed in well-stoppered tubes and should be placed
with adequately absorbent material, preferably in a closed leakproof con-
tainer, for delivery.
2. Specimens should be maintained at room temperature (18 to 22⬚C) during
transportation and storage.
3. Testing must be performed within 4 h from the time of collection.
C. Specimen labeling and request submission
1. Each patient sample must be properly labeled.
2. Each sample should be accompanied by some type of procurement form
which should contain appropriate identifiers, e.g., patient’s name, date of
birth, and gender; date and time of collection; clinician; and other relevant
patient information.
D. Rejection criteria
1. Inadequate sample volume
2. Clotted specimen
3. Hemolyzed blood specimen
4. Specimen more than 4 h old
5. Specimen exposed to cold temperatures

11.18.1
11.18.2 Immunology

III. MATERIALS A. Reagents (see appendix 11.18–1 for B. Supplies


reagent concentrations and prepara- 1. Adjustable-volume pipette and tips
tion) 2. 12- by 75-mm polystyrene tubes
Include QC information on 1. Ammonium chloride lysing solu- (BD Biosciences, Franklin Lakes,
reagent container and in tion N.J.)
QC records. a. NH4Cl (Sigma Chemical Co., 3. Liquid-waste container with bleach
St. Louis, Mo.) 4. Disposable gloves
b. NaHCO3 (Fisher Scientific, Fair 5. Protective eyewear
Lawn, N.J.) C. Equipment
c. EDTA (Fisher Scientific) 1. Vortex
2. Hanks balanced salt solution 2. Timer/stop watch
(HBSS) without calcium, magne- 3. Centrifuge with swinging bucket
sium, or phenol red (Invitrogen, rotor, capable of centrifugation at
Grand Island, N.Y.) 300 ⳯ g and equipped with aerosol
3. DHR-123 (Molecular Probes, Eu- containment canisters
gene, Oreg.) 4. CO2 incubator (5% CO2)
4. Dimethyl sulfoxide (DMSO) 5. Vacuum source with pipette at-
(Sigma) tached
5. PMA (Sigma) 6. Class II biosafety cabinet
6. Flow cytometer calibration beads 7. Flow cytometer
or other calibration reagents (fol-
low manufacturer’s directions)

ANALYTICAL CONSIDERATIONS

IV. QUALITY CONTROL Controls

A. Cells from a healthy donor. These cells should be treated in the same manner
Observe standard precautions.
as the patient specimens. Values should fall within established normal ranges.
B. If for some reason the results for the healthy donor are abnormal (e.g., the
neutrophil oxidative index [NOI]), then the patient’s results should not be re-
ported. Instruments and reagent integrity should be checked, and if necessary,
service on the flow cytometer should be requested. A new patient sample should
be submitted.

V. CALIBRATION A. Optical alignment


Flow cytometer must be optically aligned. Follow manufacturer’s directions.
(Some cytometers require a company engineer.)
B. Calibration
Depending on the flow cytometer manufacturer, either standardized fluorescent
beads or chicken RBCs may be used for calibration and to assess instrument
function. Follow manufacturer’s procedures.

VI. CGD ASSAY A. Procedure


1. Set two tubes per run for both a healthy donor and the patient, labeled as
It is imperative that these follows.
cultures be handled in a a. DHR-123
biosafety hood. b. DHR-123 Ⳮ PMA
2. Add 100 ll of whole blood to each tube.
3. Add 3 ml of lysing solution (prewarmed to 37⬚C) to each tube. Vortex and
let stand at room temperature for 5 min.
Observe standard precautions.
4. Spin at 300 ⳯ g for 5 min. Aspirate supernatant.
5. Wash twice with 2 ml of HBSS. Aspirate supernatant.
6. Add 400 ll of HBSS. Vortex tubes lightly.
Flow Cytometric Test for CGD 11.18.3

VI. CGD ASSAY (continued) 7. Add 10 ll DHR-123. Vortex lightly. Incubate in 5% CO2 incubator at 37⬚C
for 5 min.
8. Add 40 ll of HBSS to tube a. Add 40 ll of PMA to tube b. Vortex lightly
and incubate in 5% CO2 incubator at 37⬚C for 15 min.
9. Collect immediately on the flow cytometer.
B. Flow cytometry analysis
1. Using forward versus side light scatter parameter, set a region around the
large granular cells (neutrophils).
2. Set the mean channel of the histogram from tube a (DHR) at around 10.0
on the log scale and collect specimen.
3. Collect 10,000 events for both tubes.
4. Using the region around the neutrophils, print the histograms from both
tubes.

POSTANALYTICAL CONSIDERATIONS

VII. REPORTING RESULTS Record the mean fluorescence intensity (MFI) from both the DHR tube and the
tube with DHR plus PMA. Calculate the NOI. The NOI is the MFI of the cells with
DHR plus PMA (stimulated) divided by the MFI of cells with DHR alone (unstim-
ulated).

VIII. INTERPRETATION Normal values

A. NOIs for at least 50 healthy donors should be ascertained by each laboratory


performing this test.
B. Compare the NOIs to established normal ranges. Patients with CGD have a lack
of oxidative burst function and will have a very low NOI (e.g., ⬃0 to 10). If
the patient has an intermediate value, repeat studies should be done. A normal
value indicates that there is no evidence of lack of oxidative burst function.

IX. LIMITATION OF TESTING Stimulation with PMA does not occur in vivo.

SUPPLEMENTAL READING Bass, D., W. Parce, L. Dechatelet, P. Szejda, M. Malech, H. L., and J. I. Gallin. 1987. Neutro-
Seeds, and M. Thomas. 1983. Flow cytometric phils in human diseases. N. Eng. J. Med. 317:687–
studies of oxidative product formation by neutro- 693.
phils: a grade response to membrane stimulation. O’Gorman, M. R. G., and V. Corrochano.
J. Immunol. 136:1910–1917. 1995. Rapid whole blood flow cytometry assay for
Centers for Disease Control and Prevention. the diagnosis of chronic granulomatous disease.
1997. Revised guidelines for performing CD4Ⳮ T- Clin. Diagn. Lab. Immunol. 2:227–232.
cell determinations in persons infected with hu- Vowells, S. J., S. Sekhsaria, H. L. Malech, M.
man immunodeficiency virus (HIV). Morb. Mor- Shalit, and T. A. Fleisher. 1995. Flow cytometric
tal. Wkly. Rep. 46 (RR-2): 1–29. analysis of granulocyte respiratory burst: a com-
Emmendorffer, A., M. Hecht, M. L. Lohmann- parison study of fluorescent probes. J. Immunol.
Matthes, and J. Roesler. 1990. A fast and easy Methods 178:89–97.
method to determine the production of reactive
oxygen intermediates by human phagocytes using
dihydrorhodamine 123. J. Immunol. Methods
131:269–275.
Hassan, N. F., D. E. Campbell, and S. D. Doug-
las. 1988. Phorbol myristate acetate oxidation of
2⬘7⬘ dichlorofluorescein by neutrophils from pa-
tients with chronic granulatomatous disease. J.
Leukoc. Biol. 43:317–322.
11.18.4 Immunology

APPENDIX 11.18–1 Reagent Preparation


A. Ammonium chloride lysing solution
Include QC information on 1. Stock solution
reagent container and in NH4Cl ........................................... 8.02 g
QC records. NaHCO3 ........................................ 0.84 g
EDTA ........................................... 0.37 g
Bring to a volume of 100 ml with distilled H2O. Store at 4⬚C. Keeps for 6 months.
2. Working solution diluted 1:10 in distilled H2O. Keeps for 1 week.
B. DHR-123
Stock solution of 5 mg/ml is prepared in DMSO, with aliquots frozen at ⳮ70⬚C. Working
solution is diluted in HBSS for a 15-lg/ml concentration. Final concentration in assay is
1 lM.
C. PMA
Stock solution of 1 mg/ml is prepared in DMSO, with aliquots frozen at ⳮ70⬚C. Working
solution is diluted in HBSS for a 1-lg/ml concentration. Final concentration in assay is
90 ng/ml.