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may cause a 40X objective with a NA of 0.65 to
read one or more dilutions lower than a 40X objec-
tive with a NA of 0.85. The numerical aperture is
printed on the side of the objective.
6. Supression filters. Supression filters reduce specific
wavelengths of excitation and may be used in
reducing sensitivity. Refer to your fluorescent mi-
croscope manual or sales representative for more
information.
7. Precision and accuracy of dilution technique,
equipment, and performance of the test proce-
dures.
IntErPrEtatIon oF PatIEnt rESultS
200X total magnification is recommended for screening
positive/negative, while 400X total magnification is
recommended for pattern recognition and viewing
mitotic cells.
negative: A serum is considered negative for antinu-
clear antibodies if nuclear staining is less than or equal
to the negative control well with no clearly discernible
pattern. The cytoplasm may demonstrate weak stain-
ing, with brighter staining of the nonchromosome region
of mitotic cells, but with no clearly discernible nuclear
pattern.
Positive: A serum is considered positive if the nucleus
shows a clearly discernible pattern of staining in a
majority of the interphase cells.
titers: When reading titers, many laboratories begin
reading with the well that contains the most dilute
sample and read "backwards" to the 1:40 dilution. The
first well in which a clearly discernible pattern is visible
is the titer end-point. We recommend this technique
for determining titer end-points. It is important that the
intensity of staining not be confused with the presence
or absence of antinuclear antibodies. The key factor to
consider in determining whether a given dilution of se-
rum is positive is the appearance of a clearly discernible
nuclear pattern, irrespective of the staining intensity.
caution: Some sera may demonstrate nuclear and
cytoplasmic staining with no apparent nuclear pattern.
This phenomenon is generally due to heterophile anti-
bodies and should be reported as negative (31).
FluorEScEnt IntEnSItY
Fluorescent intensity may be semi-quantitated by fol-
lowing the guidelines for fluorescent antibody reagents
established by the Centers for Disease Control and
Prevention, Atlanta, Georgia (CDC).
4+ Brilliant yellow-green (maximal fluorescence):
clear-cut cell outline; sharply defined cell center.
3+ Less brilliant yellow-green fluorescence: clear-cut
cell outline; sharply defined cell center.
2+ Definite cell pattern but dim fluorescence: cell
outline less well defined.
1+ Very subdued fluorescence: cell outline almost
indistinguishable from cell center in most in-
stances.
A standard slide for the determination of these fluores-
cent intensities, FITC QC Slide™, catalog number 1900,
is available from Immuno Concepts, N.A. Ltd.
rEPortIng oF rESultS
Screening: Results should be reported as strongly
positive or positive at the 1:40 dilution, and the nuclear
staining pattern should be reported.
titering: Results should be reported as the last serial dilu-
tion in which clearly discernible staining is seen. Results
with a strong reaction at the 1:2560 dilution should be
reported as greater than 1:2560. Titers of 1:40 to 1:80
are considered low titers; 1:160 to 1:320 are considered
medium titers; and 1:640 and greater are considered
high titers.
PattErn DEtEctIon
Homogeneous: A solid staining of the nucleus with or
without apparent masking of the nucleoli. The chro-
mosome region of metaphase mitotic cells is clearly
positive with a smooth or peripheral staining intensity
greater than, or equal to, interphase nuclei.
Synonyms: Diffuse; solid.
Nuclear antigens: dsDNA; nDNA; DNP; histone.
Disease association: High titers are suggestive of SLE.
Lower titers are suggestive of SLE or other connec-
tive tissue diseases (32).
Peripheral: A solid staining, primarily around the outer
region of the nucleus, with weaker staining toward
the center of the nucleus. The chromosome region of
metaphase mitotic cells is clearly positive with a smooth
or peripheral staining intensity greater than, or equal to,
interphase nuclei.
Synonyms: Rim, shaggy, membraneous.
Nuclear antigens: dsDNA, ssDNA, nDNA, DNP,
histone.
Disease association: High titers suggestive of SLE;
lower titers suggestive of SLE or other connective
tissue diseases (32).
Speckled: A coarse or fine granular staining of the
nucleus generally without fluorescent staining of the
nucleoli. The nonchromosome region of metaphase mi-
totic cells demonstrates staining, while the chromosome
region is negative for stain.
Nuclear antigens: Sm; RNP; Scl-70; SSA; SSB; and
other antigen/antibody systems not yet charac-
terized.
Disease association: High titers are suggestive of SLE
(Sm antigen), mixed connective tissue disease
(RNP antigen), scleroderma (Scl-70 antigen), or
Sjögren's syndrome-sicca complex (SSA or SSB
antigen). Lower titers may be suggestive of other
connective tissue disease (33).
nucleolar: Large coarse speckled staining within the
nucleus, generally less than 6 in number per cell, with or
without occasional fine speckles, 5-10 in number. The
nonchromosome region of metaphase mitotic cells
demonstrates strong staining, while the chromosome
region may demonstrate faint staining. Anaphase and
telophase cells may demonstrate similar staining to
interphase nuclei.
Nuclear antigens: Generally referred to as 4-6s RNAs
and other nuclear antigens such as fibrillarin, RNA
Polymerase I, NOR 90, and PM/Scl.
Disease association: High titers prevalent in sclero-
derma and Sjögren's syndrome (34).
centromere: A discrete speckled staining pattern
highly suggestive of the CREST* syndrome variant of
progressive systemic sclerosis (25). The nuclear speckles
are very discrete and usually some multiple of 46 (usu-
ally 23-46 speckles per nucleus). Because centromeres
are constrictions where spindle fibers attach on chro-
mosomes, mitotic cells will show the same speckling
reaction in the chromosome region (12).
Synonyms: ACA; discrete speckled.
Nuclear antigens: Chromosomal centromere
(Kinetochore).
Disease association: Highly suggestive of the CREST*
syndrome variant of progressive systemic sclerosis
(25).
*CREST is a form of PSS with prominent calcinosis,
Raynaud's phenomenon, esophageal dysfunction,
sclerodactyly, and telangiectasia.
mItotIc cEllS
DEtEctIon
Mitotic cells should be visible on every field when
viewed at 200X magnification or lower. To verify
whether a cell is in mitosis, view at 400X magnification.
Mitotic cells show a characteristic round cell shape with
no detectable nuclear membrane. The chromosome
region of mitotic cells will generally show an irregular
shape within the cell, due to the lack of nuclear mem-
brane, and extreme constriction of the chromosomes.
Sera positive for DNA and/or DNP and/or histone (such
as the IC homogeneous positive control) will show
bright staining of the chromosome region of these cells.
In samples negative for DNA and/or DNP and/or histone
(such as the IC speckled positive control), the mitotic
cells will not show chromosome staining and may be
difficult to see.
uSE oF mItotIc cEllS
Discerning Speckled vs. Homogeneous antibody: A
fine speckled pattern of staining is sometimes difficult
to differentiate from homogeneous staining. If the pat-
tern is homogeneous, there will be solid staining of the
chromosomes of the mitotic cells. If the pattern is strictly
speckled, the region outside of the chromosomes will
show a fine speckled reaction.
NOTE: If fine speckling of the entire mitotic cell occurs
along with solid staining of the chromosome region,
it is highly probable that two or more antibodies are
present. Report the screening dilution as speckled/
homogeneous and titer each antibody to endpoint.
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