immuno concepts Colorzyme ANA Für Professionellen Gebrauch Seite 5

antibodies. The key factor to consider in determining
whether a given dilution of serum is positive is the ap-
pearance of a clearly discernible pattern, irrespective
of the intensity of the staining.
This titratable control will show the typical speckled
pattern associated with the RNP antibody. Also present
may be a second pattern of NSp I (several discrete
speckles in the nucleus of interphase cells), however, it
is the typical RNP speckled pattern that is to be used for
the purpose of reading end-point.
The values obtained in our laboratory may differ from
your values. Some of the many factors that can affect
your results may include, but are not limited to:
1. Proper alignment of the microscope light path.
Refer to your microscope manual for instructions.
2. The numerical aperture of the objective. The
numerical aperture is related to the light gathering
capability and the resolution of the objective. This
number is printed on the side of the objective.
3. 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).
ENZYME STAINING
The degree of staining has no proven clinical value and
only limited value as an indicator of titer (29). To simplify
interpretation, record screening results as strongly posi-
tive or positive, and titer accordingly.
Strongly Positive Reaction: Dark to very dark blue-
purple staining, clear-cut cell outline, sharply defined
nuclear pattern.
Positive Reaction: Dim or subdued blue-purple staining
with greater variability of staining between cells; cell
outline may be less well defined in some cells with a
majority of cells still demonstrating a clearly discernible
pattern of staining.
NOTE: Because the cells are grown directly on the
slide surface, all cells are not in the same phase of the
cell cycle. It is not unusual to see differential staining
intensities from cell to cell due to the differences in the
concentration and location of different antigens during
the cell cycle.
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 staining of the nucleoli. The
nonchromosome region of metaphase mitotic cells
demonstrates staining, while the chromosome region is
negative for stain.
Nuclear antigens: Sm; RNP; Scl-70; SS-A; SS-B; 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 (SS-A or SS-B antigen).
Lower titers may be suggestive of other connec-
tive 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 Immuno Concepts homogeneous positive con-
trol) 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 con-
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