This document discusses antinuclear antibody (ANA) testing. It describes how ANA tests are commonly used to assess autoimmune diseases and how the testing methods have evolved over time. Specifically, it notes that HEp-2 cells have largely replaced rodent tissues as the substrate in ANA testing due to advantages like increased sensitivity and uniform antigen distribution. The document also outlines different ANA immunofluorescence patterns seen on HEp-2 cells and their relationship to various autoimmune conditions.

1. ANTINUCLEAR ANTIBODI
MELDA SITANGGANG

7.  Identification of antinuclear antibodies
(ANA) is an important part of clinical
medicine and clinical immunology. ANA
testing for the assessment of systemic and
organ-specific autoimmune disease has
increased progressively since
immunofluorescence techniques were first
used to demonstrate antinuclear antibodies
in 1957. ANA tests are amongst the most
commonly performed antibody tests world-
wide and the most frequently performed test
in many clinical immunology laboratories.

8.  Rodent tissues, comprising liver, kidney and
stomach, were the traditional ANA
substrates. Over the last 20 years HEp-2 cells
have largely replaced the use of rodent
tissues and more recently commercial ANA
enzyme immunoassays (EIA) have become
popular. HEp-2 cells, originally considered to
originate from a human laryngeal carcinoma,
are now known to have been established
from a HeLa cell contamination (HEp-2 cells:
CCL-23, from the American Tissue Type
Collection).

9.  HEp-2 cells have many advantages over rodent
tissues:
1. They are a more sensitive substrate that allows
identification of many patterns.
2. Human origin ensures better specificity than
animal tissues.
3. The nuclei are much larger so complex nuclear
details can be seen.
4. The cell monolayer ensures that all nuclei are
visible.
5. Cell division rates are higher so that antigens
produced only in cell divisionare easily located eg.
centromere and mitotic spindle patterns.
6. There is no obscuring of the intercellular matrix.
7. Antigen distribution is uniform

10.  Antinuclear antibody tests have their origin
in the lupus erythematosus cell (L.E.cell)
phenomenon. This was first demonstrated in
patients with SLE.
 Showed that the L.E. cell phenomenon was
caused by a gamma globulin protein which
was probably an antibody.

11. Structure of HEp-2 cells
 Immunofluorescence patterns seen on
individual HEp-2 cells are related to the cell
cycle. For instance, patterns that are unique
to cells in mitosis will be restricted to cells in
that phase of the cycle. Since most patterns
are seen with cells in interphase, HEp-2 cells
should be largely at this stage. Synchronised
cultures might not contain cells at all stages
in the cycle and should generally be avoided.

12.  HEp-2 patterns also depend upon the optimal
use of certain fixatives because some
antigens, such as SSA and actin, are easily
denatured. Care must therefore be exercised
when preparing the cells or when selecting a
manufacturer's product. The following is a
very brief outline of various components in
eukaryotic cells which should assist in
understanding autoantibody patterns

13.  In interphase, the chromosomes form a fibrillar
network of chromatin, more or less uniformly
distributed throughout the nucleoplasm and
delimited by the nuclear membrane. Only the
nucleoli are well differentiated. Cytoplasmic
organelles and fibrous structures are most visible at
this stage and tend to largely disappear or change
their appearance during mitosis

14. Figure 5. The cell during interphase.

15. Figure 7. Structure of the nuclear membrane.

16. ELISA
 Anti-Nuclear Antibodies (ANA) ELISA kit is based on
binding of ANA from serum samples to extracted
nuclear antigen immobilized on microtiter wells.
After a washing step, goat anti-human IgG-HRP
conjugate is added. After another washing step, to
remove all the unbound enzyme conjugate,
chromogenic substrate (TMB) is added and color
developed. The enzymatic reaction (color) is directly
proportional to the amount of ANA present in the
sample. Adding stopping solution terminates the
reaction. Absorbance is then measured on a
microtiter well ELISA reader at 450 nm and the
concentration of ANA in samples is calculated as ANA
index (AI) which is defined as the ratio of net
absorbance of the test sample and net absorbance of
the negative or endpoint-cutoff control.

17.  Negative samples <0..90
 Equivocal (borderline) >0.91-0.99
 An OD ratio greater than or equal to 1.00 is
interpreted as positive for IgG ANA. An OD
ration of less than or equal to 0.90 is
interpreted as negative for IgG ANA.

18. IFA
 Tahap 1 : serum direaksikan dengan substrat
ag dan akan megikat ab membentuk komplek
ag-ab
 Tahap 2: fluorescen yang dilabel ab
antihuman akan teriakt dengan kompleks
dan hasilnya adalah fluorescen hijau cerah
 Jika tidak terbentuk kompleks maka
fluorescen negatif

19. interpretasi
 Pola utama :
1.Periferal
2.Homogenous
3.Speckled
4.nucleolar

20. PERIFERAL
 Antigen nuklear ; n DNA atau histones
 Hubungan penyakit : SLE
HOMOGEN
Antigen nuklear ; n DNA, DNP atau histones
Hubungan penyakit : SLE,drug induced lupus

21.  SPECKLED
 Ab Sm dan n RNP tampak sbg speckled kasar
dengan regio kromosom dari mitotik sel negatif
(SLE)
 Ab SSA/Ro/SSB : speckled ukuran kecil( Sjogren
syndrom)
 Ab SCL-70 speckled ukuran halus) scleroderma)
 NUCLEOLAR
Antigen nuklear : Om, Scl ( sjogren syndrome)