Immunofluorescence Guide

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The identification of autoantibodies 
(AAbs), together with other serological 
and clinical data has developed into an 
indispensible component the diagnosis 
of Autoimmune Diseases (AIDs). 
Indirect immunofluorescence (IIF) is the 
. major screening method used for AAbs 
· detection. This method allows the 
identification of a wide spectrum of 
AAbs in a single assay, providing 
sensitive, fast and inexpensive results. 
Anti-nuclear antibodies (ANAs) 
[antibodies directed against nuclear 
and cytoplasmatic components of the 
ce ll] are a serological hallmark in the 
diagnosis of systemic autoimmune 
rheumatic diseases (SARD) and are 
also found in many other disorders, as 
we ll as in some healthy individuals. 
The IIF assay on HEp-2 cells is a 
commonly used test for the detection 
of ANAs and has been recently 
recommended as the screening test of 
choice by a task force of the American 
College of Rheumatology. (*) 
Most liver-related AAbs, diagnostically 
relevant to liver disease, can be 
detected by IIF when using a triple 
(kidney, stomach, liver -KSL) rodent 
(rat/mouse) tissue substrate. 
Another important area in the diagnosis 
Introduction I 01 
of AIDs is the serological diagnosis of 
systemic autoimmune vasculitis, such 
as granulomatosis with polyangiitis or 
microscopic polyangiitis. This is 
achieved through detection of AAbs 
against the cytoplasm of neutrophil 
granulocytes with the ANCA test (ANCA 
= Anti-Neutrophil Cytoplasmic Anti-
bodies). 
Diagnosis by means of IIF continues to 
demand experience and expertise of 
the laboratory personnel and clinicians. 
The purpose of this atlas is to help 
laboratory -staff recognize a huge 
variety of AAbs staining patterns. 
In addition to the photographs 
illustrating the typical staining patterns 
we have also included the autoantigens 
involved, as well as their clinically 
relevant associations. 
(*) Meroni, P L; Schur, P H. ANA 
screening, an old test with new 
recommendations. Ann Rheum Dis 
2010 69: 1420-1422 doi:10.1136/ 
ard. 2009.127100 
- --------····························· 
02 ( Table of contents 
••• • •••••••••••••·• • •·••••••••••••• _______ t_a_b-le_o_f_c_o_nt_e_n-tsl 03 
Introduction . . . ... . .. . ....... . ........... . ..... . . . ... . . 01 
HEp-2 Patterns 
Nuclear Patterns 
Smooth Nuclear Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 06 
Mitochondria Type 2 (AMA-M2) ..... ... . .. .. .... .... . . . . .. ... 42 
Multiple Dots (P bodies/GW bodies) . . . . . . . . . . . . . . . . . . . . . . . . . . 43 
Golgi Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 
F-Actin ......... .. . . . . . . . .. . . . . . . . . .. . ...... .. . ... .. .. 45 
Vimentin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 
Punctuate Nuclear Membrane ... .. . ... .. . . . .. .. . . . . . . . .. . . . 07 
Homogeneous . .... . . ........ ...... . ... .. .. . ... . . . . .... 08 KSL Tissues (Kidney/Stomach/Liver) 
Fine Speckled/Homogeneous (Scl-70) ..... . .. .. .. . .. . . . . .... . 09 Gastric Panetal_Cells Ant1bod1es (GPA, APCA) . . . ... . . . . ......... 48 
Large Speckled (Nuclear Matrix) .... . ... . ..... . ..... . .. . . .. . 10 A_nt1-M1tochondnal Ant1bod1es (AMA-M2) .... ...... . ..... .. . .. . .. 49 
Coarse Speckled . . . .... .. . ......... . . . . .'. . . . . . . . . . . . . . . 11 Liver Kidney Microsomal Ant1bod1es (LKM-1) . . . . . . . . . . . . . . . . . . . . 54 
Fine Speckled . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Heteroph1le Ant1bod1es . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 7 
Dense Fine Speckled (DFS-70/LEDGF 75). . . . . . . . . . . . . . . . . . . . . . 16 Anti-F-Actin Antibodies (ASMA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 
Few Nuclear Dots .. . . .... . .. ... ...... . . . . . . . . ... . . ... . .. 17 Liver Cytosol Antibodies (LC-1) .. ..... . .... . .. . . . . . .. . .. ... . 62 
Multiple Nuclear Dots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Anti-Elastin Antibodies .... .. ... .. . . .. . . _. . . . . . . . . . . . . . . . . . . 63 
Centromere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Anti-Ribosomes Antibodies (Rib P) . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 
Anti-S ignal Recognition Particle (SRP). . . . . . . . . . . . . . . . . . . . . . . . . 69 
Nucleolar Patterns 
Nucleolar Homogeneous (PM-Scl) ..... . . . . . . . ... . .. ........ . 22 ANCA Patterns 
Nucleolar Homogeneous (Th/To) . ....... .... . .... . . . ... . ... . 23 C-ANCA (Ethanol/Formalin Fixed Slides) .... . ...... . . .. . .... .. . 72 
Nucleolar Clumpy (Fibrillarin) . ............ .. .. . ..... .. .... . . 24 P-ANCA (Ethanol/Formalin Fixed Slides) .. ............ ... . ..... 73 
Nucleolar Speckled/Punctate (RNA Polymerase 1/NOR) ... . . . ... . . 25 Atypical-ANCA (A-ANCA) (Ethanol/Formalin Fixed Slides) .... . . ... . . . 74 
Cell Cycle Related Patterns Anti-Endomysium Antibodies - EMA 
Cell Cycle Ags (S-phase) Anti-Endomysium Antibodies (lgA, lgG) EMA . .. . . ....... . . . . . . .. 76 
Proliferating Ce ll Nuclear Antigen (PCNA) ...... .. . .. . . ... . . .. . . 28 Crithidia luciliae (dsDNA) 
Mitotic Spindle Apparatus Ags (M-phase) Crithidia luci liae (dsDNA) .... . . . . ....... . . . . . . . . ..... . . .. .. 78 
NuMA-1/MSA-1. .... . ........ ... ...... . ... .. . ..... .. . . .. 32 
NuMA-2 .... ..... .. . . .. . . . . ..... . . ... .... _ . . . . . . . . . . . . 33 References . . ............ .. .. . .. . .. . . . . . .. . .... . . . .. .. 80 
Midbody (MSA-2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Algorithm .... . .... . ..... . ....... . ................. .. . 84 
CENP-F (MSA-3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 
Centriole/Centrosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Frequently Asked Questions (FAQ's) .... . ......... . .. .. .. . ... 88 
Cytoplasmatic Patterns 
Fine Granular (Jo-1) . . . . .. ... .. .. ......... . .. ... ... .... . .. 38 
Fine Dense Granular (PL-7 /PL-12) . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 
Fine Dense Granular to Homogeneous (Rib-P) .... . . . . . . . . .... . .. 40 
Very Fine Granular (SRP) . .... ........ . ......... . ... . ...... 41 
HEp-2 Patterns 
06 ( Smooth Nuclear Membrane 
··················~················· ---------------Punctate Nuclear Membrane 
I ! I , ' i 
! I { 
I ; t 
I , • I , 
i l 
4oox l 
• lnterphase • lnterphase 
Fine, linear fluorescence and sta ining of folds in the nuclear membrane Discontinuous punctate (granu-
with a light homogeneous staining of the nucleus. lar/ speckled) staining along the 
• Nucleoli 
Negative 
• Mitosis 
nuclear membrane. Enhanced 
fluorescence when two nuclear 
membranes touch. 
· . • Nucleoli 
Metaphase: Chromat_in plate negative. Post telophase: Nuclear Negative 
membrane reconst1tut1on. Newly formed nuclei are surrounded by a fine 
membranous fluorescence . • Mitosis 
A f Metaphase cells show a diffuse 
• n igens . . . . . . . . staining throughout the cyto-
Lamins A, Bl , B2, and C. Spec1f1c1t1es against lamin associated proteins plasm without chromosomal 
(LAP lA and LAP 2) have been also 1dent1f1ed. stain i~g. 
• Disease Associations . . • Antigens 
Systemic Lupus Erythematosus, Systemic Sclerosis, Chronic Active Gp210, nucleoporin p62 
Hepatitis 
• Disease Associations 
(Nuclear pore complex) 
400x 
lOOOx 
Primary Biliary Cirrhosis (patients with anti-Gp210 antibodies may have 
a more active liver disease) 
............................. ------------------Possible follow-up tests 
AESKUBLOTS® Liver Pro 
07 
• lnterphase 
Uniform diffuse staining of the nucleoplasm, whose inten-
sity can vary depending on concentration of antibodies in 
the serum. In some cases a more intense staining of the 
inner edge of the nucleus (nuclear rim) can be seen. 
• Nucleoli 
Nucleolar staining is variable, 
positive/ negative. Some samples 
may show a peripheral nucleolar 
staining. 
• Mitosis 
In all phases, a homogeneous or 
peripheral chromatin staining can 
be seen. 
• Antigen 
dsDNA, nucleosomes, histones 
• Disease Associations 
Systemic Lupus Erythematosus, 
Drug-induced Lupus 
Possible follow-up tests 
DANA-Pro/Nucleo-h/dsDNA-check/Histone-C/AESKUBLOTS® ANA-17 Pro/ 
AESKUBLOTS® ANA-12 Pro 
• lnterphase 
Fine Speckled/Homogeneous 
(Scl-70) 
Intense fine speckled staining (may appear homogeneous) covering the 
entire nucleoplasm. 
• Nucleoli 
May or may not be positive. 
• Mitosis 
Metaphase and telophase cells show staining of the chromatin plate with 
2-5 discrete dots (nuclear organizing region). 
• Cytoplasm 
May have weakly speckled staining. 
• Antigen 
DNA topoisomerase 1 
• Disease Associations 
Diffuse Systemic Sclerosis 
Possible follow-up tests 
ANA-8Pro/ENA-6Pro/DANA-Pro/Sclero-Pro/Sc/-70/ 
AESKUBLOTS® ANA-1 lPro/AESKUBLOTS® ANA-l 2Pro 
09 
10 
11 
Large Speckled 
(Nuclear Matrix) 
• lnterphase 
Variable large speckles arranged 
as a network. 
• Nucleoli 
Negative 
• Mitosis 
No staining of the condensed 
chromatin in mitotic cells . 
• Antigen 
Heterogeneous 
proteins (hnRNP) 
• Disease Associations 
ribonuclear 
Mixed Connective Tissue Disease. 
Can also occur in Rheumatoid 
Arthritis, Systemic Lupus 
Erythematosus and Systemic 
Sclerosis . 
................. '• .. .............. . 
lnterphase 
Dense intermediate sized speckles, 
together with large speckles 
throughout the nucleoplasm. 
Mitosis 
Ce lls in mitosis without staining 
of the condensed chromosomal 
• Antigen 
Ribonucleoproteins Sm (from U2 
to U6 without U3) and RNP (Ul 
RNP) 
• Disease Associations 
Coarse Speckled j 11 
Systemic Lupus Erythematosus, Mixed Connective Tissue Disease 
............................. ----------,~--------
Possible follow-up tests 
ANA-8Pro/ENA-6Pro/DANA-Pro/Sclero-Pro/Sm/U 1-70/snRNP-C/ 
AESKUBLOTS® ANA-l 7Pro/AESKUBLOTS® ANA-l 2Pro/AESKUBLOTS® Myositis Pro 
( Fine Speckled 
SSA/SSB 
................. ·· ··············· --------------
Fine Speckled l 13 
SSA/SSB I 
• lnterphase 
Fine to discrete speckled staining of the nuclei in a uniform distribution 
• Nucleoli 
Are mainly negative. Frequent staining of a few nucleoli may be observe( 
in presence of anti-SSB/La and anti-Ku antibodies. 
• Mitosis 
Meta phase cells show fluorescence in the cytoplasm, with condensatio1 
around the chromatin plate which itself is negative. 
• Antigen 
Nuclear proteins: SSA/Ro, Ro60, Ro52, SSB/La, Ku (70 KDa and 80 kD, 
heterodimer) and Mi-2 (240kDa) 
• Disease Associations 
Systemic Lupus Erythematosus, Neonatal Lupus, Cutaneus Lupus Ery 
thematosus, Sjogren's Syndrome, Systemic Sclerosis, Dermatomyosith 
............................. ----------------
Possible follow-up tests 
ANA-8Pro/ENA-6Pro/DANA-Pro/Sclero-Pro/SS-A/SS-A52/SS-A60/SS-B 
AESKUBLOTS® ANA-l 7Pro/AESKUBLOTS® ANA-l 2Pro 
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AESKUBLOTS® ANA-l 7Pro/AESKUBLOTS® ANA-l 2Pro 
II 
II 
I 
I 
14 ( Fine Speckled 
I Mi2 
· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · --------F-in_e_S_p_e_c_k-le-d ""\ 15 
Ku I 
............................. ---------------- ............................ . 
Possible follow-up tests 
AESKUBLOTS® ANA-17Pro/AESKUBLOTS® Myositis Pro 
Possible follow-up tests 
AESKUBLOTS® ANA-17Pro/AESKUBLOTS® Myositis Pro 
15 ( Dense Fine Speckled 
I DFS70 
• lnterphase 
Dense fine speckles that are somewhat uniformly distributed throughou 
the nucleus. 
• Nucleoli 
Negative 
• Mitosis 
Metaphase and telophase cells show a strong speckled fluorescence o 
the chromosomes. 
• Antigen 
LEDGF-p75 (lens epithelium-derived growth factor 75) initially terme 
DFS70 (70kDa protein) 
lnterphase 
Two to six bright dots in the 
nucleoplasm, often adjacent to 
the nucleoli. 
Nucleoli 
Negative 
Mitosis 
No staining of metaphase or 
telophase chromatin plates. 
Antigen 
Coiled bodies 80kDa (nuclear 
• Disease Associations protein designated p80 coilin) 
Although a distinctive clinical association is still unclear, sera witl . . . 
only DFS70 antibodies may help to identify patients who do no Disease Associations 
have a rheumatologic disease despite a positive ANA IF test. DFS7( No clear clinical fe_atures _have 
represent a potentially important biomarker that can be clinically use1 bee_n associated. with anti-p80 
to discriminate Systemic Autoimmune Rheumatic Diseases from ANA coilm autoantibodies. 
positive healthy individuals and/or other inflammatory conditions sucl 
as autoimmune diseases . 
Few Nuclear Dots I 11 
............................. ----------------- ............................ . 
Possible follow-up tests 
AESKUBLOTS® Liver Pro (to exclude splOO) 
1s ( Multiple Nuclear Dots 
• lnterphase 
Five to twenty differently sized 
dots which are spread over the 
cell nucleus. 
• Nucleoli 
Negative 
• Mitosis 
Chromosomes of metaphase or 
telophase cells are negative. 
• Antigen 
Sp-100 protein, PML (promye-
locytic leukemia protein) 
• Disease Associations 
Primary Biliary Cirrhosis 
.................... ............... 
lnterphase 
40-60 speckles distributed 
throughout the entire nucleus. 
Mitosis 
A "block" of closely associated 
speckles is found in the 
condensed nuclear chromatin of 
the metaphase, anaphase and 
telophase cells. 
Antigen 
CENP-A, B, C, D, E and even CENP-G and H 
Disease Associations 
Limited Systemic Sclerosis - CREST (Calcinosis, Raynaud's Syndrome, 
Esophogeal dysmotility, Sclerodactyly, Telangiectasia), Primary Biliary 
1 Cirrhosis, Raynaud's Syndrome and in a minority of patients Sjogren's 
Syndrome 
····························· ---------------- ............................ . 
Possible follow-up tests 
AESKUBLOTS® Liver Pro 
Possible follow-up tests 
ANA-8Pro/DANA-Pro/Sclero-Pro/Cenp-B 
AESKUBLOTS® ANA-17Pro/AESKUBLOTS® ANA-12Pro 
19 
1-
I 
I 
Notes 
HEp-2- Patterns 
-
22 ( Nucleolar Homogeneous I PM-Scl 
............ ····-. ... . ............. ---------------
Nucleolar Homogeneous, 23 
• lnterphase 
Homogeneous fluorescence of 
the nucleoli, with a weaker fine 
granular reaction of the nucleo-
plasm. 
• Nucleoli 
Positive 
• Mitosis 
In mitotic cells the region of 
condensed chromosomes is not 
stained. 
A fine granular staining outside of 
chromosomes can be seen. 
• Antigen 
PM-Scl 75 and PM-Scl 100 
• Disease Associations 
Polymyositis/Systemic Sclerosis 
Overlap syndrome, Myositis, Sys-
temic Sclerosis 
lnterphase 
Homogeneous fluorescence 
of the nucleoli, and granular 
nucleoplasm granular. 
Nucleoli 
Positive 
Mitosis 
In mitotic cells the region of 
condensed chromosomes is not 
stained. 
A fine granular staining outside of 
the chromosomes can be seen 
Antigen 
Rpp25 (a component of the Th/ To 
complex) 
Disease Associations 
Systemic Sclerosis limited 
cutaneous form 
Th/To I 
............................. ----------------- ............................ . 
Possible follow-up tests 
Sc/ero-Pro/PM-ScVAfSKUBLOTS® ANA-1 7Pro/AESKUBLOTS® Myositis Pro 
24 
I 
I 
LI 
Nucleolar Clumpy 
Fibrillarin 
.. ............. 
Nucleolar Speckled/Punctated 
RNA polymerase 1/NOR 
• lnterphase • lnterphase 
Granular fluorescence of the nucleoli. The nucleoplasm is dark althoug Speckled nucleolar fluorescence of the nucleoli, frequently associated 
the coiled bodies may be stained as few nuclear dots. with fine speckled staining of the nucleoplasm. 
• Nucleoli 
Positive 
• Mitosis 
Metaphase and telophase plates show reticular staining. 
• Antigen 
U3-nRNP fibrillarin• Disease Associations 
Systemic Sclerosis (high specificity) 
, Nucleoli 
Posit ive 
, Mitosis 
In mitotic cells the region of condensed chromosomes is not stained. 
A few dots corresponding to the nucleolar organizing regions (NOR) 
may fluoresce. Outside the chromosomes a fine granular to smooth 
fluorescence can be seen. 
•Antigen 
RNA polymerase 1/ NOR 
, Disease Associations 
Systemic Sclerosis particularly associated with the diffuse cutaneous 
form 
····························· ----------------- ............................ . 
25 
Notes 
28 
I I 
I 
PCNA 
................ ', ... ............. . 
(Proliferating cell nuclear antigen) 
• lnterphase 
The fluorescence pattern of Abs against PCNA depends on the cell cycl 
Only (30-60%) of the lnterphase (S-phase) cells show a characteristic fi 
to coarse speckled nuclear staining. Resting G-phase cells are negativ 
• Nucleoli 
Are stained in some cells. 
• Mitosis 
In mitotic cells the region of condensed chromosomes is not stained. 
• Antigen 
DNA polymerase 6 (cyclin) 
• Disease Associations 
Anti-PCNA antibodies are historically considered to be highly specif 
for SLE, although antibodies to PCNA have also been described 
patients infected with the hepatitis B virus and hepatitis C virus. An 
PCNA antibodies are not specific for systemic autoimmune disease 
The clinical significance is not well understood, although an associatio 
with Lupus nephritis has been described. 
PCNA 
(Proliferating cell nuclear antigen) 
............................. --------....-------- ............................ . 
Possible follow-up tests 
AESKUBLOTS® ANA-17Pro 
Notes 
I I 
I 1 
I 
I 
32 fNuMA-1 I (MSA-1) 
• lnterphase 
Fine speckled staining of the 
nucleus. 
• Nucleoli 
Negative 
• Mitosis 
Staining of the spindle poles in 
metaphase and anaphase. 
• Antigen 
Nuclear mitotic apparatus, type 1 
(centrophilin -230kDa protein) 
• Disease Associations 
Sjbgren's Syndrome, Systemic 
Lupus Erythematosus and Mixed 
Connective Tissue Disease 
............... .. 
401ih 
.... ······ ·· ···· --------------
NuMA-21 33 
i 
! , 
l 
! 
I 
I 
l 
i 
' 
lnterphase 
0 staining of the nucleus. 
ucleoli 
egative 
itosis 
Staining of the spindle apparatus 
during mitosis extending from 
pole to pole. 
#1,ntigen 
~uclear mitotic apparatus, type 
2 (HsEg5 - Kinesin like protein of 
130 KDa) 
llisease Associations 
fystem ic Lupus Erythematosus 
and Sjbgren's Syndrome 
400x 
····························· _______ .....;,:. ______ _ 
34 ( Midbody I (MSA-2) 
• lnterphase 
Nuclear staining in G2 phase cells. 
• Nucleoli 
Negative 
• Mitosis 
Metaphase cells 
fluorescence as fine 
perpendicular to 'the edge of 
the metaphase plate. There is 
staining of the cleavage furrow 
and midbody in anaphase and 
telophase cells. 
• Antigen 
Aurora kinase B complex 
• Disease Associations 
Systemic Sclerosis and Cancers 
nterphase 
ine speckled staining of G2 phase cells. 
itosis 
etaphase cells show two sets of dense large granules surrounding the 
chromosomal metaphase plate as a grip. The surrounding cytoplasm is 
tliffusely stained. In prophase cells a dense punctate decoration of the 
chromosomes is seen. There is often staining of the midbody region of 
telophase cells (although it is not seen in all anti-CENP-F positive serum 
samples). 
ntigen 
Centromere protein F (350/ 400kDa, mitosin) 
Disease Associations 
Anti-CENP-F are associated with malignancies and might help in the early 
diagnosis of cancer. 
............................. -------"'---------
35 
36 ( Centriole/Centrosome 
• lnterphase 
One to two bright spots are seen 
in the cytoplasm adjacent to the 
nucleus. 
• Nucleoli 
Negative 
• Mitosis 
In metaphase, a fluorescent spot 
at each of the spindle po-les is 
seen. 
• Antigen 
Pericentrin, ninein, Cep250, 
enolase 
• Disease Associations 
The centrosome pattern is a very 
rare pattern that appears to be 
associated with systemic auto-
immune diseases at higher titers 
(~ 1:320). 
................ 
38 ( Fine Granular I Jo-I 
.............. . ', ...... .. ....... . 
• lnterphase 
Fine granular to homogeneous 
cytoplasmic staining. The nucleus 
also shows a few dots. 
• Nucleoli 
Negative 
• Mitosis 
Chromosomal material in meta-
phase cells is negative. 
• Antigen 
Jo-1 Histidyl-tRNA synthetase 
• Disease Associations 
Polydermatomyositis 
lr terphase 
tytoplasm fine dense granular 
PL-7) or very fine dense granular 
ith perinuclear accentuation 
PL-12). No staining of the nucleus. 
ucleoli 
egative 
itosis 
Chromosomal material in meta-
hase cells is negative. 
ntigen 
Threonyl-tRNA synthetase (PL-7), 
Alanyl-tRNA synthetase (PL-12) 
isease Associations 
olydermatomyositis 
............................. ________ ....;.;--------
Possible follow-up tests 
ANA-8Pro/ENA-6Pro/DANA-Pro/5clero-Pro/Jo-1 
AESKUBLOTS® ANA-17Pro/AESKUBLOTS® ANA-12Pro/AESKUBLOTS® Myositis 
Fine Dense Granular) 39 
PL-7 /PL-12 I 
40 Fine Dense Granular to Homogeneo~~ · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Very Fine Granular\ 41 
SRP I Rib-P 
• lnterphase 
Cytoplasm shows a very fine 
dense granular to homogeneous 
staining with vacuoles. No 
staining of the nucleus. 
• Nucleoli 
Homogeneously stained (rRNA 
proteins) or negative. 
• Mitosis 
Chromosomal material in meta-
phase cells is negative. 
• Antigen 
Phosphorilated proteins (P pro-
teins): PO (35kD), Pl (19kD) and 
P2 (17kD) 
• Disease Associations 
Systemic Lupus Erythematosus 
• lnterphase 
Cytoplasm very fine granular with vacuoles. 
No staining of the nucleus. 
• Nucleoli 
Negative 
• Mitosis 
Chromosomal material in metaphase cells 
is negative. 
• Antigen 
Signal recognition particle (7SL RNA + 6 proteins -
SRP 9, 14, 19, 54, 68, 72) 
• Disease Associations 
Myositis, Polymyositis, Necrotizing Myopathy 
............................. ------~-------····························· 
Possible follow-up tests 
Rib-P /AESKUBLOTS® ANA-l 7Pro/AESKUBLOTS® ANA-l 2Pro 
42 ( Mitochondria Type 2 
AMA-M2 
• lnterphase 
Fluorecence of large irregular 
granules organized as a network 
of filaments extending around 
the nucleus and throughout the 
cytoplasm. No staining of the 
nucleus. 
• Nucleoli 
Negative 
• Mitosis 
Chromosomal material in meta-
phase cells is negative. 
• Antigen 
Pyruvate dehydrogenase complex 
............... · ....... ......... . 
I terphase . . 
q aining of multiple dots through-
ut the cytoplasm. No staining of 
the nucleus. 
itosis 
O:hromosomal material in meta-
JPhase cells is negative. 
ntigen 
Wl 82, Su/Ago2, RAP55 (RNA 
associated protein 55), Ge-1/ 
edls 
(PDC). The PDC-E2 antigen is the I IDisease Associations 
main antibody target in PBC. O:erebellar ataxia, motor and sen-
• Disease Associations sory neuropathy, Sjiigren's Syn-
Primary Biliary Cirrhosis, less frequent in Systemic Sclerosis, CRE:tllrome, Sys_temic Lupus _Erythe-
Systemic Lupus Erythematosus and Sjiigren's Syndrome lillatosus, Primary B1l1ary C1rrhos1s 
Multiple Dots 
(P bodies/GW bodies) 
............................. ------- ·-------- ............................ . 
Possible follow-up tests 
AMA-M2-check/AESKUBLOTS® ANA-17Pro/AESKUBLOTS® LiverPro 
43 
44 ( Golgi Apparatus 
• lnterphase 
Cytoplasm shows a reticular 
granular staining adjacent to one 
side of the nucleus. No staining of 
the nucleus. 
• Nucleoli 
Negative 
• Mitosis 
Chromosomal material in meta-
phase cells is negative. 
• Antigen 
Macrogolgin/ giantin, golgin-67, 
golgin-95 (GM130), golgin-97, 
golgin-160, golgin-245 (p230) 
• Disease Associations 
Unclear. Described in patients with 
Sjogren's Syndrome, Systemic 
Lupus Erythematosus and other 
Chronic Rheumatic Diseases . 
.............. . ' ....... ......... . 
terphase . . 
Staining of straight cytoplasmic 
c.ables (microf1laments) running 
he length of the cell. No staining 
f the nucleus. 
in meta-
ntigen 
f ilamentous (polymerized) actin 
(F-actin) 
isease Associations 
1 hronic AutoimmuneHepatitis 
,ype 1 
. ............................ --------,;--------
45 
46 
• lnterphase 
Fine net of fibres (intermediate 
filaments) in the cytoplasm. 
Aggregated bundles around the 
nucleus which is not stained. 
• Nucleoli 
Negative 
• Mitosis 
In mitotic cells numerous round 
fluorescing droplets can be seen 
outside the dark chromosomes. 
• Antigen 
Vimentin 
• Disease Associations 
Unclear 
............................. --------
K9I. Tissues 
48 
-------------- .............. . 
Gastric Parietal Cells Antibodies 
(GPA, APCA) 
', ..... ... ....... . 
Anti-Mitochondrial Antibodies 
(AMA-M2) 
• Kidney (idney 
Negative ,ranular fluorescence in the cytoplasm of distal and proximal renal 
• Stomach ubules cell s. Renal medulla (composed only by distal tubules) has 
Fine granular fluorescence of the parietal cells. . m intense staining. _Proximal tubules_ show a differential staining 
Confuse heterophile staining can be seen when using rat stomachPl>P2>P3). Glomeruli wrth granular staining. 
substrate. It is also useful to test on kidney and liver sections. ,tomach 
• Liver iranular fluorescence of parietal (+++) and chief cells (+). 
Negative .iver 
• Antigens lepatocytes show cytoplasmic granular staining. 
a (92kDa) and ~ (60-90kDa) subunits of the gastric H+/ K• ATPase (prC\ntigens 
pump) 'DH-E2, BC0ADC-E2, OGDC-E2 
• Disease Association lisease Association 
Type A atrophic gastritis, Pernicious Anemia (90%) 'rimary Biliary Cirrhosis (90-95%) 
............................. -------~',i-------- ............................. 
Possible follow-up tests 
Parietal ce/VAESKUBLOTS" Gastro Pro 
Possible follow-up tests 
AMA-M2-check/ AESKUBLOTS® ANA-17Pro/AESKUBLOTS" LiverPro 
49 
················ ··· ·············· --------------
Anti-Mitochondrial Antibodies 
(AMA-M2) 
Pl 
External cortex 
P2+P3 
Inner cortex 
D 
Medulla 
Kidney 
D>Pl>P2>P3 
Possible follow-up tests 
AMA-M2-check/AESKUBLOTS® ANA-I 7Pro/ AESKUBLOTS® LiverPro 
4ox l 
t 
' ~ 
l 
t 
i 
l 
t 
li 
f t; 
f 
' • ,i ' • Kidney 
Anti-Mitochondrial Antibodies 
(AMA-M2) 
External cortex 
Dots in the glomeruli 
Kidney 400x 
P2+P3 
Inner cortex 
D 
Medulla 
Liver lOOx 
Possible follow-up tests 
AMA-M2-check/AESKUBLOTS® ANA-I 7Pro/AESKUBLOTS® LiverPro 
51 
I 52 
.... ············ · ·············· .... -------------Anti-Mitochondrial Antibodies 
(AMA-M2) Anti-Mitochondrial Antibodies 
(AMA-M2) 
Dots in the chief ce lls 
Stomach 
Liver 
400x 
400x 
Possible follow-up tests 
AMA-M2-check/AESKUBLOTS® ANA-1 7Pro/AESKUBLOTS® LiverPro 
Differences between APCA and AMA-M2 
APCA 
Chief cells 
Parietal cells 
Stomach 
Dots in the chief cells 
Parietal cells 
Stomach 
400x 
AMA-M2 
400x 
Possible follow-up tests 
AMA-M2-check/AESKUBLOTS® ANA-17Pro/AESKUBLOTS® LiverPro 
53 
54 
. . . . . . . . . . . . . . . . . . . ................ . 
Liver/Kidney Microsomal Antibodies 
(LKM-1) 
• Kidney 
Proximal tubules in the inner cortex (P3) show a diffuse homogeneous 
intense staining. Medulla (distal tubules) is negative. Some proximal 
tubules close to the glomeruli are negative or weakly positive. Glomeruli 
are negative. 
• Stomach 
Negative 
• Liver 
Intense homogeneous staining of the hepatocytes. 
• Antigens 
Cytochrome P450 2D6 
• Disease Association 
Autoimmune Hepatitis Type II (80-95%). LKM-1 Abs can also be detected 
in certain patients with Hepatitis C Virus. 
Possible follow-up tests 
LKM-1/AESKUBLOTS® LiverPro 
Liver/Kidney Microsomal Antibodies 
(LKM-1) 
-------- ····························· 
Possible follow-up tests 
LKM-1/AESKUBLOTS® LiverPro 
55 
56 
liver/Kidney Microsomal Antibodie~ • • • • • • • • • • • • • • • • · · • · • • • • • · • • • • • • • • • _____ H_e_t_e-ro_p_h-il_e_A_n_t-ib_o_d_ie-s '\ 57 
(LKM-1) I 
Differences between AMA-M2 and LKM-1 
Possible follow-up tests 
LKM-1/ AESKUBLOTS® LiverPro 
• Kidney 
Only the brush border of the tubules is stained. 
• Stomach 
Staining between the gastric glands similar to the smooth muscle, but 
blood vessels are negative. Parietal cells are positive. 
• Liver 
Sinusoides stained 
• Antigens 
Various antigens 
• Disease Association 
Unclear 
-------- ····························· 
58 ( Heterophile Antibodies 
............................. ---------
• ••••••••••••••••• _____ A_n_t-i--F--A-c-ti_n_A_n_t_ib_o_d-ie-s) 59 
(ASMA) I 
• Kidney 
Staining of mesangial cells of the glomeruli, intracellular fibrils around 
renal tubules and peritubular areas and vessel walls. 
• Stomach 
Staining of the muscularis mucosae, muscle layers of blood vessels and 
the interglandular actin fibers. 
• Liver 
Staining of sub-membranous actin around hepatocytes with a polygonal 
pattern (not always seen) and smooth muscle fibers of hepatic portal 
tracts. 
• Antigens 
Filamentous (polymerized) actin (F-actin) 
• Disease Association 
Autoimmune Hepatitis Type 1 (50-85%) and Primary Biliary Cirrhosis 
(20%) 
"Jote: 
Anti-smooth muscle antibodies (ASMA) have several target antigens found in the filaments 
of smooth and striated muscle (actin, myosin, tropomyosin, vimentin, desmin, cytokeratin, 
tubulin). Anti -Actin are the most clinically relevant for Autoimmune Hepatitis Type 1. 
60 (_A_n_ti--F---A-ct-in-A-nt-ib_o_d-ie_s ____ ······· ·········· 
(ASMA) 
............................. -------
· · · · · · · · · · · · · · · · · · -----A-n-ti--F--A-c-t-in_A_n_t-ib_o_d-ie-s) 61 
(ASMA) I 
Smooth muscle 
fibers 
Smooth muscle 
fibers 
Kidney 
Liver 
400x 
200x 
------- ............................ . 
I I 
62 ( Liver Cytosol Antibodies 
I (LCl) 
• Kidney 
Negative 
• Stomach 
Negative 
• Liver 
Homogeneous cytoplasmic staining of the hepatocytes. The 
hepatocyte layer around the central vein is spared . 
• Antigens 
Formiminotransferase cyclodeaminase 
• Disease Association 
Autoimmune Hepatitis Type 2 (20-30%) and Hepatitis C (2-10%) 
............................. ---------
Possible follow-up tests 
LC-1/ AESKUBLOTS® LiverPro 
· •·••••···•·••··•• -----A-nt-i--E-la_s_f_,n_A_n_t_ib_o_d-ie-sl 63 
Kidney 
Negative 
• Stomach 
The elastic fibers of the muscle are stained. 
• Liver 
The vessels rich in elastin, show the internal and external elastic 
membranes stained, the smooth muscle cells are negative. 
• Antigens 
Elastin, elastic fibers (unclear) 
• Disease Association 
Unclear 
............................. 
64 I Anti-Elastin Antibodies 
Elastic fibers 
Ela_stic fibers 
of the muscle 
Stomach 
Internal elastic 
membrane 
Liver 
200x 
400x 
............................. --------
· · • ·· •• · •·•••• · •· · -----A-n-ti--E-la_s_t-in_A_n_t_ib_o_d-ie-sl 65 
External elastic 
membrane 
Stomach 
Kidney 
Elastic fibers 
of the muscle 
are stained 
400x 
Internal elastic 
membrane 
400x 
............................. 
66 ( Anti-Elastin Antibodies 
.................. 
Differences between Anti-E/astin and F-Actin 
Muscularis 
mucosae 
Anti-Elastin 
Elastic 
fibers 
Elastic fibers 
of the muscle 
are stained 
Stomach 400x 
F-Actin 
lnterg landular 
actin fibers 
Stomach 400x 
. ............................ --------
···•·•·••••••·•••• -----A-n-ti--E-1-a-st_i_n_A_n-ti_b_o_d_ie--.sl 67 
Differences between Anti-Elastin and F-Actin 
External elastic 
membrane 
Smooth muscle 
ne ative 
Kidney 
Smooth 
muscle cells 
Kidney 
Anti-Elastin 
Internal elastic 
membrane 
400x 
F-Actin 
400x 
-------- . ........................... . 
68 Anti-Ribosomes Antibodies 
(Rib-P) 
• Kidney 
Negative 
• Stomach 
Staining of the chief cells. 
• Liver 
Cloudy cytoplasmic staining of the hepatocytes. 
• Antigens 
Phosphorylated proteins (P proteins): PO (35kD), Pl (19kD) and P2 (17kD) 
• Disease Association 
Systemic Lupus Erythematosus 
............................. --------
Possible follow-up tests 
Rib-P /AESKUBLOTS® ANA-1 7Pro/AESKUBLOTS® ANA-l 2Pro 
. ................. --------------
• Kidney 
Negative 
• Stomach 
Anti-Signal Recognition Particle 
(SRP) 
Staining ofthe chief cells . 
• Liver 
The cytoplasm of the hepatocytes show irregular granular perinuclear 
staining. 
• Antigens 
Signal recognition particle (?SL RNA + 6 proteins - SRP 9, 14, 19, 54, 
68, 72) 
• Disease Association 
Myositis, Polymyositis, Necrotizing Myopathy 
............................. 
69 
Notes 
ANCA Patterns 
C-ANCA 
Ethanol/Formalin Fixed Slides 
• C-ANCA pattern on ethanol fixed substrate 
Granular staining of neutrophil cytoplasm. 
................. . 
The staining is most intense in the center of the cell, between the lobes 
of the nucleus (central accentuation). 
Staining usually fades gradually at the outer edge of the cytoplasm. 
• C-ANCA pattern on formalin fixed substrate 
Is similar to the pattern seen on ethanol fixed slides. 
The granules often appear a little larger and more distinct than on the 
ethanol fixed substrate. 
• Antigens 
Proteinase 3 (PR3), Bactericidal permeability increasing protein (BPI; 
CAP57) and other unknown antigens 
• Disease Association 
Granulomatosis with Polyangiitis = Wegener's granulomatosis 
Occasionally positive in patients with microscopic polyangiitis or 
Eosinophilic Granulomatosis with Polyangiitis = Churg Strauss Syndrome 
····························· ---------
Possible follow-up tests 
Vasculitis-Screen/PR3 sensitive/ANCA-Pro/AESKUBLOTS® Vasculitis Pro/ANCA Pro 
·················· ---------------P-ANCA 
Ethanol/Formalin Fixed Slides 
• P-ANCA pattern on ethanol fixed substrate 
Neutrophils have perinuclear pattern. Due to ethanol fixation, the anti-
gens migrate to the margins of the nucleus. 
• P-ANCA pattern on formalin fixed substrate 
Formalin fixation dramatically changes the staining pattern associated 
with P-ANCA. Staining is cytoplasmic because the antigens are fixed 
at their original sites in the neutrophil granules, and do not migrate 
to the periphery of the nucleus. The overall staining intensity may be 
decreased. 
• Antigens 
Myeloperoxidase (MPO), is the most common antigen associated with 
the P-ANCA pattern, but a variety of other antigens can produce P-ANCA 
staining (Elastase, Cathepsin G, Lactoferrin, BPI (CAP57), a-Enolase, 
~-Glucuronidase, Lysozyme, Azurodicine (CAP37) and other unknown 
antigens). 
• Disease Association 
Microscopic Polyangiitis, some patients with Granulomatosis with 
Polyangiitis, Systemic Lupus Erythematosus, Goodpasture's Syndrome, 
Inflammatory Bowel Disease, Rheumatoid Arthritis ............................. 
Possible fo llow-up tests 
Vasculitis-Screen/MPO/ANCA-Pro/AESKUBLOTS® Vasculitis Pro 
73 
74 Atypical-ANCA (A-ANCA) [X-ANCA, NANA] 
Ethanol/formalin Slides 
• A-ANCA pattern on ethanol fixed 
substrate 
Show a very perinuclear staining 
of the nucleus. 
• A-ANCA pattern on formalin fixed 
substrate 
Negative 
• HEp-2 cells 
Negative 
• Antigens 
Lactoferrin , Cathepsin G, Lyso-
zyme 
• Disease Association 
Ulcerative Colitis, Crohn's Disease 
and Autoimmune Liver Diseases 
Differences between Atypica/-ANCA (A-ANCA} and P-ANCA 
............................. --------
Possible follow-up tests 
AESKULISA® ANCA Pro 
Anti-Endomysium Antibodies 
I 
I I 
76 Anti-Endomysium Antibodies (EMA) 
(lgA, lgG) 
Endomysial staining of 
the muscularis mucosae 
• Substrate 
Monkey esophagus 
• Pattern 
400x 
Endomysial staining of the muscularis mucosa seen as a network of 
fibers surrounding the smooth muscle cells (looking like a honeycom). 
• Antigen 
Enzyme Tissue Transglutaminase (tTG) 
• Disease Association 
Coeliac Disease, Dermatitis Herpetiformis 
Possible follow-up tests 
Ce/iCheck New Generation/tTg-GA New Generation/tTg-A New Generation/tTg-G New 
Generation/DGP Check/DGP-G.IVDGP-.IVDGP-G/Gliadin-Check/AESKUBLOTS® Gastro Pro 
Crlltddla luclllae 
1s ( Crithidia luciliae I (dsDNA) 
• Substrate 
Haemoflagellate Crithidia /uci/iae 
• Pattern 
Homogeneous fluorescence of kinetoplast. 
Fluorescence of the flagellum basal body is of no significance. 
Reaction of the cell nucleus is not relevant. 
• Antigen 
dsDNA (nDNA) 
• Disease Association 
Systemic Lupus Erythematosus 
............................. --------
Possible follow-up tests 
DANA-Pro/ dsDNA-check/ AESKUBLOTS® ANA-17 Pro/ AESKUBLOTS® ANA-12 Pro 
Notes 
84 ( Algorithm 
( 
ANA Test not 
indicated 
CYTOPLASMIC 
Fine Dense 
Granular/ 
Homogeneous 
Rib-P 
Fine Granular 
Jo-1, PL-7, PL-12, 
SRP 
Granular/ 
filamentous 
AMA-M2 (PDCE2) 
Filamentous 
F-Actin, Vimentin 
Multiple Dots 
P bodies/ 
GW bodies 
Reticular Granu-
lar on one Site of 
the Nucleus 
Golgi apparatus 
Cllnlcal Suspicion of Systemic 
Rheumatic Diseases 
NO 
Smooth Nuclear 
Membrane 
Lamins,A,B,C 
MCPl-r 
Smooth Nuclear 
Membrane 
Lamins A,8,C 
MCP(-J' 
YES 
NUCLEAR 
Homogeneous 
(dsDNA), 
nucleosomes 
histones 
MCP (+) .. 
"quasi" 
Homogeneous 
MCP(+) .. 
RequestlFA 
on HEp-2 cells 
Positive 
Fine Speckled 
SSA/SSB 
Ku, Mi2 
MCP/-)' 
Large/Coarse 
Speckled 
hnRNP 
RNP/Sm, Sm 
MCP(-)' 
Dense Fine Speckled 
DFS70/LEDGF 75 
MCP(+)'' 
Scl-70 
Topoisomerase I, MCP (+) .. 
Mixed Patterns 
'MCP (-) Metaphase Chromosomal Plate 
negative 
.. MCP {+) Metaphase Chromosomal Plate 
positive 
) 
RequestlFA 
on HEp-2 cells 
Clumpy 
U3-RNP 
(Fibrillarin) 
MCP /+!" 
Punctated 
RNAP-1 /NOR 
MCP (-)' 
_) 
Legend: 
Algorithm I 
,- -- . I 
: Test for SSA/SSB, Jol, Rib P by : 
-------- ----- , ELISA/DOT If negative, no further , 
:-: autoant1bod1es testing 1s 1nd1cated : Negative 
- - - - - - - - - - - - - : Fo llow patient clm1cally and con- : 
NUCLEAR 
Multiple Nuclear 
Dots 
SpIOO, PML 
MCP (-)' 
Few Nuclear 
Dots 
p80 coilin 
MCP 1-r 
: siderrepeat testing _________ : 
Cyclin 
MCP/-)' 
CENPF-(MSA-3) 
Mitosin 
MCP(+)'' 
MITOSIS 
ASSOCIA7ED 
PATTFRNS 
Centrosome 
(centriole) 
Pericentrin, 
enolase 
NuMA-1 
(NuMA) 
NuMA-2 
HsEg 5 
Midbody 
(MSA-2) 
Aurora kinase B 
complex 
Mixed Patterns 
Adapted and modified from: "Nuclear pattern (true ANA), proposal for a classification tree" 
K. Conrad (Germany) & J. Damoiseaux (The Nederlands) on ICAP (International Concensus on ANA 
patterns) 2014, S. Paulo, Brazil 
85 
Notes 
88 ( FAQ's 
Problem Possible Causes Possible Solutions 
Both positive and nega-
tive controls not visible 
through the fluorescence 
microscope. 
Nothing seen on slide. 
Cells visible but negative 
staining with all tests in-
cluding positive control. 
Negative control stains 
positive and/or positive 
control stains negative. 
Negative control stains 
positive and/or repeated 
specimens give no repro-
ducible results. 
Variation of end point 
titer of known positive 
control either weaker or 
stronger than expected 
and/or specimen results 
inconsistent. 
Fluorescence microscope may 
not be working properly. 
Antigen wiped off during remov-
al from pouch or during testing. 
Overly vigorous use of wash 
buffer (WB) squeeze wash bot-
tle during rinses. 
Microbial contamination of 
serum specimen will digest off 
the antigen substrate. 
Reagents used in wrong 
sequence or one or more re-
agent not added. 
Check microscope with previously 
stained slide. If there is still noth-
ing visible, check filters for correct 
wavelength and/or replace or 
realign the bulb. 
Do not touch antigen surface with 
hands or pipettes at any time. · 
Follow rinse directions carefully. 
Do not focus wash buffer (WB) 
stream directly onto the antigen 
well. 
Take care to avoid contamination 
during specimen collection, sepa-
ration and handling. 
Review procedure and repeat test 
following staining steps precisely. 
Prepare and use abbreviated 
checklist of steps. 
Use of wrong control or Check reagent labels and repeat 
reagents. test. 
Cross-contamination of con-
trols and/or specimens. 
Running of controls and/or 
specimens between wells. 
Splashing of excess control 
and/or specimens from well to 
well due to over vigorous rin-
sing with wash bottle. 
Inconsistency in timing inter-
vals and incubation tempera-
tures. 
Always change pipettes or pipettortips between use of controls and 
specimens. Always return proper 
caps to reagent vials and speci-
men containers. 
Use smaller drops to stay within 
well perimeters. 
Follow rinse directions carefully. Al-
ternative rinse method is to shake 
off excess of reagent and gently 
put the slide in jar of fresh WB. 
Use consistent, uniform technique 
run to run and person to person. 
Re-check times and temperatures 
on your abbreviated checklist. 
FAQ's! 89 
Problem Possible Causes Possible Solutions 
Positive control stains at 
a weaker reaction than 
expected (all specimens 
show decreased fluores-
cence intensity/. 
Positive control stains at 
a weaker reaction than 
expected. (All specimens 
show decreased fluores-
cent intensity./ 
Serum dilutions made incor-
rectly. Serum added at top of 
test tube and not mixed well 
into sample buffer (SB). Serum 
amount not correct for dilution. 
Pipettor delivery not accurate. 
Reagents outdated or mixed 
between kits. 
Check fluorescence micro-
scope. Most common error is 
an improperly aligned bulb. 
Deterioration of all reagents 
particularly the antigen sub-
strate due to improper storage 
such as excessively hot room 
temperature. 
Deterioration of serum speci-
men due to improper storage 
(excessive heat or· multiple 
freeze/thaw cycles). 
Conjugate activity lost due to 
excessive exposure to strong 
light and/or heat during stor-
age or use in the test or due 
to contamination (e.g. with 
serum). 
Antigen deterioration from 
punctured pouch or slides 
removed from pouch too long 
before testing. 
WB not prepared correctly. 
Re-check serum dilution scheme. 
Prepare fresh dilutions with correct 
technique (mixing serum and sam-
ple buffer (SB) well at each step). 
Repeat test. 
Pipettor calibration must be perio-
dically checked. 
Always check expiry dates of 
reagents and never mix reagents 
from kit to kit or substitute from 
another manufacturer. 
Re-check filter wavelengths. 
Replace or realign bulb. 
Store kit with all components as 
instructed in refrigerator. 
Handle serum specimens sterile 
and refrigerate until tested. For 
long term storage aliquot and 
freeze at -20°C, or below. 
Obtain fresh conjugate. Always 
store away from direct light. 
Cover moist chamber with paper 
towel to block light during restai-
ning. 
Check pouch for punctures. Puffi-
ness indicates integrity not com-
promised. Remove slides from 
sealed pouch 5 to 30 minutes 
before use in test. Restain. 
Follow directions for WB prepara-
tion carefully. Distilled water gives 
more consistent results than deion-
ized water. 
90 ( FAQ's 
.................. 
Problem Possible Causes Possible Solutions 
Positive control stains at 
a weaker reaction than 
expected. (All specimens 
show decreased fluores-
cent intensity.) 
Increased incidence of 
weak positive speci-
mens (all specimens 
show increased fluores-
cent intensity). 
Nonspecific staining with 
all cells appears green. 
Nonspecific staining with 
entire antigen substrate 
appears green. 
Reagents not brought to room 
temperature prior to use in the 
test. 
Substrate is not incubated 
long enough with specimen or 
conjugate. 
Excessive wash times may 
leach out antigenic material. 
Two little mounting medium 
between slide and coverslip. 
Excess mounting medium 
does not allow tight contact 
between antigen substrate 
and coverslip for best light 
pathway. 
Slides left overnight before 
reading may show deteriora-
tion in fluorescent intensity, 
especially if exposed to exces-
sive light and/or heat. 
Over-reading - some speci-
mens may fluoresce more than 
the negative control, but less 
than I+, or nonspecific fluores-
cence misinterpreted as true 
staining. 
Some sera may have hetero-
phile or autoantibodies against 
nuclear or cytoplasmic cellular 
components that may add to 
or obscure the specific stain-
ing pattern (i.e. ANA, AMA, 
ASMA). 
Check microscope filter for 
wavelength band. 
Allow time for reagents to equili-
brate to room temperature. Cold 
reagents take longer to react. 
Re-check correct time for all steps. 
Do not shorten incubation periods. 
Re-check wash times. Do not 
leave substrate in WB longer than 
instructed. 
Add additional mounting medium 
and/or recoverslip. 
Drain excess mounting medium by 
holding edge of slide against ab-
sorbent paper. Do not push down 
on coverslip. 
Store slides in refrigerator, protect 
from direct light and read as soon 
as possible. 
Experienced personnel must be 
available to read and interpret 
results of fluorescent tests. 
Try to tiler out beyond the non-
specific staining interference. May 
have to report results as "unable 
to interpret due to the presence of 
interfering antibodies or nonspeci-
fic fluorescence". 
Replace broad band filter with a 
narrower band more suitable for 
FITC conjugates. 
FAQ's I 91 
Problem Possible Causes Possible Solutions 
Nonspecific haze or film 
over entire antigen sub-
strate in all controls and 
specimens. 
Nonspecific haze or film 
with only certain speci-
mens. 
Nonspecific dense stai-
ning particularly around 
perimeter of well. 
Antigen substrate cells 
distorted or nucleus of 
cells with punched out 
appearance. 
Antigen substrate inadequately 
rinsed and washed between 
incubation steps. 
Drying of antigen substrate 
after rinse and wash steps. 
Check fluorescence micro-
scope. Optical pathway may 
be dirty. 
Check if coverslip is too thick 
or if two coverslips are stuck 
together. 
Lipemic serum may bind 
nonspecifically to antigen sub-
strate masking specific anti-
gen/antibody reaction. 
Many other proteins and anti-
bodies are present in the se-
rum and may react. 
Hemolyzed serums may have 
nonspecific fluorescing por-
phyrins. 
Drying out of serum specimen 
or conjugate on antigen sub-
strate during incubation steps 
before excess is removed. 
Conjugate may run off slide 
edge due to capillary action. 
Pushing down with excessive 
pressure of coverslip on sub-
strate to eliminate bubbles or 
excess mounting medium. 
Frequent changes of fresh WB 
for the rinse and wash steps are 
necessary. 
Do not allow antigen to dry at 
any time during or between stain-
ing steps. Handle only one or 
two slides at a time from wash 
step before applying conjugate 
and returning to moist chamber. 
Clean objectives and eyepiece lens. 
Use #I coverslip. Carefully remove 
double coverslip and remount with 
single coverslip. 
Obtain fasting specimen to reduce 
lipids. 
Obtain a fresh fasting specimen. 
Obtain fresh non-hemolyzed serum 
specimen. 
Increase size of reagent drop to 
well area and/or increase amount 
of water in moist chamber. 
Shake off excess WB and carefully 
wipe around edges of slide before 
adding conjugate. 
Drain off excess mounting medium 
by holding edge of slide against al>-
sorbent paper. ij bubbles are exces-
sive, remove coverslip and remount. 
Microbial contamination of Take care to avoid contamination 
serum specimen. during specimen collection, sepa-
ration and handling. 
92 ( FAQ's 
Problem Possible Causes Possible Solutions 
Antigen substrate cells 
distorted or nucleus of 
cells with punched out 
appearance. 
Uneven staining within 
well. 
Scrapes or tears on anti-
gen surface. 
Nonspecific dense stain-
ing particularly around 
perimeter of well. 
Irregular fluorescent stai-
ning pattern not closely 
resembling that seen 
with the positive control. 
Results differ from those 
reported by another 
laboratory. 
WB not prepared correctly. 
· Use of water instead of SB/ 
WB. 
Fluorescent staining in bubbles 
of air in mounting medium be-
tween substrate and coverslip. 
Excessive movement of cover-
slip or scraping with pipe! 
tips. 
Drying out of serum specimen 
or conjugate on antigen sub-
strate during incubation steps 
before excess is removed. 
Conjugate may run off slide 
edge due to capillary action. 
Foreign matter or bacterial/ 
fungal contamination of buffer 
and reagents may obscure or 
distort antigensubstrate. 
Microscope optics, filters, 
light sources, reagents and 
personnel may vary enough to 
result in differences of two-fold 
or more. 
Follow directions for WB prepara-
tion carefully. Always mix well to 
dissolve. 
Use SB/WB as described in instruc-
tion for use. 
Do not agitate mounting medium 
bottle, it causes bubbles. 
Do cover-slipping procedure care-
fully. 
Handle coverslips gently. Do not 
touch antigen surface. 
Increase size of reagent drop to 
well area and/or increase amount 
of water in moist chamber. 
Shake off excess WB and carefully 
wipe around edges of slide before 
adding conjugate. 
Check WB (particularly the squeeze 
wash bottle) and other reagents lor 
precipitate or turbidity. Change WB 
rinse/wash solutions frequently. 
Each laboratory must establish 
their own normal ranges. Results 
between laboratories should not 
be compared. 
HELMED® IFA, ELISA, BLOT - ALL IN ONE 
COLOR CODED TROUBLESHOOTING 
The HELMED® was the first machine to 
introduce a built-in barcode reader allowing 
automated detection and scanning of 
sample tube IDs eliminating transcription 
errors. Communication between LIS and 
instrument can be done directly or via our 
middleware AESKU.LAB. 
OUTSTANDING REAGENT CAPACITY 
AND PRACTICABILITY 
In the outer ring for IFA, ELISA 
and Blot, several reagent racks can 
be combined, each with a capa-
city of up to 16 reagent bottles. 
AESKUBLOTS®, AESKULISA® and 
AESKUSLIDES® reagent, calibrator 
and control vials fit directly into the 
corresponding positions. 
PLUG & PLAY MODULAR SYSTEM 
The HELMED® is capable of 
performing the 3 major lab tech-
niques: IFA, ELISA and BLOT. This is 
achieved by simply changing racks 
and rings and using dedicated 
software. The proprietary built-in 
barcode scanner allows automatic 
identification of the correct rack. 
Up to 3 machines can work in 
parallel with one computer. 
REVOLUTIONARY DESIGN WITH THE 
SMALLEST FOOTPRINT AVAILABLE 
The HELMED®' s compact architecture is 
intended to give the technician direct and 
easy access to all working areas . The main 
instrument parts are magnetically mounted, 
allowing easy and fast replacement by techni-
cal service or by the operating technician . An 
optimal footprint (57 cm x 62 cm x 75 cm) 
combined with the light weight (25 kg) makes 
it suitable for any lab space. 
SAFE HANDLING AND SECURE RESULTS 
The HELMED®· s green cover auto-
matically locks before the assay starts 
running. This creates a perfect repro-
ducible environment inside of the 
machine, keeping a constant temperature 
inside and . avoiding interference from 
external factors, like light or dust. 
FEWER CONSUMABLES, 
MORE FREE SPACE 
The HELMED® has not only a minimal 
footprint, but also has a sample 
requirement as low as 50 µL. The 
amount of reagents used and the dead 
volume are considerably reduced, as 
are consumables like plates. Due to its 
needle technology, no plastic tips are 
needed.