Fluorescent Antinuclear Antibody - PDF document

1 Fluorescent Antinuclear Antibody (FANA)
Fluorescent Antinuclear Antibody (FANA) Presented by Shekarabi.M, Ph.D of Immunology Iran University of Medical Sciences (IUMS) Massoud Clinical Lab The 12 th International & 17 th National Congress on Quality Improvement in Clinical Laboratories 1 Overview • Introduction • FANA SOP and Troubleshooting • Clinical value of FANA in Rheumatic dise

2 ases • Interpretation of FANA results
ases • Interpretation of FANA results in Autoimmune diseases • Automation and Novel methods to assay ANA • challenges for different patterns • Panel discussion 2 SLE Description, ( 1941 ) LE Cell, (Hargraves, 1948 ) Fluorescent conjugated Ab, (Holborow, 1957 ) ENA ( ≈ 1960 ) Animal Tissues ( ≈ 1970 ) Historical notes Hep - 2 Cells ≈

3 1980 Automation & Multiplex ( ≈ 200
1980 Automation & Multiplex ( ≈ 2000 - 2019 ) ?? ?? LE Cell 3 Systemic Lupus Erythematosus Systemic Lupus International Collaborating Clinics (SLICC) - 2012 4 A nti N uclear A ntibodies ( ANA ) • ANA test is the most commonly antibody detection test that performed in many clinical and immunology laboratories in the worldwide; • for exampl

4 e, 8854 tests per year ( 1397 : 740 p
e, 8854 tests per year ( 1397 : 740 per month) are performed in Massoud Lab. • Routinely ANA test performed in fluorescent (IFA) and Enzymatic (EIA) methods. • Based on the confirmation of all guidelines, the gold standard method for evaluation anti - nuclear antibodies is Indirect fluorescent assay in human cell lines or animal tissues

5 as substrate; that called FANA 5 FANA su
as substrate; that called FANA 5 FANA substrate • There are two major types of substrate for ANA testing. animal substrates, such as mouse kidney or rat liver and cultured human cells (HEp - 2 cells) . • Sera of some patients with SLE were reportedly negative on such substrates. False negativity on animal substrates. • Presently ,

6 the vast majority of laboratories use a
the vast majority of laboratories use a specific type of cultured human cells referred to as HEp - 2 cells. • It is essential to pay attention to the substrate being used by each of the various laboratories from which a physician may receive different results . 6 Hep - 2 as substrate • These are obtained from cultured laryngeal esoph

7 ageal squamous cell carcinoma cells.
ageal squamous cell carcinoma cells. The cells are available commercially, prefixed on glass slides (CCL - 23 ,ATTC). 7 ANA Positive Hep - 2 cells 8 Sample for ANA test: blood serum . The common tests used for detecting and quantifying ANAs are indirect immunofluorescence (IFA) and enzyme - linked immunosorbent assay ( ELISA ). 9 How i

8 s the test reported? The results of ANA
s the test reported? The results of ANA testing are reported in three components : ° Reactivity ° Titer (intensity) ° Pattern Titer The quantitation of an ANA is most commonly reported as a titer, Although the widely accepted cutoff for FANA positivity has remained 1:40 , greater clinical significance has generally been thought to correlat

9 e with higher titers. 1:20 1:40 1:80 1:
e with higher titers. 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560 … FANA cut off, which Titer is better 1:40 or 1:80 ? • Generally, the ANA test is negative or very low in young and healthy persons. high in patients with systemic Connective Tissue Diseases (CTD). • The ANA titer is intermediate in some patients with CTD as well as in pe

10 rsons with a wide variety of conditions
rsons with a wide variety of conditions . Old age, pregnancy, close relatives of patients with systemic CTD, and patients taking drugs 10 Positive fluorescent ANA test in healthy persons (on HEp - 2 cells) ANA patterns. The patterns of fluorescence of the nuclei of Hep - 2 cells in an ANA test are usually associated with specific antin

11 uclear antibodies www.medscape.com 11 A
uclear antibodies www.medscape.com 11 A standard operating procedure for FANA assay • Hep - 2 substrate • Serum dilution and PBS Buffer • FITC conjugated antibody • Mounting media and coverslips • Fluorescent microscope 12 HEp - 2 substrate 1 - Expression of more antigens , which increase the sensitivity of the FANA test. 2 . The huma

12 n origin of HEp - 2 cells ensures a be
n origin of HEp - 2 cells ensures a better specificity . 3 . The size of the HEp - 2 cells is much larger . 4 . The amount of cell division (mitosis) is much greater than that of animal tissues, so cell - cycle and centromere - dependent antibodies are more easily identified. 5 . HEp - 2 cells grow in single - layer form , so all nuclei are

13 readily available and its microscopic e
readily available and its microscopic examination is simpler. 6 . The size of the nuclei is bigger, so the details of the nuclei and patterns are better distinguished. 7 . Distribution of antigens is uniform and the difference in color intensity is not seen. 8 . The intracellular matrix is ​​readily seen. 9 . The cell source is available

14 as an immortal and standardized cell li
as an immortal and standardized cell line. 10 . Non - use of damaging fixative solutions that damage some intracellular antigens. 13 14 The ideal HEp 2 cell, how should it look like? Therefore, an ideal HEp 2 cell substrate has to be up to a certain standard: • The cell nuclei have to be large and uniform in size. • The distribution of the

15 cells should not be too dense, the opti
cells should not be too dense, the optimum number of cells varies between 70 and 100 cells in the visible area at a magnification of 400 ( 40 X) , less than 50 or more than 110 cells is not adequate • Broad cytoplasms with clearly definable antibody patterns and definite discrimination of the cell nucleus are required. • A considerabl

16 e number of cells should be in differen
e number of cells should be in different phases of the cell cycle . 15 • A limited number of chromosomes ( 10 ) should be clearly visible. Two to six chromosomes of which one to three are in metaphase is the optimum. • Minimum background fluorescence desired. 40 X Cell Cycle of Hep - 2 Cells: Interphase and Mitotic Phase 16 Serum Seria

17 l Dilution 17 1:20 1:40 1:80 1:160 1:320
l Dilution 17 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560 … PBS Buffer (Phosphate Buffered Saline) pH= 7.2 - 7.4 Which is better? 18 PBS Buffer (Phosphate Buffered Saline) pH= 7.2 - 7.4 • Adjusted pH by HCL, 1 N ر̴شن̯او Salt یارب زاین دروم ریداϘم لولحم 1X (mmol/Lit) لولحم ییاϬن تظل 1X (gr

18 /Lit) لولحم ییاϬن تظل 10X
/Lit) لولحم ییاϬن تظل 10X (gr/Lit) NaCl 137 8 80 KCl 2.7 0.2 2 Na 2 HPO 4 Without H 2 O 10 1.44 14.4 2 H 2 O 10 1.8 18 7 H 2 O 1 2.7 27 12 H 2 O 10 3.6 36 KH 2 PO 4 1.8 0.24 2.4 FITC conjugated secondary antibodies 19 Ready to use or Commercial FITC Antibodies ( Chessboard or

19 Chequerboard Titrations ) High backgrou
Chequerboard Titrations ) High background during IFA, what's your suggestions ? 20 • No optimal concentration of FITC conjugated Ab or excess of incubation time • Non - appropriate of wash time or shaking • Non - specific binding of FITC conjugated Ab • Drying the substrate in the wells during the process **Pay a lot of attention; The h

20 igh background of the conjugated antib
igh background of the conjugated antibodies around the wells are greater than its center Mounting medium and Coverslips • MOUNTING MEDIUM; P hosphate buffered glycerol of pH 7.4 ± 0.2 . Stable at 2 - 8 ° C until labeled expiration date. Many fluorophores such as FITC are brighter at higher pH (pH= 8.5 to 9.2 ). • For FITC it

21 should not be lower than pH 7.2 beca
should not be lower than pH 7.2 because FITC fluorescence decreases rapidly below this pH. Oxidation and absorption of CO 2 occur in stored mounting medium and thus decrease the pH of the glycerol in the mounting fluid. Therefore, pH of the mounting fluid should be at least pH 8.0 , preferably between pH 8.5 and 9.0 . The pH should be

22 checked at least once a month, prefe
checked at least once a month, preferably weekly . • Mounting Media and Anti - fade reagent 21 Choose the best size and transparency coverslips Fluorescent Microscope 22 One of the important reasons for differences in results between laboratories is the fluorescence microscope, and in particular the quality of its fluorescent light source. Mec

23 hanisms of fluorescent imaging 23 Light
hanisms of fluorescent imaging 23 Light microscopic Diascopic microscopic Episcopic microscopic Fluorescent Microscope 24 25 FITC excitation and emission wavelength 495 - 520 nm 26 Focus and Alignment of Mercury and Xenon Arc Lamps https:// www.microscopyu.com/tutorials/arclamp 27 تسا Ϫتخوس پم� تسا Ϫتخوس ϊیذغت ϊب

24 نم زویف م� یور یÙ
نم زویف م� یور یمشچ یسدϋ ( Ϫنومن ) تسین میظنت Ϫتسب پو̰سور̰یم مگارفاید دین̯ زاب ار مگارفاید دیاب و تسا !!!!!!!!!!!!!!!!!!!!!! ϩدش م̯ یدایز نازیم Ϫب روسنادن̯ دوش میظنت روسنادن̯ ωافترا و تسا !!!!!!!!!!!!! یم نÛ

25 ŒØ¨Ø±ÙˆØ¯ تمس Ϫب رون یجورخ
ŒØ¨Ø±ÙˆØ¯ تمس Ϫب رون یجورخ دوش تیادϫ یمشچ یسدϋ تمس Ϫب رون یجورخ و دور رد پو̰سور̰یم تنسرولف پم� رون یمن دϫد و ای پم� رون یم دϫد اما نادیم دید ̮یرات تسا : Quality Control • For quality control and confirmation of the process o

26 f reporting the results should be combi
f reporting the results should be combined with positive and negative samples . • the serum samples ( external quality control ) of the controlled pattern are commercially available. • It is important to use weak controls to control the sensitivity of the test, in addition to using a typical pattern control. • In some laboratories, usin

27 g native (regional) samples, determine t
g native (regional) samples, determine the reference interval (home made) between positive and negative results. Typically, 200 negative control samples and 100 patient samples are used with definitive SLE diagnosis. 28 Quality Assurance • There are several international and commercial programmes for quality assurance, two of which are li

28 sted here. These programs are widely us
sted here. These programs are widely used and cover the most common antibodies. Typically, samples are sent several times a year, and laboratories are able to compare their performance with other laboratories using a consensus report. 29  A scheme for autoimmune serology is available from The College of American Pathologists (CAP)  The Unite

29 d Kingdom National External Quality Ass
d Kingdom National External Quality Assessment Schemes for Autoimmune Serology are available from the Department of Immunology 30 DIAGNOSTIC VALUE OF THE FLUORESCENT ANA TEST 31 Diseases and Related Conditions Associated with Anti - nuclear Antibodies.  Fluorescent ANA (FANA) testing is appropriate as a first screening step & should be perfor

30 med only in the context of clinical sig
med only in the context of clinical signs when rheumatic diseases are clinically suspected .  Among the rheumatic diseases, the presence of anti - nuclear antibodies (ANAs) is characteristic of SLE, SSc , inflammatory myositis, and Sjogren ’ s syndrome. It is required for the diagnosis of some syndromes, such as MCTD or drug - induced lu

31 pus(DIL). 32 Anti - nuclear Antibodies
pus(DIL). 32 Anti - nuclear Antibodies in Systemic Lupus Erythematosus ° SLE is prototype of autoimmune diseases ° More than 180 different autoantibodies ( 2016 ) ° past studies have reported FANA frequencies as low as 90 % , the test is positive in more than 99 % of patients with use of current methods . 33 Major Antibodies in SLE 34

32 Drugs reported with drug - induced SLE Â
Drugs reported with drug - induced SLE ° Allopurinol ° Captopril ° Chlorpromazine ° Clonidine ° Danazol ° Diphenylhydantoin ° Ethosuximide ° Griseofulvin ° Hydralazine ° Isoniazid ° Lithium ° Lovastatin 35 ° Penicillamine ° Penicillin ° Phenothiazines ° Phenylbutazone ° Piroxicam ° Practolol ° Primidone ° Procainamide ° Propylthiour

33 acil ° Quinidine ° Streptomycin ° Sul
acil ° Quinidine ° Streptomycin ° Sulfasalazine ° Sulfonamides ° Tetracycline ° Thiamazole ° Trimethadione ° Valproate ° Mesalazine ° Methyldopa ° Minocycline ° Oral contraceptives ° Mephenytoin Histone: Homogeneous, rim Antigens: H 1 , H 2 A/B, H 3 , H 4 Anti - nuclear Antibodies in Systemic Sclerosis ( SSc )  ANAs against nucleolar a

34 ntigens characterize the autoantibody r
ntigens characterize the autoantibody response in SSc .  Positive FANAs, sometimes speckled in appearance, appear in up to 97 % of sera, although percentages vary depending on the substrate used for detection ..  Unlike SLE sera, however, SSc sera typically, but not always , contain monospecific autoantibody specificities, targeting

35 such structures as the kinetochore (c
such structures as the kinetochore (centromere) , topoisomerase I , or RNA polymerases. Scl - 70 , Diffuse, grainy nuclear or Nucleolar ( fine speckled, nucleoli speckled) RNAP: nucleolar Centromere 36 Anti - nuclear Antibodies in Systemic Sclerosis Centromere pattern Nucleolar pattern (anti - Scl - 70 ) 37 Anti - nuclear Antibodies in Sj

36 ogren ’ s Syndrome  ANA positivity
ogren ’ s Syndrome  ANA positivity as low as 40 % has been reported , many studies report frequencies of 90 % to 96 %, with a diverse range of autoantibodies.  the most well - characterized remain Ro (SS - A) and La (SS - B ) Antigen Nuclear Staining Mitotic Cells SSA fine - coarse speckled negative SSB fine - coarse speckled negative

37 RNP coarse speckled; nucleoli negative
RNP coarse speckled; nucleoli negative negative Fine speckled Coarse speckled 38 Anti - nuclear Antibodies in Inflammatory Muscle Diseases • Diverse group of illnesses often characterized by autoantibody responses against cytoplasmic antigens . Although between 40 % and 80 % of patients with polymyositis / dermatomyositis have positive A

38 NA, as many as 90 % of patients with
NA, as many as 90 % of patients with all types of inflammatory muscle diseases have autoantibodies to cellular antigens . • Myositis autoantibodies are generally categorized into myositis - specific autoantibodies, which are considered exclusive to inflammatory myositis , and those associated with overlap syndromes that include myos

39 itis. • The most well - characterized
itis. • The most well - characterized myositis - specific autoantibodies include the antisynthetases , which target different aminoacyl - tRNA synthetases . 39 Polymyositis appears more commonly with anti – Jo - 1 , and dermatomyositis is more common with the other synthetases . Other myositis - specific autoantibodies include specifi

40 cities against Mi - 2 , a component of
cities against Mi - 2 , a component of the nucleosome remodeling – deacetylase ( NuRD ) complex involved in chromatin remodeling and transcription Regulation . 40 A: Anti - Jo - 1 b: anti - Jo - 1 and anti - Ro 52 , c: anti - PL - 7 , d: anti - PL - 12 , e: anti - KS , f: anti - Mi - 2 , g anti - SAE - 1 , h anti - MDA 5 , i: anti -

41 NXP - 2 j: anti - NXP - 2 with coile
NXP - 2 j: anti - NXP - 2 with coiled bodies , k: anti - TIF 1 γ, l: anti - SRP 41 Antigen Nuclear Staining Mitotic Cells dsDNA homogeneous positive Histone homogeneous positive Nucleosome homogeneous positive SSA fine - coarse speckled negative SSB fine - coarse speckled negative RNP coarse speckled; nucleoli negative negative Sm coarse spec

42 kled negative Nuclear matrix variable la
kled negative Nuclear matrix variable large speckles, nucleoli negative negative PCNA variable staining of nuclei -- Scl - 70 fine speckled, nucleoli speckled positive PmScl - 100 fine speckled, nucleoli homogeneous negative Fibrillarin nucleoli clumpy positive NOR 90 (RNA - Polymerase I) nucleoli speckled dots in the metaphase plate Nuclear Dots (p 80

43 coilin) one to six dots negative Multi
coilin) one to six dots negative Multiple Dot (soluble acidic protein) average of ten dots negative Centromere Proteins 46 dots positive – centromeric dots Fluorescence patterns of autoantibodies to different nuclear antigens in Hep 2 cells 42 the presence of high - versus low - titer FANA results may not be of sufficient clinical significa

44 nce to warrant differential subsequent
nce to warrant differential subsequent evaluation. Rather, a positive screening FANA of any titer requires clinical correlation. • Limitation of number of antigens being in coated in wells. • Because the ELISA technique can denature autoantigens, ELISAs may produce false - positive results, and confirmation may warrant further testing, which

45 is not always clinically available. 43
is not always clinically available. 43 Algorithm for the use of anti - nuclear antibodies (ANAs) in the diagnosis of connective tissue disorders. 44 Main conditions which may be diagnosed from ANA, ENA testing 45 Panel Discussion 46 International Consensus on Antinuclear Antibody (ANA) Patterns (ICAP) https :// www.anapatterns.org 47 48 49 Autoanti

46 bodies Negative# AC= 0 50 Anti - Scl - 7
bodies Negative# AC= 0 50 Anti - Scl - 70 (AC - 29 ) 51 Golgi apparatus pattern (AC - 22 ) ل̰شم ل̰شم ندومن فرطرب یارب ن̰مم لح ϩار تفرگ رظن رد یفنم ای Ϫ̯ فیόض تبثم Ϫجیتن ای و ϩدش Ϫ تسا ل̰شم نآ ریسفت  دشاب فیόض رایسب Ϫگوژنو̯  تسا دایز وÙ

47 „ب سناوا لولحم یفنم یاÏ
„ب سناوا لولحم یفنم یاϫ لولس دح زا شیب یزیمآ ̲نر  تسا یوϗ رایسب Ϫگوژنو̯ فیόض یژوϟوفروم  لاچخی زا ϩدش جراخ یاϫ م� یفا̯ ϩزادنا Ϫب ندشن مرگ  را̯ ماجنا نیح رد اϫدی�سا ندش ̮شخ Ϫباشم یو̴ϟا ا

48 ب تبثم رواجم یاϫ ̮ϫاچ ï
ب تبثم رواجم یاϫ ̮ϫاچ  یاراد Ϫ̯ ییاϫ دی�سا رد ϩژیوب اϫ ̮ϫاچ ریاس Ϫب Ϫنومن ̮ی ندش ϩدیشاپ 24 دنتسϫ ̮ϫاچ  یتϗد یب اب ندر̯ تپیپ اϫ لولس Ϫب ی̰یزیف Ώیسآ  لم� نداد رارϗ ،وشتسش ،ندومن تپیپ ؛یرØ

49 §Ì¯ دنور رد یتϗد یب ϪطϘن
§Ì¯ دنور رد یتϗد یب ϪطϘن ϪطϘن دی�سا ای ϩداد Ώیسآ یاϫ لولس یا  رد Ϫنومن ترارح ای و Ϫنومن ندومن رتلیف ،ϩزات Ϫنومن زا ϩدافتسا ؛ϩدوϟآ Ϫنومن 65 یتناس Ϫحرد دارگ ( دوش یم اϫ یرت̯اب میزنآ نتفر نیبزا ΏبØ

50 ³ ) تنسرولف ϊیرس ندش وحÙ
³ ) تنسرولف ϊیرس ندش وحم  دننام یگدش وحم دض لولحم زا ϩدافتسا DABCO پو̰سور̰یم Ϫب دی�سا ندیبسچ  Ϫتنوم رفاب زا دح زا شیب ϩدافتسا لم� ریز Ώابح لی̰شت  لم� ندر̯ Ϫتنوم تϗد اب و لم� ϩر

51 ابود ندومن ادج اϫ لولس
ابود ندومن ادج اϫ لولس یور رب تنسرولف رونزا یا Ϫϟاϫ  یرا̯ لحارم یارجا نیح رد اϫ ̮ϫاچ رد ارتسبوس ندش ̮شخ  نیئتورپ Ϫب Ϫگوژنو̯ لاصتا ؛یصاصتخاری دناب ( دنن̯ ̭اپ لماوϋ ندر̯ Ϫفاضا رفاب Ϫب ϩ ) ی̰تسϫ یو̴

52 ϟا رد فیόض یزیمآ ̲نر 
ϟا رد فیόض یزیمآ ̲نر  ی̰من تافسف رفاب اب ندومن ϖیϗر ؛Ώآ اب اϫ Ϫنومن ندومن ϖیϗر ( PBS )  یا Ϫشیش فورظ رد یفیث̯ Ù¾ رایسب اϫ لولس ،یزوتیم لا̰شا ندوب یفا̯ان و ϩدن̯ار رشتنم  توافتم دیϟوت یرس اب یدی�