DCM099-7

Ed. 10/2021

ANA Screen for routine analysis

Qualitative determination of IgG class antibodies against nuclear antigens in human serum or plasma

LOT
RUO REF DKO099
See external label Σ = 96 tests

1. INTENDED PURPOSE 3. PRINCIPLE OF THE METHOD

The ANA Screen assay is a manual in vitro device intended The ANA Screen test is an indirect enzyme immunometric
for the qualitative determination of IgG antibodies directed assay (ELISA) based on the binding of present antibodies
against Sm (Smith), RNP/Sm, Scl-70, SS-A (Ro) (52kDa with Sm, RNP/Sm, SS-A (Ro), SS-B (La), Scl-70, Jo1, U1-
e 60kDa), SS-B (La), Jo1, U1-SmRNP, CENP-B, dsDNA SmRNP, CENP-B, dsDNA and Histones antigens coated on
and Histones in human serum or plasma. ANA Screen the microplates. Any antibodies present in calibrators,
Kit is intended for research use only. controls or prediluted patient samples bind to the inner
surface of the wells. After a 30 minutes incubation the
microplate is washed with wash buffer for removing non-
reactive serum components.

An anti-human-IgG horseradish peroxidase conjugate

solution recognises IgG class antibodies bound to the
immobilised antigens. After a 30 minutes incubation any
2. CLINICAL SIGNIFICANCE excess enzyme conjugate, which is not specifically bound is
Anti-nuclear autoantibodies (ANAs) are a heterogeneous washed away with wash buffer.
group of autoantibodies that recognise nuclear
macromolecules and their complexes; antibodies to certain A chromogenic substrate solution containing TMB is
cytoplasmic proteins might also fall into this category1-6. dispensed into the wells. After 15 minutes of incubation the
These antibodies commonly occur in the sera of patients with colour development is stopped by adding the stop solution.
systemic autoimmune rheumatic diseases (SARDs) and can The solution turns yellow at this point. The level of colour is
bind to DNA, RNA and proteins, as well as complexes of directly proportional to the concentration of IgG antibodies
nucleic acids with proteins that are essential for different present in the original sample.
intracellular functions such as replication and transcription.

ANAs’ targets can be divided into two groups in the context 4. REAGENTS, MATERIALS AND INSTRUMENTATION
of systemic rheumatic autoimmune diseases: antibodies that 4.1. Reagents and materials supplied in the kit
recognise DNA, histones and nucleosomes1,2,5,6; and those 1. Calibrator (1 vial, 1.2 mL)
that bind to complexes of RNA with RNA-binding proteins Phosphate buffer 0.1M, NaN3 < 0.1%, human serum
(RBPs) and small nuclear ribonucleoproteins (snRNPs)1. CAL1 REF DCE002/09906-0
2. Controls (2 vials, 1.2 mL each, ready to use)
Autoantibodies directed to nuclear antigens are serological Phosphate buffer 0.1M, NaN3 < 0.1%, human serum
hallmarks of SARDs and their detection is used by clinicians Negative Control REF DCE045/09901-0
to support the diagnosis of disorders such as SLE, Sjögren Positive Control REF DCE045/09902-0
syndrome, systemic sclerosis, polymyositis and mixed 3. Sample Diluent (1 vial, 100 mL)
connective tissue disease. It is well documented that these Phosphate buffer 0.1M, NaN3 < 0.1%
are complex disorders and characterised by the presence of REF DCE053-0
a wide spectrum of autoantibodies. Furthermore, it has 4. Conjugate (1 vial, 15 mL)
widely been shown that ANAs are also frequently found in Anti h-IgG conjugated with horseradish peroxidise (HRP),
the sera of patients with non-rheumatic diseases and healthy BSA 0.1%, ProClin™ < 0.0015%
subjects that do not develop a rheumatic disease.
REF DCE002/09902-0
5. Coated Microplate (1 breakable microplate)
Due to the complexity of SARDs, ANA tests should always
Microplate coated with ANA antigens
be considered together with the evaluation of the patient’s
REF DCE002/09903-0
history, clinical manifestations and other laboratory tests to
diagnose these conditions. 6. TMB Substrate (1 vial, 15 mL)
H2O2 -TMB (0,26 g/L) (avoid any skin contact)
REF DCE004-0
7. Stop Solution (1 vial, 15 mL)
Sulphuric acid 0.15M (avoid any skin contact)
REF DCE005-0
8. 10X Conc. Wash Solution (1 vial, 50 mL)
Phosphate buffer 0.2M pH 7.4 REF DCE054-0
4.2. Materials required but not provided on automatic systems is recommended to increase the
Distilled water number of washes.
• It is important that the time of reaction in each well is held
4.3. Auxiliary materials and instrumentation constant for reproducible results. Pipetting of samples
Automatic dispenser. should not extend beyond ten minutes to avoid assay
Precision Pipetting DevicesMicroplate reader (450 nm, 620- drift. If more than 10 minutes are needed, follow the
630 nm) same order of dispensation. If more than one plate is
used, it is recommended to repeat the dose response
curve in each plate.
5. WARNINGS • Addition of the TMB Substrate solution initiates a kinetic
• This kit is intended for in vitro use by professional reaction, which is terminated by the addition of the Stop
persons only. Not for internal or external use in Humans Solution. Therefore, the TMB Substrate and the Stop
or Animals. Solution should be added in the same sequence to
• Use appropriate personal protective equipment while eliminate any time deviation during the reaction.
working with the reagents provided. • Observe the guidelines for performing quality control in
• Follow Good Laboratory Practice (GLP) for handling medical laboratories by assaying controls and/or pooled
blood products. sera.
• Material of animal origin used in the preparation of the kit • Maximum precision is required for reconstitution and
has been obtained from animals certified as healthy and dispensation of the reagents.
the bovine protein has been obtained from countries not • Samples microbiologically contaminated, highly lipemic
infected by BSE, but these materials should be handled or haemolysed should not be used in the assay.
as potentially infectious. • Plate readers measure vertically. Do not touch the
• Some reagents contain small amounts of Sodium Azide bottom of the wells.
(NaN3) or ProClin™ 300 as preservative. Avoid the • WARNING: the conjugate reagent is designed to
contact with skin or mucosa. ensure maximum dose sensitivity and may be
• All human source material used in the preparation of the contaminated by external agents if not used
reagents has been tested and found negative for properly; therefore, it is recommended to use
antibody to HIV 1&2, HbsAg, and HCV. No test method disposable consumables (tips, bottles, trays, etc.). For
however can offer complete assurance that HIV, HBV, divided doses, take the exact amount of conjugate
HCV or other infectious agents are absent. Therefore, needed and do not re-introduce any waste product into
the Calibrators and the Controls should be handled in the the original bottle. In addition, for doses dispensed with
same manner as potentially infectious material. the aid of automatic and semi-automatic devices,
• Sodium Azide may be toxic if ingested or absorbed before using the conjugate, it is advisable to clean the
through the skin or eyes; moreover, it may react with lead fluid handling system, ensuring that the procedures of
or copper plumbing to form potentially explosive metal washing, deproteinization and decontamination are
azides. If you use a sink to remove the reagents, wash effective in avoiding contamination of the conjugate. This
through large amounts of water to prevent azide build- procedure is highly recommended when the kit is
up. processed using analysers which are not equipped with
• The TMB Substrate contains an irritant, which may be disposable tips.
harmful if inhaled, ingested or absorbed through the skin. For this purpose, DiaMetra supplies a separate
To prevent injury, avoid inhalation, ingestion or contact decontamination reagent for cleaning needles.
with skin and eyes.
• The Stop Solution consists of a diluted sulphuric acid
solution. Sulphuric acid is poisonous and corrosive and 7. REAGENT STORAGE AND STABILITY
can be toxic if ingested. To prevent chemical burns,
avoid contact with skin and eyes. Store the kit at 2÷8°C in the dark.
• Avoid the exposure of reagent TMB/H2O2 to directed • The kit is stable at 2-8°C until the expiry date stated in
sunlight, metals or oxidants. Do not freeze the solution. the external kit label.
• Once opened, the kit is stable at 2-8°C for 6 months.
• The diluted wash solution is stable for 30 days at 2-8°C.
6. PRECAUTIONS
• Please adhere strictly to the sequence of pipetting steps Important note: open the bag containing the Coated
provided in this protocol. The performance data Microplate only when it is at room temperature and close it
represented here were obtained using specific reagents immediately after use.
listed in this Instruction For Use.
• All reagents should be stored refrigerated at 2-8°C in
their original container. Any exceptions are clearly 8. SAMPLE COLLECTION AND STORAGE
The assay should be performed using serum (standard sampling
indicated.
tubes or tubes containing serum separating gel) or plasma (lithium
• Allow all kit components and specimens to reach room heparin, sodium heparin or potassium EDTA) samples.
temperature (22-28°C) and mix well prior to use.
• Do not interchange kit components from different lots.
The expiry date printed on box and vials labels must be Sample Storage Duration
observed. Do not use any kit component beyond their 2-8°C 96 hours
expiry date.
• If you use automated equipment, the user has the Freeze/thaw cycles 3 cycles
responsibility to make sure that the kit has been
appropriately tested.
• The incomplete or inaccurate liquid removal from the
wells could influence the assay precision and/or increase
the background. To improve the performance of the kit
 Sample/
Reagent Calibrator Blank
9. PROCEDURE Controls
Calibrator C1 100 µL
9.1. Preparation of the Calibrators (C1)
Calibrators are ready to use; the concentration of the
Controls 100 µL
Calibrator is printed on the label.
Diluted
9.2. Preparation of the Wash Solution 100 µL
Sample
Dilute the contents of each vial of the buffered wash solution
concentrate (10X) with distilled water to a final volume of Incubate for 30 minutes at room temperature (22-28°C).
500 mL prior to use. For smaller volumes respect the 1:10 Remove the content from each well, wash the wells 3
dilution ratio. The diluted wash solution is stable for 30 days times with 300 µL of diluted wash solution.
at 2-8°C. Important note: during each washing step, gently shake
It is possible to observe the presence of crystals within the the plate for 5 seconds and remove excess solution by
concentrated wash solution; in this case mix at room tapping the inverted plate on an absorbent paper towel.
temperature until the complete dissolution of crystals. For
greater accuracy, dilute the whole bottle of concentrated Automatic washer: if you use automated equipment,
wash solution to 500 mL, taking care also to transfer crystals wash the wells at least 5 times.
completely by rinsing of the bottle, then mix until crystals are
completely dissolved. Conjugate 100 µL 100 µL

9.3. Preparation of Samples Incubate for 30 minutes at room temperature (22-28°C).

For determination of ANA antibodies, human serum or Remove the content from each well, wash the wells 3
plasma are the preferred sample matrixes. times with 300 µL of diluted wash solution.
All serum and plasma samples must be prediluted with Washing: follow the same indications of the previous
sample diluent 1:100; for example, 10 μL of sample may be point.
diluted with 990 μL of sample diluent. TMB
Fasting samples are not necessary and no special 100 µL 100 µL 100 µL
Substrate
preparations are required. Collect blood by venepuncture
into vacutainers and separate serum (after clot formation) or Incubate for 15 minutes in the dark at room temperature
plasma from the cells by centrifugation. (22-28°C).

Neither Bilirubin nor Haemolysis have significant effect on Stop

100 µL 100 µL 100 µL
the procedure. Solution
Shake the microplate gently.
The Controls are ready to use. Read the absorbance (E) at 450 nm against a reference
wavelength of 620-630 nm or against Blank within 5
9.4. Procedure minutes.
• Allow all reagents to reach room temperature (22-
28°C) for at least 30 minutes. At the end of the assay
store immediately the reagents at 2-8°C: avoid long 10. QUALITY CONTROL
exposure to room temperature. Good Laboratory Practice (GLP) requires the use of quality
• Unused coated microwell strips should be released control specimens in each series of assays in order to check
securely in the foil pouch containing desiccant and stored the performance of the assay. Controls should be treated as
at 2-8°C. unknown samples, and the results analysed with appropriate
• To avoid potential microbial and/or chemical statistical methods.
contamination, unused reagents should never be
transferred into the original vials. The kit controls provided in the kit should be tested as
• As it is necessary to perform the determination in unknowns and are intended to assist in assessing the validity
duplicate in order to improve accuracy of the test results, of results obtained with each assay plate.
prepare two wells for each point of the calibration curve
(C1), two for each Control, two for each sample, one for The mean concentration of each control level is documented
Blank. in the QC report included with each kit. These mean
concentration levels are determined over several assays
which are run in duplicate in multiple locations across each
plate.

DiaMetra recommends the users to maintain graphic records

of the control values generated with each assay run,
including the running means, SDs and %CVs. This
information will facilitate the controls trending analysis
relating to the performance of current and historical control
lots relative to the supplied Quality Control data. The
trending will assist in the identification of assays which give
control values significantly different from their average
range.
 DKO099
When interpreting control data, users should note that this Total
product was designed and developed as a manual product. Positive Negative
The range stated on the QC certificate should be appropriate Positive 53 0 53
for assays that are performed manually and with strict True
adherence to the Assay Procedure described above. It is state Negative 0 50 50
recognised by Quality Control professionals, that as a result
Total 53 50 103
of differences in conditions and practices, there will always
be variability in the mean values and precision of control
measurements between different laboratories7. Diagnostic sensitivity: 100%

Diagnostic specificity: 100%

11. CALCULATION OF RESULTS
Determine the mean absorbance for each duplicate sample.
14.2. Precision
To obtain sample value: divide the mean absorbance of the Precision of the ANA Screen kit was determined by
sample by the absorbance of the Calibrator, then multiply by performing a complex precision study.
the concentration of the Calibrator, as shown below:
Repeatability: A total of A total of 3 serum samples were
𝐷𝐷𝐷𝐷 𝑚𝑚𝑚𝑚𝑚𝑚𝑆𝑆𝑆𝑆𝑚𝑚 assayed using 2 lots of reagents in 10 replicates per sample,
𝑆𝑆𝑚𝑚𝑚𝑚𝑆𝑆𝑆𝑆𝑚𝑚 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶. = × 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶. 𝐶𝐶1 by 3 operators, once a day for 5 days. Data from one
𝐷𝐷𝐷𝐷 𝐶𝐶1 representative lot is shown below:
Reactivity is not connected in a linear way to the amount of Repeatability results from one representative lot:
antibodies present. Although an increase or a decrease in
+’ve -‘ve % +’ve % -‘ve
the concentration of antibodies results in increased or Sample n
results results results results
decreased responsiveness, the change is not in proportion
(e.g., doubling the concentration of antibodies does not lead Cut off 150 82 68 54.7 45.3
to a doubling of responsiveness).
Negative 150 1 149 0.7 99.3
To achieve greater accuracy in the determination of
antibodies it is recommended to test serial dilutions of the Positive 150 150 0 100.0 0.0
sample. The final dilution that is positive in the test is the
antibody concentration of the patient.
Reproducibility results for the combined data from two lots is
shown below:
12. METROLOGY AND TRACEABILITY +’ve -‘ve % +’ve % -‘ve
Sample n
The ANA Screen assay is traceable to the Antinuclear results results results results
Antibodies (ANA) Reference Standards by Autoantibody Cut off 300 163 137 54.3 45.7
Standardisation Committee (ASC) - Center for Disease
Control (CDC) and WHO anti-dsDNA 15/174.
Negative 300 3 297 1.0 99.0

Positive 300 300 0 100.0 0.0

13. INTERPRETATION OF RESULTS

ANA Screen Interpretation 14.3. Serum-plasma study

(AU/ mL) The ANA Screen matrix comparison study was performed to
The sample should be considered evaluate the difference across tube types (serum separator
< 25
negative tubes (SST), lithium heparin plasma, sodium heparin plasma
The sample should be considered and K2 EDTA plasma) versus the control samples (red top
≥ 25
positive serum, without additive) following CLSI GP34-A “Validation
and Verification of Tubes for Venous and Capillary Blood.
The above index and interpretation should be considered as Specimen Collection”. A total of 20 samples (16 native, 4
guidelines only. Positive results should be verified contrived) were evaluated.
concerning the entire clinical status of the patient. Also,
every decision for therapy should be taken on an individual Results vs Serum
patient basis. No.
Sample Type False False
It is recommended that each laboratory establishes its own Samples
normal and pathological ranges of serum anti-ANA. pos neg
SST 20 0 0

14. PERFORMANCE CHARACTERISTICS K2 EDTA 20 0 0

Representative performance data are shown. Results Lithium heparin 20 0 0
obtained at individual laboratories may vary.
Sodium heparin 20 0 0
14.1. Diagnostic Sensitivity and Specificity
The sensitivity and specificity were determined with
guidance from CLSI EP-24 “Assessment of the Diagnostic
Accuracy of Laboratory Tests Using Receiver Operating
Characteristic Curves” using 50 negative and 53 positive
samples run on a single reagent lot.
14.4. Interferents 19. PRODUCT COMPLAINTS AND TECHNICAL
The following substances do not interfere in the ANA Screen SUPPORT
assay when the concentrations presented in the following For a patient/user/third party in the European Union and in
table are below the stated threshold. countries with similar regulatory regime (Regulation
2017/746/EU on IVD Medical Devices); if, during the use of
Potentially Interfering Threshold this device or as a result of its use, a serious incident has
Reagent Concentration occurred, please report it to the manufacturer and/or its
Bilirubin, conjugated 15 mg/dL authorised representative and to your national regulatory
authority.
Bilirubin, unconjugated 15 mg/dL The manufacturer can be contacted through their customer
service or technical support team. The contact details can be
Haemoglobin 200 mg/dL found below and on the company website:
www.diametra.com.
Total Protein 10 g/dL

Triglyceride 500 mg/dL

Ed. 10/2021 DCM099-7

Legal Manufacturer
15. LIMITATIONS OF USE Dia.Metra Srl
- Heterophilic antibodies in human serum can react Via Pozzuolo 14
with reagent immunoglobulins, interfering with in vitro 06038 SPELLO (PG) Italy
immunoassays8. Patients routinely exposed to Tel. +39-0742-24851
animals or to animal serum products can be prone to Fax +39-0742-316197
this interference and anomalous values may be E-mail: [email protected]
observed.

16. WASTE MANAGEMENT

Reagents must be disposed of in accordance with
local regulations.

17. BIBLIOGRAPHY
1. Pisetsky DS. Antinuclear antibody testing -
misunderstood or misbegotten? Nat Rev Rheumatol.
2017 Aug;13(8):495-502.
2. Pisetsky DS. Antinuclear antibodies in rheumatic
disease: a proposal for a function-based
classification. Scand J Immunol. 2012
Sep;76(3):223-8.
3. Fritzler MJ. Clinical relevance of autoantibodies in
systemic rheumatic diseases. Mol Biol Rep.
1996;23(3-4):133-45.
4. von Mühlen CA, Tan EM. Autoantibodies in the
diagnosis of systemic rheumatic diseases. Semin
Arthritis Rheum. 1995 Apr;24(5):323-58.
5. Defendenti C, Atzeni F, Spina MF, Grosso S, Cereda
A, Guercilena G, Bollani S, Saibeni S, Puttini PS.
Clinical and laboratory aspects of Ro/SSA-52
autoantibodies. Autoimmun Rev. 2011
Jan;10(3):150-4.

6. Aggarwal A. Role of autoantibody testing. Best Pract

Res Clin Rheumatol. 2014 Dec;28(6):907-20.
7. Basic QC Practices On-line Course;
http://www.Westgard.com.
8. Boscato, LM. and Stuart, MC., ‘Heterophilic
antibodies: a problem for all immunoassays’. Clin
Chem, 34, 1988, pp 27–33

18. REVISION IDENTIFIER

Additions or changes to the IFU are indicated by grey
highlighting.