Instructions for use

Ref. IMG-289

Manufactured by:
HEALTH IN CODE, S.L.
Calle de la Travesía s/n, 15E Base 5, Valencia 46024, España
+34 963 212 340 - [email protected]
healthincode.com

Code: HIC-PT-KIT 03-F-03 V.01

 Health in Code S.L. guarantees that all its products are free of defects, in both materials
used and its manufacturing process. This warranty is extended until the expiration
date, if the storage conditions specified in this manual are met.

Our products are designed for in vitro diagnostic use. Health in Code S.L. does not
offer any other warranty, express or implied, which extend beyond the proper
functioning of the components of this kit. Health in Code S.L. only obligation in respect
to the previous guarantees, will be to replace the product or return the purchase price,
when desired by the customer, if the existence of a defect in the materials or in the
manufacturing of its products is proven.

Health in Code S.L. is not liable for any damage, direct or indirect, resulting in economic
loss or resulting from the use of this product by the purchaser or user.

All products sold by the Health in Code S.L. are subjected to rigorous quality control. The
has passed all internal validation tests, ensuring the reliability and
reproducibility of each assay.

If you have any questions about the use of this product or its protocols, please contact
our Technical Department:

* Imegen® is a registered trademark in Spain of the Health in Code group

Instructions for Use (IFU) modifications

Modification of the storage and shipping temperature of the GENERAL MASTER
Version 07 DEC 2022
MIX reagent (Section 5).
Change in manufacturer’s address: Health in Code S.L., Calle de la Travesía s/n,
Version 06 NOV 2022
15E Base 5, Valencia 46024, Spain.
Version 05 SEP 2022 Change in manufacturer’s identification: from Imegen to Health in Code S.L.
Version 04 NOV 2019 Section 2. Intended Use.
Version 03 OCT 2018 Update by certified CE/IVD

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 index
General information 4
Intended use 5
Technical characteristics 6
Safety warnings and precautions 7
Content and storage conditions of the kit 8
Equipment, reagents, and material not included in the kit 9
Assay protocol 10
07.1 | PCR reactions preparation 10

07.2 | Setup of the real-time PCR programme 10

Analysis of results 12
Troubleshooting 14
Limitations 15
10.2 | Equipment 15
10.3 | Reagents 15
10.5 | Product stability 15

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 General information
The major histocompatibility complex (MHC) region, located on 6p21, contains hundreds of
human leukocyte antigen (HLA) genes that encode glycoproteins that present exogenous
and/or endogenous peptides to the immune cells stimulating apoptosis when the peptide
is recognized as foreign.

Genes in this complex are categorized into three basic groups: Class I, Class II, and Class
III. In humans, the HLA-B gene and two related genes, HLA-A and HLA-C, are the main
genes in MHC class I.

The HLA-B gene has many possible variations, allowing each person's immune system to
react to a wide range of foreign invaders. Hundreds of versions (alleles) of the HLA-B gene
are known, each of which is given a particular number (such as HLA-B*27). Additionally,
over 190 subtypes have been described for the HLA-B*27 allele.

The HLA-B*27 allele is related to autoimmune inflammatory diseases of the spine, rachis
and sacroiliac joints known as spondyloarthropathies, such as Ankylosing Spondylitis (AS).

Additionally, the presence of the HLA-B*27 allele is also associated with low viral load
and long-term non-progression in HIV infection as well as spontaneous clearance of
hepatitis C virus (HCV) infection.

References

› Chen B, Li J, He C, Li D, Tong W, Zou Y, et al. Role of HLA-B27 in the pathogenesis of ankylosing

spondylitis. Mol Med Rep. 2017;15: 1943–1951.

› Dashti N, Mahmoudi M, Aslani S, Jamshidi A. HLA-B*27 subtypes and their implications in the
pathogenesis of ankylosing spondylitis. Gene. 2018;670: 15–21.

› Neumann-Haefelin C. HLA-B27-mediated protection in HIV and hepatitis C virus infection and

pathogenesis in spondyloarthritis. Curr Opin Rheumatol. 2013;25: 426–433.

› Robinson J, Halliwell JA, Hayhurst JH, Flicek P, Parham P, Marsh SGE The IPD and IMGT/HLA database:
allele variant databases Nucleic Acids Research (2015) 43:D423-431

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 Intended use
consists of a qualitative real-time PCR assay aimed at detecting the
presence of the HLA-B*27 allele. This kit serves as a screening method for purposes
related to autoimmune inflammatory diseases of the spine, rachis and sacroiliac joints
(spondyloarthropathies).

IMPORTANT: The purpose of kit is not the tissue typing for

histocompatibility test prior to transplantation.

This assay employs a combination of oligonucleotides and fluorescent hydrolysis

probes in a diagnostic test directed to amplify and detect the presence of the HLA-B*27
allele and the endogenous gene, β-globin, used as a positive internal control of the DNA
sample. The results obtained from this assay will confirm the diagnosis of the patient.
The molecular design is directed to target polymorphisms on exon 2 of HLA-B gene,
offering greater sensitivity and specificity.

The assay studies the genotype in the germline, thus the optimal type of sample required
for this analysis is genomic DNA (gDNA).

kit has been designed for in vitro diagnostics and it is directed to

professionals from the molecular biology sector.

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 Technical characteristics
This kit has been validated using diagnostic reference material obtained from Coriell
Institute for Medical Research. Additionally, diagnostic samples were kindly provided by
C.H.U. Insular Materno-Infantil (Las Palmas de Gran Canaria, Spain) and internal material
from the Medical Genetic Unit of Health in Code S.L. was included in the verification and
validation assays, as well as certified synthetic vectors (GenScript) containing the sequences
of interest. This vector is provided as a positive control to ensure the correct functioning of
the PCR system. The thorough validation provides a robust and specific diagnostic method.
The type of sample required for this analysis is genomic DNA extracted from peripheral
blood or saliva, a total quantity of 50 ng will be necessary.
Health in Code S.L. is certified under UNE-EN ISO 13485:2018 Medical Devices: Quality
Management Systems - Requirements for regulatory purposes standard by the SPANISH
AGENCY OF MEDICINES AND MEDICAL DEVICES (AEMPS) for the Design, development, and
production of medical devices for in vitro diagnostic use:
Genetic testing kits
Software for the bioinformatics analysis of genetic data

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 Safety warnings
and precautions
Strictly follow the instructions of this manual, especially regarding the handling
and storage conditions.

Do not pipette by mouth.

Do not smoke, drink, or eat in areas where specimens or kit reagents are being handled.

You must properly protect any skin condition, as well as cuts, abrasions, and other
skin lesions.

Avoid discharge of reagents waste to the sink drinking water. Use waste containers
established by the legislation and manage their treatment through an authorized
waste manager.

In case of an accidental release of any of the reagents, avoid contact with skin, eyes
and mucous membranes and clean with abundant water.

The materials safety data sheets (MSDS) of all hazardous components contained in this
kit are available on request to Health in Code S.L.

This product requires the handling of samples and materials of human and animal origin.
You should consider all human and animal source materials as potentially infectious and
handled in accordance with OSHA Biosafety Level 2 of blood borne pathogens or must
use other relevant biosafety practices for materials containing or suspect that they may
contain infectious agents.

Reagents included in this kit are non-toxic, neither explosive, infectious, radioactive,
magnetic, corrosive nor environmental polluters.

This kit has been validated with specific equipment under certain conditions, which
could be different in other laboratories. It is recommended that each laboratory
performs an internal validation when the kit is used for the first time.

The manufacturer is not responsible for the malfunction of the assay when one or
more reagents included in the kit are replaced by other reagents not supplied by
Health in Code S.L.

The manufacturer does not guarantee the reproducibility of the assay when the user
employs reagents not validated by Health in Code S.L., considering them equivalent
to those provided in the Kit.

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 Content and storage
conditions of the kit
The kit includes the following reagents, enough to analyze 48 reactions:

HLA-B27 Master Mix: PCR Master Mix containing the primers to perform the amplification
of the target region and two probes labelled with FAMTM and VICTM fluorophores which
allow the differentiation between HLA-B*27 allele and β-Globin gene.
General Master Mix: PCR Master Mix containing MgCl2, nucleotides and Buffer solution,
required for the PCR reaction.
Positive control: Positive control for the HLA-B*27 target allele and the endogenous
gene, β-Globin.

Reagents Color indicator Quantity Conservation

HLA-B27 Master Mix Blue pad 2 x 180 µl -20ºC

General Master Mix White pad 600 µl -20ºC*

Positive Control Blue pad 1 x 100 µl -20ºC

Table 1. Components of the Imegen® HLA-B27 kit

(*) General Master Mix: Recommended to be kept frozen until first use, protected from light, and stored
between 2- 8 ºC after first use.

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 Equipment, reagents
and material not
included in the kit
Equipment:

Real-time PCR thermocycler

Micropipette (10 µL, 20 µL and 200 µL)
Vortex
Centrifuge

Reagents:

Nuclease-free water

Materials:

Optical PCR tubes 0.2 mL

Optical lids for the PCR tubes
Filter tips (10 µL, 20 µL and 200 µL)
Sterile tubes 1.5 mL
Dust-free gloves

Complementary kits
For sensitive and specific detection of other HLA alleles with different clinical targets,
Health in Code S.L. has developed (ref IMG-306) and
(ref IMG-307).

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 Assay protocol

07.1 | PCR reactions preparation

The recommended protocol for preparation of amplification reactions is showed below:

01 Thaw the HLA-B27 Master Mix, the Positive Control and the genomic DNA samples.
02 Vortex and spin each reagent to mix thoroughly and keep on ice.
03 Add into a 1.5 mL tube, the following reagents:

Reagent Volume per reaction

HLA-B27 Master Mix 7.5 µL

General Master Mix 12.5 µL

NOTE: In order to estimate the amount of necessary reagents, we recommend making calculations
taking into account the number of samples to be included in the assay, and add additionally 10% of
the volume of each reagent.

04 Vortex the PCR Master Mix tube and dispense 20 µL in the corresponding wells.
05 Once the master mixes have been dispensed, add the following into the
corresponding wells:
5 µL of the genomic DNA sample (10 ng/µL).
5 µL of the positive control (included).
5 µL of nuclease-free water (no template control, NTC).

NOTE: It is recommended to add a PCR negative control (NTC) in order to rule out PCR contamination,
and also positive controls to ensure the correct set up and functioning of the PCR reaction.

06 Place the samples in the Real Time PCR thermal cycler and set the PCR programme
according to the following section.

07.2 | Setup of the real-time PCR programme

The following instructions must be followed in order to setup the amplification programme:

Recommended experiment for 7500 FAST or StepOne: Quantitation-Standard Curve

Reference ROXTM for 7500 FAST or StepOne: included
Ramp rate: Standard

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 Reaction volume: 25 µL
TaqMan® probes fluorophores:

Probe Fluorophore Target Quencher

HLA-B27-P FAM TM
HLA-B*27 allele MGB
β-Globin-P VICTM β-Globin MGB
Table 2. Hydrolysis probe information

Optimal real-time PCR programme:

Phase 1 Phase 2
Fields
Enzymatic activation PCR
40 cycles
No. of cycles 1 initial cycle
Denaturation Annealing & Extension
Temperature 95ºC 95ºC 60ºC
Time 10 minutes 15 seconds 1 minute*
Table 3. Optimal PCR programme for the 7500 FAST, StepOne (Thermo Scientific) or CFX96 (BioRad)

(*) Fluorescence detection

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 Analysis of results
For the correct interpretation of the results, the following recommendations are given:

Confirm that there is not amplification in the no template control. If amplification

signal is detected, the analysis should be repeated to rule out accidental
contamination.
Confirm that the positive controls produced an amplification signal in both the FAM
and VIC channels.
Ensure that every sample, positive or negative, shows a positive signal in VIC
proceeding from the amplification of the endogenous gene, β-globin.
To analyze the samples, the specific software of the Real-Time PCR thermal cycler
has to be used to analyze the samples in this type of analysis.

In the following represent expected results obtained with the kit:

Figure 1. Fluorescent signal resulting from the correct amplification of the positive control. Amplification in FAM (blue)
and VIC (pink) channel.

Figure 2. Fluorescent signal resulting from the correct amplification of a DNA samples lacking the HLA-B27 allele.
Amplification is detected in VIC channel (pink) from β-globin gene.

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 Figure 3. Fluorescent signal resulting from the correct amplification of the NTC. There is no amplification signal.

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 Troubleshooting
The table below represents the results that could be obtained using the positive
and negative controls and the samples. In case an unexpected result is obtained, the
interpretation of the result and the cause most likely reason for such result is given
in the table below.

HLA-B27 β-Globin
Control Result / Interpretation
(FAM) (VIC)

+ + Expected result

- -
Positive control
+ - Fail in the PCR setup 1

- +
+ +
Expected result
- +
Sample
- - Fail to amplify the sample 2

+ - Fail in the PCR setup 1

- - Expected result
Negative control (NTC) Contamination with human DNA or
+ + with the positive control 3

Table 4. Interpretation of the possible results obtained using Imegen® HLA-B27

(1) Fail in the PCR setup: An error in the amplification might be due to a technical issue during the configuration
of the PCR setup. Check the amplification program and the setup of the fluorescence detection.

(2) Fail to amplify the sample: An error to amplify might suggest the quantity or the quality of the sample is
compromised. In this situation, a second analysis or new extraction of DNA would be recommended before
an interpretation of the results is made.

(3) Contamination with human DNA or with the positive control: PCR contamination might be caused by an
inappropriate handling of the sample, the use of contaminated reagents or caused by an environmental
contamination. To solve this issue, a thorough cleanse of the laboratory where the PCRs are prepared, including
the equipment and material used is recommended. If necessary, use fresh aliquots of the PCR reagents and
prepare last, the PCR reactions containing the positive controls in order to avoid any cross contamination.

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 Limitations
10.1 | Equipment

has been validated using the following real-time PCR systems:

StepOnePlus™ Real-Time PCR System (ThermoFisher Scientific)

7500 FAST Real-Time PCR System (ThermoFisher Scientific)
CFX96 Real-Time PCR System (BioRad)

Technically, this kit is compatible with any real-time PCR systems that enable the
detection of the fluorescence emitted by FAMTM and VICTM fluorophores.

If a real-time PCR cycler different from the systems described in this section is going to
be used, it is possible that the PCR programme might need to be readjusted. In this case,
please contact our Technical Support team for more details.

10.2 | Reagents

has been validated using the reagents included in the kit and the
ones recommended in the section 6 of this manual (Equipment and materials not
included in the kit).

10.3 | Product stability

The optimal analytical functioning of this product is confirmed if the recommended storage
conditions are applied as specified on Section 5 (Contents and storage conditions of the kit)
from the reception of the kit until the expiry date assigned to each production batch.

IMG-289 V.07 REVISION DATE 02.12.2022 HIC-PT-KIT 03-F-03 V.01 PAGE 15 OF 16

 For any questions about the applications
of this product or its protocols, please
contact our Technical Department:

Discover all our

diagnostic kits

Manufactured by:
HEALTH IN CODE, S.L.
Calle de la Travesía s/n, 15E Base 5, Valencia 46024, España
+34 963 212 340 - [email protected]
healthincode.com

Code: HIC-PT-KIT 03-F-03 V.01