DESIGNATION REF.

CLONE PACKAGING
ANTI-Cw (RH8) 78013 MS110 1 x 3 mL
ANTI-Fyb (FY2) 78015 SPA264LBG1 1 x 3 mL
ANTI-Jka (JK1) 78003 P3HT7 1 x 3 mL
ANTI-Jkb (JK2) 78004 P3 143 1 x 3 mL
ANTI-Lea (LE1) 78009 13643B9 1 x 3 mL
ANTI-Leb (LE2) 78010 GX336 1 x 3 mL
ANTI-M (MNS1) 78005 2514E6 1 x 3 mL
ANTI-N (MNS2) 78006 1422C7 1 x 3 mL
ANTI-P1 78011 650 1 x 3 mL

INTRODUCTION
These reagents are in vitro diagnostic medical devices (IVDMD) for professional use. They are intended for the
analysis of human biological specimens. They are used in the phenotyping of human red blood cells. The reagents
are designed to determine the presence of antigens Cw (RH8), Fyb (FY2), Jka (JK1), Jkb (JK2), Lea (LE1), Leb
(LE2), M (MNS1), N (MNS2) and P1 on the surface of red blood cells.

PRINCIPLE
The manual tube method used is based on the principle of haemagglutination. Red blood cells bearing an antigen
agglutinate in the presence of the reagent containing the corresponding antibody:
- either using the direct haemagglutination method,
- or using papain.
Papain, a proteolytic enzyme derived from the papaya (Carica papaya) induces a marked decrease in the
electronegative charge on the surface of red blood cells, enabling their agglutination by normally 'non-agglutinating'
antibodies in saline medium.

COMPOSITION
The reagents are produced by DIAGAST from red cell monoclonal antibodies and presented in a storage medium.
The monoclonal antibodies ANTI-Cw, ANTI-Fyb, ANTI-Jka and ANTI-Jkb of IGM type, are derived from the in vitro
culture supernatant of hybridomas of human origin.
The monoclonal antibodies ANTI-Lea, ANTI-Leb and ANTI-P1 of IGM type, are derived from the in vitro culture
supernatant of hybridomas of murine origin.
The monoclonal antibodies ANTI-M and ANTI-N of IgG type are derived from the in vitro culture supernatant of
hybridomas of murine origin.
All the reagents contain sodium azide (< 0.1%). All the reagents except ANTI-Cw (RH8), ANTI-P1 and ANTI-Fyb
(FY2) contain sodium arsenite (0.02 %). All the reagents except ANTI-Jkb (JK2) contain bovine albumin.
ANTI-Fyb (FY2) contains Proclin at 0.05%.
The reagents are packaged in 3-mL vials fitted with calibrated droppers.

PRECAUTIONS
WARNING (Only for ANTI-Fyb (FY2) containing Proclin at 0.05%)
Particulars of danger :
H317 May cause an allergic skin reaction.
Advice of caution :
P261 Avoid breathing dust/fume/gas/mist/vapours/spray.
P280 Wear protective gloves/protective clothing/eye protection/face protection.
P333+P313 If skin irritation or rash occurs: Get medical advice/attention.
P362+P364 Take-off contaminated clothing and wash it before reuse
P501 Dispose of contents/container in accordance with the local regulations, regional, national and international.

STORAGE
All the reagents except ANTI-Lea (LE1) antibody are to be stored between +2ºC and +8ºC.
ANTI-Lea (LE1) antibody is to be stored between –18ºC and –30ºC. Do not refreeze after thawing. After thawing,
ANTI-Lea (LE1) can be stored between +2ºC and +8ºC for up to 6 months.
The performance of these reagents is guaranteed for the recommended methods from first use to the expiry date
indicated on the label. The reagents must not be used after the expiry date. It is advisable to minimize their time left
outside the refrigerator and to avoid leaving them at room temperature between uses.

REAGENTS AND MATERIALS NECESSARY

- Plastic and glass test tubes, tube rack.
- Automatic pipettes with adjustable precision.
- Centrifuge of relative force from 100 to 1200 g.
- Isotonic saline solution (0.9 % NaCl).
- PALERM (DIAGAST ref.: see catalogue).

ENGLISH DIA03809
- Positive control blood samples of guaranteed phenotype are required carrying the corresponding heterozygous
antigen (except for the Lewis system) and similarly for a negative control, blood samples should be used which
lack the antigen corresponding to the reagent used.
- Negative control: NEG CONTROL (DIAGAST ref.: see catalogue).

SAMPLES – CONTROLS
TEST BLOOD SAMPLE
The blood sample, collected in EDTA, heparin or citrate anticoagulant, in a stoppered sterile tube and stored
between +2ºC and +8ºC, should be tested within 72 hours, providing that no haemolysis is visible.
At the time of testing, centrifuge the blood sample at 1200 g for 3 minutes.

BLOOD SAMPLES OF GUARANTEED PHENOTYPE

The use of these control samples ensures the detection of anomalies with (handling, reagents, apparatus and the
environment) and enables the user to implement any corrective actions.
The analytical system must be validated using control samples of guaranteed phenotype :
- sample expressing the antigen corresponding to the reagent antibody used,
- sample lacking the antigen corresponding to the reagent antibody used.
For each blood sample to be tested, a reagent control is performed alongside using the same conditions but
replacing the reagent with NEG CONTROL.

PROCEDURE FOR ANTI-Cw (RH8), ANTI-Fyb (FY2), ANTI-Jka (JK1), ANTI-Jkb (JK2),
ANTI-M (MNS1) AND ANTI-N (MNS2)
- In a plastic tube, prepare a 5 % unwashed red blood cell suspension in isotonic saline solution.
- Using the vial dropper, transfer 1 drop of reagent to a glass tube.
- Add 50 µL of erythrocyte suspension.
- Shake to mix, then centrifuge at 500 g for 1 minute.
- Gently swirl the tube to detach the erythrocyte pellet, observe macroscopically to detect the appearance of any
agglutinates.
- Read and record the reaction immediately.

PROCEDURE FOR ANTI-Lea (LE1)

- In a plastic tube, prepare a 5 % unwashed red blood cell suspension in isotonic saline solution.
- Using the vial dropper, transfer 1 drop of reagent to a glass tube.
- Add 50 µL of erythrocyte suspension.
- Using the vial dropper, transfer 1 drop of PALERM.
- Incubate the tubes between +18ºC and +25ºC for 5 minutes.
- Shake to mix, then centrifuge at 500 g for 1 minute.
- Gently swirl the tube to detach the erythrocyte pellet, observe macroscopically to detect the appearance of any
agglutinates.
- Read and record the reaction immediately.

PROCEDURE FOR ANTI-Leb (LE2)

- In a plastic tube, prepare a 5 % unwashed red blood cell suspension in isotonic saline solution.
- Using the vial dropper, transfer 1 drop of reagent to a glass tube.
- Add 50 µL of erythrocyte suspension.
- Incubate the tubes between +18ºC and +25ºC for 15 minutes.
- Shake to mix, then centrifuge at 500 g for 1 minute.
- Gently swirl the tube to detach the erythrocyte pellet, observe macroscopically to detect the appearance of any
agglutinates.
- Read and record the reaction immediately.

PROCEDURE FOR ANTI-P1

- In a plastic tube, prepare a 5 % unwashed red blood cell suspension in isotonic saline solution.
- Using the vial dropper, transfer 1 drop of reagent to a glass tube.
- Add 50 µL of erythrocyte suspension.
- Shake to mix, then centrifuge at 120 g for 1 minute.
- Gently swirl the tube to detach the erythrocyte pellet, observe macroscopically to detect the appearance of any
agglutinates.
- Read and record the reaction immediately.
- If no agglutination is visible, incubate the tubes between +2ºC and +8ºC for 30 minutes.
- Shake to mix, then centrifuge at 120 g for 1 minute.
- Gently swirl the tube to detach the erythrocyte pellet, observe macroscopically to detect the appearance of any
agglutinates.
- Read and record the reaction immediately.

ENGLISH DIA03809
INTERPRETATION
If agglutination is present (the red blood cells form one or several clump(s)), the reaction is positive and the antigen
corresponding to the reagent used is present on the test red blood cells.
If there is no agglutination (the red blood cells go back into homogeneous suspension), the reaction is negative and
the antigen is absent from the test red blood cells.
The reaction can only be interpreted if :
- the result of the NEG CONTROL with the subject's red blood cells is negative,
- the analytical system has been validated with samples of guaranteed phenotype.

LIMITATIONS OF THE METHOD

Only suitably qualified personnel should use the devices.
It is advisable to gently shake the reaction tubes with ANTI-Jka (JK1), because the agglutinations created by
ANTI-Jka (JK1) are more brittle.
Temperatures > 25 ºC can disrupt results obtained with ANTI-M (MNS1) and/or ANTI-N (MNS2). It is therefore
advisable to take these reagents out of the refrigerator just before use and to read the reactions immediately after
centrifugation.
It is imperative to use the calibrated dropper of the IVDMD vial to transfer 1 drop of reagent.
Only use the complementary reagents cited in the section entitled 'Reagents and materials necessary'.
The reactions are to be read immediately after centrifuging and resuspending.
It is imperative to work with clean apparatus and uncontaminated products (bacterial or other contamination).
Strict compliance with the following is required:
- storage conditions and expiry date,
- the procedure indicated above,
- equipment calibration is recommended.
No reagent can guarantee the detection of all the antigenic profiles rare, weak or variants.

PERFORMANCE DATA
A performance assessment of the reagents was conducted on a random samples panel of known common
phenotypes including clinical and neonatal samples. The samples were drawn in the recommended anticoagulants
(EDTA, heparin, citrate). The expert assessment demonstrated 100 % specificity for each of the reagents with
respect to the expected results.
ANTI-M (MNS1) can recognize unspecifically some HENSHAW red blood cells (M , He  is an extremely rare
phenotype).
On 25 blood samples from blood donors having a FYX phenotype, the ANTI-Fyb (FY2) PK has detected 100% of
these red blood cells.
Performances of devices are guaranteed for the method when used in combination with reagents recommended in
this leaflet (for instance : PALERM Papain).
Use and validation of other reagents used in combination with the devices, other than those indicated in paragraph
titled 'Reagents and materials necessary' is possible but only on the user’ s responsibility.

DIAGAST denies all responsibility in cases where the devices are not used in accordance with this leaflet.

ENGLISH DIA03809
REVISION HISTORY
Impact on the Verification of method
Description of the change according to standard NF EN ISO
15189
§ PRECAUTIONS : Updating of risk phrases according CLP regulation
N°1272/2008 No
§ PERFORMANCE DATA : Correction HENSCHAW by HENSHAW

The identifier, '0459', of the notified organization (LNE/G-MED) which validated the conformity
evaluation procedure, cited in appendix IV of Directive 98/79/EC, is only applicable to the ANTI-Jka
(JK1), ANTI-Jkb (JK2) and ANTI-Fyb (FY2) antibodies. The ANTI-Jka (JK1), ANTI-Jkb (JK2) and
0459
ANTI-Fyb (FY2) antibodies are included in list B of appendix II to that directive.

DIAGAST 251, AV. AVINEE - 59120 LOOS – FRANCE

www.diagast.com
key-code : DIA03809

Revision date : March 2022

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ENGLISH DIA03809