BGA00010 BGB00010 BGAB0010 BGD00010 BGABD030

For In Vitro Diagnostics Use Only

Lot Number
1 x 10ml 1 x 10ml 1 x 10ml 1 x 10ml 3 x 10ml
Catalogue Number
BGA01010 BGB01010 BGAB1010 BGD01010 BGABD040 Storage Temperature

MONOCLONAL BLOOD 10 x 10ml 10 x 10ml 10 x 10ml 10 x 10ml 4 x 10ml

Expiry Date (Year / Month)

GROUPING REAGENTS INSTRUCTIONS FOR USE

Warning, Read Enclosed Documents
Instructions For Use
Manufactured By
FOR IN-VITRO DIAGNOSTIC USE ONLY / STORE AT 2-8°C

 Samples collected into EDTA or Heparin should be tested within 48 Quality Control:
MONOCLONAL GROUPING hours, clotted samples within 14 days and those drawn into ACD,CPD
or CPDA-1 up to their expiry dates or within 3 weeks of
It is recommended that appropriate antigen-positive and antigen-negative
cells be tested with the reagents on each day of use in order to confirm the
(ANTI-A, ANTI-B AND ANTI-A,B) withdrawal. reactivity and specificity of the blood grouping reagents.
Tube, Slide and Microplate Tests Test Procedure: Reverse grouping should be carried out on individuals who are older than 6
Principle: The test is based on the principle of agglutination. Red cells Bring reagents and samples to room temperature (18-25oC) before testing. months of age.
with the antigen will agglutinate when tested against the Tube Technique: Reactions:
corresponding antibody. 1. Prepare a 2-3% red cell suspension using isotonic buffered saline with a ABO
Presentation: pH of 6.9. Anti-A Anti-B Anti-AB A1 A2 B 0 Group
2. Place in a glass test tube 1 volume of ABO Grouping Reagent and 1 + - + - - + - A
Reagent Code Size volume of the Red Cell Suspension and mix well. - + + + + - - B
Anti-A Monoclonal BGA00010 1 x 10 ml 3. Centrifuge at 900 to 1000 rpm for 60 seconds or incubate at room + + + - - - - AB
temperature for 20-30min. - - - + + + - O
Anti-A Monoclonal BGA01010 10 x 10 ml 4. Gently resuspend the cell button and examine macroscopically for
If the results obtained with the serum do not correlate with the red cell test,
Anti-B Monoclonal BGB00010 1 x 10 ml signs of agglutination.
further investigation is required.
5. Record the results.
Anti-B Monoclonal BGB01010 10 x 10 ml Limitations of the procedure:
Microplate Technique:
False positive and negative results may occur due to contamination of test
Anti-A,B Monoclonal BGAB0010 1 x 10 ml 1. Prepare a 2 – 3 % Red Cell Suspension using isotonic buffered saline
materials, improper cell concentrations, incorrect centrifugation, incubation
Anti-A,B Monoclonal BGAB1010 10 x 10 ml with a pH of 6.9.
and temperature times.
2. Place in the appropriate well of a V-bottom microplate 1 volume (30 -
Grouping Kit (A, B, D) BGABD030 3 x 10 ml Any deviation from the test procedure could result in inaccurate results.
50l) of ABO Grouping Reagent and 1 volume of the 2 – 3 % (30 - 50l)
Weaker reactions may be observed with stored blood.
Grouping Kit (A, AB, B, D) BGABD040 4 x 10 ml Red Cell Suspension.
Weaker reactions may be observed with cord blood or neonatal red cells as
3. Mix well, preferably using a microplate shaker, taking care to avoid
ABO Antigens are not fully developed at birth.
Composition: Monoclonal ABO Grouping Reagents are prepared using in cross-well contamination.
Cord samples contaminated with Whartons Jelly may give false positive
4. Tilt the plate at an angle of 70° to the bench top and observe over the
vitro culture supernatants from hybridised immunoglobulin-secreting mouse results.
next three minutes for signs of streaming. Negative reactions allow the
cell lines. Each antibody is then diluted in a phosphate buffer which Blood samples of weak A or B subgroups may result in false negative or
contains sodium chloride, EDTA and bovine albumin resulting in a reagent cells to flow downwards in a uniform stream. Positive reactions remain
weak reactions. Extending the incubation times to 30 minutes might
as distinct buttons either on the bottom of the well or occasionally
which is optimised for use in slide, tube and microplate test methods. improve the reaction strength.
 Anti-A is coloured with acid blue dye. sliding down the side.
5. As an alternative to tilting the plate, the cell buttons can be
 Anti-B is coloured with acid yellow dye. Reference:
 Anti-AB is uncoloured. 6. resuspended using carefully controlled agitation on a
BCSH Blood Transfusion Task Force Guidelines for microplate techniques in
microplate shaker, then examined for agglutination either
Although all our components which have been derived from human origin liquid phase blood grouping. Clin. Lab. Haem, 1990: 12, 437 – 460
visually or by means of a validated automated reader.
have been tested and found to be negative for the presence of anti-HIV, Voak D., et al. (1982) Monoclonal anti-A and anti-B development as cost
Slide Technique:
anti-HCV as well as HbsAg, it is recommended that they be handled effective reagents. Med. Lab. Sci. 39, 109 – 122.
1. Prepare a 35 – 45% red cell suspension using either their own group or
cautiously and treated potentially infectious. Kholer G., Milstein C., (1975) Continuous culture of fused cells secreting ab
Storage: group compatible plasma or serum.
predefined specificity. Nature 256, 495 – 497
2. Onto a slide which is at room temperature (18 – 25°C) place 1 volume
 Store components at 2-8°C. Mollison P.L. Blood Transfusion in Clinical Medicine, 8 th Ed, Oxford: Blackwell
 Do not freeze or expose to elevated temperatures. of ABO Grouping Reagent and 1 volume of the 35 – 45% Red Cell
Scientific, 1987, Chapter 7.
Suspension.
 Do not use beyond the expiry date. Issitt P.D. Applied Blood Group Serology, 3rd ed. Miami: Montgomery
3. Using a clean applicator stick, mix the reagent and cell suspension
 Marked turbidity may indicate reagent contamination or Scientific, 198 Chapter 6
deterioration. over an area of approximately 20 x 40 mm.
Race RR., Sanger R., Blood Grps in Man 6 th Ed. Oxford, Blackwell Sci 1987:
Samples: 4. Slowly tilt the slide back and forth for no longer than two minutes and
Chapter 7.
observe for signs of agglutination.
 Blood samples which have been drawn with or without anti-coagulant
5. Record the results.
may be used.
Reaction Stability: Following centrifugation all tube and microplate tests
 Testing should be performed as soon as possible to avoid false
reactions occurring due to contamination or incorrect storage. should be read immediately and results interpreted without delay. Slide tests
should be interpreted within two minutes to avoid drying of the reagents
 Samples may be stored at between 2 and 8°C and tested within two
days provided there is no evidence of haemolysis. which might result in negative reactions being called positive.

Fortress Diagnostics Limited, Unit 2C Antrim Technology Park, Antrim, BT41 1QS (United Kingdom)
Tel: +44 (0) 2894 487676 | Fax: +44 (0) 2894 469933 | Website: www.fortressdiagnostics.com BGABD – MONOCLONAL GROUPING | Revision No. 13 S E P T /16 | Page 1 of 2

ANTI-D IgG/IgM
BLENDED MONOCLONAL Rho (D)
Tube, Slide and Microplate Tests
Principle:
The test is based on the principle of agglutination. Red cells which the
antigen will agglutinate when tested against the corresponding
antibody.
Presentation:
Reagent Code Size
Anti-D Blend BGD00010 1 x 10 ml
Anti-D Blend BGD01010 10 x 10 ml
Composition:
The Anti-D IgG / IgM Blend has been prepared from carefully blended
monoclonal IgM and IgG anti-D’s. Monoclonal Anti-D IgG/IgM
antibodies are derived from hybridoma cell lines, created by fusing
mouse antibody producing B lymphocytes with mouse myeloma cells
or are derived from a human B cell line through EBV transformation.
The IgG anti-D directly agglutinates D positive red cells, including the
majority of D variants, with the exception of DVI , and a high proportion
of weak D (Du) phenotypes. The IgG anti-D agglutinates DVI and low
grade weak D (Du) phenotypes by the indirect antiglobulin test
method. The antibodies are diluted in a phosphate buffer which
contains sodium chloride, bovine albumin and macromolecular
potentiators to give a reagent which is optimised for use in tube, slide
or microplate tests. Contains 0.1% sodium azide.
Although all our components which have been derived from human
origin have been tested and found to be negative for the presence of
anti-HIV, anti-HCV as well as HbsAg, it is recommended that they be
handled cautiously and treated potentially infectious.
Storage:
 Store components at 2-8°C.
 Do not freeze or expose to elevated temperatures.
 Do not use beyond the expiry date.
 Marked turbidity may indicate reagent contamination or
deterioration.
Samples:
 Blood samples which have been drawn with or without anti-
coagulant may be used.
 Testing should be performed as soon as possible to avoid false
reactions occurring due to contamination or incorrect storage.
 Samples may be stored at between 2 and 8°C and tested within
two days provided there is no evidence of haemolysis.
 Samples collected into EDTA or Heparin should be tested within
48 hours, clotted samples within 14 days and those drawn into
ACD, CPD or CPDA-1 up to their expiry dates or within 3 weeks of
withdrawal.
 Prolonged storage of red cells may result in weaker reactions.
 Bacterial contamination may cause false test results.
Fortress Diagnostics Limited, Unit 2C Antrim Technology Park, Antrim, BT41 1QS (United Kingdom)
Tel: +44 (0) 2894 487676 | Fax: +44 (0) 2894 469933 | Website: www.fortressdiagnostics.com
Test Procedure:
Bring reagents and samples to room temperature (18-25oC) before
testing.
Tube Technique:
1. Prepare a 2-3% red cell suspension using isotonic buffered saline
with a pH of 6.8 – 7.2.
2. Place in a glass test tube 1 volume of Anti-D Blend and 1 volume of
the Red Cell Suspension and mix well.
3. Centrifuge at 900 to 1000 rpm for 60 seconds or 3400rpm for 20
second or incubate at room temperature for 20-30min.
4. For apparently negative results which are to be tested for DVI and
other weak D phenotypes, proceed to step 3 of the indirect
antiglobulin test.
Microplate Technique:
1. Prepare a 2 – 3 % Red Cell Suspension using isotonic buffered
saline with a pH of 6.8 – 7.2.
2. Centrifuge the microplate at 140rcf for 1 minute.
3. Tilt the plate at an angle of 70° to the bench top and observe over
the next three minutes for signs of streaming. Negative reactions
allow the cells to flow downwards in a uniform stream. Positive
reactions remain as distinct buttons either on the bottom of the
well or occasionally sliding down the side.
4. For apparently negative results which are to be tested for DVI and
other weak D phenotypes, retest using the indirect antiglobulin test
procedure.
Note: If performing indirect antiglobulin tests by validated microplate
techniques, tests should be washed a minimum of 3 to 4 times due to
the small volume of saline which can be added to each microwell.
Slide Technique:
1. Prepare a 35 – 45% red cell suspension using either their own group
or group compatible plasma or serum.
2. Onto a slide which is at room temperature (18 – 25°C) place 1
volume of Anti-D Blend and 1 volume of the 35 – 45% Red Cell
Suspension.
3. Using a clean applicator stick, mix the reagent and cell suspension
over an area of approximately 20 x 40 mm.
4. Slowly tilt the slide back and forth for no longer than two minutes
and observe for signs of agglutination. Record the results.
5. For apparently negative results which are to be tested for D VI and
other weak D phenotypes, retest using the indirect antiglobulin test
procedure.
BGABD – MONOCLONAL GROUPING | Revision No.13 SEPT/16 | Page 2 of 2
Indirect Antiglobulin Test:
1. Prepare a 2-3% red cell suspension using isotonic buffered saline
with a pH of 6.8 – 7.2.
2. Place in a glass test tube 1 volume of Anti-D Blend and 1 volume
of the Red Cell Suspension.
3. Mix well and incubate at 37°C for 15 – 30 minutes.
4. After incubation wash the cells once in isotonic buffered saline,
decanting the saline completely.
5. Add two volumes of polyspecific anti-human globulin or anti-IgG
to the dry cell button and mix gently to resuspend the cells.
6. Centrifuge at 900 – 1000 rcf for 15 seconds.
7. Gently resuspend the cells and examine macroscopically for signs
of agglutination. Record the results.
8. Confirm validity of negative tests with IgG sensitised cells.
Reaction Stability:
Following centrifugation all tube and microplate tests should be read
immediately and results interpreted without delay. Slide tests should
be interpreted at the end of two minutes.
Quality Control:
 It is recommended that appropriate antigen-positive and
antigen-negative cells be tested with the reagents on each day
 of use in order to confirm the reactivity and specificity of the
blood grouping reagents. It is recommended that AB serum, 5%
BSA in PBS or autologgous serum be used in parallel with Anti-D
Blend reagent for all the cells tested as red cells which have been
coated in vivo with antibody (positive DAT) may show false
positive reactions when potentiators are used in red cell diluent
(e.g. albumin, ficoll, dextran). Even if cells are typed as negative
DAT, such potentiatiors can sometimes cause difficulties in
microplate techniques.
Limitations of the procedure:
 False positive and negative results may occur due to
contamination of test materials, improper cell concentrations,
incorrect centrifugation, incubation and temperature times.
 Any deviation from the test procedure could result in inaccurate
results.
 Stronger direct reactions with weak D phenotypes will be
observed in tube tests than in slide and microplate tests.
 Red cells showing a positive direct antiglobulin test cannot be
typed by the indirect antiglobulin test.
 Weaker reactions may be observed with stored blood.
Reference:
Kohler G., Milstein C. (1975) Continuous culture of fused cells secreting ab of
predefined specificity. Nature 256, 49497
Walker RH., ed. Technical Manual, 11th Ed. Bethesda, MD: American
Association of Blood Banks, 1993: Ch11
Issitt PD., Applied Blood Group Serology, 3rd Ed. Miami: Montgomery
Scientific, 1985. Ch. 10
Jones j., Scott ML., Voak D. Monoclonal anti-D specificity and Rh D structure:
criteria for selection of monoclonal anti=D reagents for routine typing of
patients and donors. Transfusion Medicine 1995: 5, 171 – 184.
Tippett P. Sub-Divisions of the Rh (D) antigen. Med. Lab. Sci. 1988. 45. 88-93.
Guidelines for compatibility testing in hospital blood banks. Cli. Lab. Haem.,
1987: 9:333-341