Figure 4: MosaiQ ™ Antigen Typing printing layout (a), typical assayed array image (b) and microscopic image of bound cells to a spot (c).

Evaluation of a Novel Serology-Based Platform for Simultaneous Blood Group Antibody Identification and Antigen Typing (Project MosaiQ™)Robb, J.S.1, Renault, N.K.1, Knowles, L.K.1, MacDonald, I.1 and Robson, D.C.1 (1Quotient, Edinburgh, UK)

Antibody Identification – Method & ResultsQuotient 10 cell panel reagent red blood cells, suspended in a proprietary print buffer, were printed onto a modified substrate. 16 grids per slide were then subdivided into wells using a super-structure. Slides were processed to yield stable cell monolayers.

Antigen Typing – Method & ResultsFormulated antibodies to blood group antigens A, B, C, c, D, E, e, and buffer were printed in arrays onto a modified substrate for this feasibility study. The 16 arrays per slide were subdivided into wells using a super-structure.

BackgroundMosaiQ™ is a new serology-based antibody-antigen microarray technology in development which has the capability to simultaneously combine blood group antigen typing, direct antiglobulin testing, reverse typing, irregular antibody screening and identification, in one step. Currently, automated systems run ABO/RhD typing (some run Rh/K also) and an initial antibody screening test. If the antibody screen is positive, an antibody identification investigation will subsequently be performed, often manually, before suitable blood can be selected for cross-matching. If required (e.g. first time donor) additional confirmatory or extended blood typing may also be performed. Identifying the antibody and performing extended antigen typing in the initial test would offer time and cost savings in the provision of blood for patients.

Aim: The technology has been developed to meet the sensitivity levels required for donor and patient testing, and of current state-of-the-art systems. This work reports an evaluation of the technology to perform blood group antigen typing and irregular blood group antibody identification on over 1000 samples.

ConclusionsThe study demonstrated that MosaiQ™ can be used for irregular blood group antibody identification and extended antigen typing simultaneously. The platform showed the required level of sensitivity against weak blood group antibodies and antigens, while demonstrating no unwanted positives. Development and optimisation work is ongoing to extend the range of blood typing specificities available on MosaiQ™. Adaptations of this format have also successfully demonstrated that direct antiglobulin testing, reverse ABO typing and virology assays can also be performed in parallel. The MosaiQ™ platform offers an efficient testing system which challenges current serologic and molecular systems.

QuotientPentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PL United Kingdomwww.quotientbd.com

1000 Random Donor (EDTA) Plasma Sample Study99.9% Correlation to Comparator Achieved (Table 1)

Table 1: 1000 samples were tested on MosaiQ™ and with comparator systems. Seven positives were identified initially on the comparator system. After retest four of them were confirmed to be positives, of which MosaiQ™ detected three of these (3 out of 4). Of the negatives, 996 were identified by comparator systems, while MosaiQ™ found 997 negative.Sample #0236 – Anti-K identified (11 cell panel) on CAT (0.5+ reaction). Not detected on MosaiQ™ although current

work is focused on optimising and automating the assay to enhance sensitivity. Sample #0313 – Anti-M identified (11 cell panel) on CAT (0.5+ to 2+ reaction). Detected by MosaiQ™ (IgM).Sample #0314 – Anti-Cw identified (11 cell panel) on CAT (2+ reaction). Detected by MosaiQ™ (IgM).Sample #0842 – Anti-E identified (11 cell panel) on CAT (4+ reaction). Detected by MosaiQ™ (IgM).

Demonstration of specificity detection of Ax cellsAn antibody known to detect Ax cells (a component of a blended anti-A,B reagent) was printed in the microarray and shown to bind red blood cells from Ax donors equally as well as red cells from A and AB donors.

Evaluation of 1022 random donor (EDTA) samplesTo evaluate the feasibility of the MosaiQ™ antigen typing platform 1022 random samples were tested and results compared with those obtained from comparator (CAT) testing carried out in parallel. The results correlated with a confidence of 98% or greater for the detection of A, B, C, c, D and E antigens. Correlation for e detection was 68%.

Table 2: Feasibility results. Anti-e antibody was yet to be fully optimised explaining the high incidence of false negative results.

ArtworkClient: Quotient Doc: 130958 ABS A0 Scientific Poster

Project: Scientific Poster Size: 1189mm x 841mm A/C: CB AW: BG

C M Y K Date: 20.11.13 Version: 3

Figure 1: Microscopic image of red blood cell spot (a), MosaiQ™ Antibody identification printing layout of cell types [1-10] (b) and typical assayed array image (c).

aRed Blood Cell Panel number

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MosaiQ™ Antibody Identification AssayDiluted patient plasma was incubated with processed, immobilised red cells. After washing a secondary antibody cocktail was applied. Upon further washing an enhancement reagent was used to visualise a positive result (grey/black). White spots indicate a negative reaction.

Figure 2: Schematic representation of the MosaiQ™ antibody identification assay.

MosaiQ™ Antigen Typing AssayRed cells were then introduced to the wells where they bound to specific antibodies. After washing these red cells were clearly visible.

Figure 5: Schematic representation of the MosaiQ™ antigen typing assay.

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Proof of detection of all available specificities• 23 specificities (IgG / IgM) detected using MosaiQ™. • Anti-A, B, D, C, c, E, e, Cw, V, K, k, Kpa, Kpb, Fya, Fyb, Jka,

Leb, Xga, Lua, S, s, M and P1.

Figure 3: Example images when samples of Anti-D, Anti-Fya, Anti-e and Anti-K were tested using the MosaiQ™ system against a printed 10 cell panel and detected with an anti-human IgG and IgM cocktail of secondary antibodies.

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Representative results for MosaiQ™ antigen typing

Figure 6: MosaiQ™ antigen typing of AR1r and Brr cells. Black spots represent positive results.

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A 358 352 6 4 99%

B 110 107 3 4 99%

D 793 788 5 3 99%

C 645 639 6 2 99%

c 855 844 11 4 99%

E 266 264 2 0 99.9%

e 996 672 324 5 68%

ArtworkClient: Quotient Doc: 130958 ABS A3 Scientific Poster

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C M Y K Date: 20.11.13 Version: 1