Transfusion – lab techniques and Setup

 

Example of my local Set up

 

Grifol’s Analyser and reagents

- Used for all routine samples

- Gel-based card assays

- Single donor source for A and B cell suspensions for reverse group

 

BioRad

- Secondary supplies / techniques

- Glass bead card assays

- Pooled donors for A and B cell suspensions for reverse group

- Assays include a ten min incubation prior to centrifuge (may increase pick up rate of weak antibodies)

 

Quality Control

- Negative control for grouping – so as to ensure can trust positive results

- Weak anti-K control for IAT – so can be sure of detecting weak antibodies

- Daily 2 cell control (manufacturer supplied) for Grifol analyser. Also run with each reagent change.

- Internal IPEX (Internal Proficiency Exercise) for lab staff IAT interpretation, supplied by NEQAS

- External NEQAS – 6x a year for ABO, Rh, IAT, FMH, Emergency issue,

- External NEQAS - 4x year ABO titres

- Currently NEQAS pilot of DAT assessments.

 

Safety

- Universal international antibody colouring for consistency

            - anti-A (blue)

            - anti-B (yellow)

            - AHG (green)

            - all other antibodies are colourless

- BGS1 x2 for all new/unknown patients (fwd + reverse group and Ab screen)

- BGS2 for known patients (reverse group)

- Standard lab PPE

 

On-site Blood Stock – critical limits

- RBC – 30 x O neg, 30 x A pos, 5 x B neg, 10 x O neg, 8 x A neg, 2 x B neg

- FFP – 20 x O, 20 x A, 10 x B, 20 x AB, 6 x neonatal

- Plt – 10 pools, delivered daily

 

Product Costs

- Used and unused stock charged to transfusion lab. Due to change – requesting departments to fund unused wastage.

- RBC – standard adult £120/unit

- FFP - £30/unit (Non-UK, e.g. MB-FFP £180/unit)

- Plt - £200/unit (HLA £240/unit)

- Other – pooled granulocytes £1100/unit, HEV neg supplement £17/unit

 

Principles and Techniques

 

Group Technique (BioRad)

- Card Assay

- Always set up control last and read it first (to ensure it has shortest incubation time – avoids false negatives)

 

1. Prepare Reverse Group

- 50 microlitres patient plasma

- 50 microlitres 0.8% A1 & B cell suspensions, always Rh neg (manuf. supplied)

- Incubate 10 min at room temperature

2. Prepare Forward Group

- Make 5% patient red cell suspension – i.e 10microl cells in 200microl of ‘Diluent 2’ in a glass tube

- Add 10microl cell suspension to each well (anti-A, anti-B, anti-D, negative control)

3. Spin for 10 min at 1000 rpm

4. Read card

 

IAT Principles

- Card assay again, Grifols 8 well card, BioRad 6 well card

- Card contains IgG (anti-human globulin (AHG)) + C3d. These act as bridges, i.e. IgG alloantibodies bind to red cells but not strong enough to then bind cells together. AHG acts as bridge to bring cells together.

- 3 cell screen covering all Rh antigens, so typically R1R1 + R2R2 + rr.

- Will also typically cover Jka, Jkb, Fya, Fyb, M, N, S, s

- May cover Cw, Lua, Kpa depending on the cells used.

- Positivity in 3 cell screen will then trigger 10 cell panel to identify antibody.

 

IAT Technique

 

1. Prepare cards

- 50 microl 0.8% cell suspension (manufacturer supplied), enzyme treated and non-enzyme treated + 25 microl patient plasma

- Auto-immune control – i.e. 50microl 0.8% suspension pt red cells with 25 microl patient plasma. To differentiate auto from allo antibodies.

- Weak anti-K control

 

2. Incubate at 37 degrees for 15 min

3. Spin for 10 min at 1000 rpm

4. Read card

 

5. If antibody detected, go on to test for presence of antigen to confirm result

If antigen present then either error in initial interpretation, or more rarely an auto-antibody is to blame (not allo)

 

DAT Technique

- Card assay as for IAT and Grouping

- 5% cell suspension as for grouping, essentially same protocol