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