Flow

Question 1

What are the 4 aspects of QA in flow?

Answer 1

1. Sample
2. Flow cytometer
3. Reagents including anti-sera
4. Analysis

Question 2

Flow sample requirements

Answer 2

The aim is to get fresh, live, intact cells:

• 3ml
• analyse within 24 hrs
• use preservative media where required

Question 3

How is flow cytometer calibration done?

Answer 3

BD CaliBRITE beads - adjust and monitor instrument settings and performance eg laser, fluidics, detectors

Rainbow beads - run in optimal analyser set up. Get target MFI, adjust voltages.
Plot on LJ charts

Question 4

How are flow reagents checked with QC?

Answer 4

Quantification - commercial cells
Lot to Lot comparisons
Check different clones for sensitivity and specificity.

Question 5

QC on analysis checks?

Answer 5

B cells = kappa + lambda 
T cells = CD4 + CD8
Internal populations - use as staining checks and for positive/negative controls
Eg MPO of neutrophils compared to blasts
CD19 of normal B cells vs CD19- blasts

Question 6

What is MRD?

Answer 6

The persistence of leukaemic cells following therapy below the limits of detection of routine morphology

Question 7

Methods of MRD assessment?

Answer 7

Multicolor flow
Molecular
- PCR (qPCR, digital PCR)
- NGS

Question 8

What are the roles of a BMT lab?

Answer 8

Receipt of HPC product
Processing 
Cryopreservation
Storage
Thawing
Post thaw release
QC on product

Question 9

Names some methods used to monitor equipment at the HPC lab

Answer 9

Continuous monitoring and alarms
Calibration
Back up generator
Contingency plan

Question 10

Labelling requirements of HPC product

Answer 10

Contents
Volume - product and anticoagulant
Date of collection
Name of donor and recipient
Address
Recommended storage temperature 
Do not irradiate
Do not leucodeplete

Question 11

Steps of cryopreservation

Answer 11

Volume reduction (plasma and RBCs)
Introduction of storage solution - culture and DMSO
Controlled rate freezing
Storage (-150)
Thawing
Post-thaw assessment (3 aliquots frozen with sample)

Question 12

Testing of the HPC product (7)

Answer 12

Volume (HPC and anticoagulant)
Visual inspection (haemolysis, labelling, integrity of bag)
Sterility check - cultures
CBC (automated)
CD34 quantification (ISHAGE) and CD3 counts for allo
Viability - 7AAD, Trypan dye exclusion test, CFU
Confirm ABO group

Question 13

Requirements for MRD testing (4)

Answer 13

Sensitive
Specific - differentiate residual disease from other cells
Quantitative
Reproducible

Question 14

Reasons for MRD assessment

Answer 14

Objective assessment of depth of remission
Change management - alter intensity of treatment, allow early intervention e.g. DLI
Allow robust post-transplant assessment

Question 15

What are the 3 main flow cytometry methods for MRD?

Answer 15

1. LAIP
2. DfN
3. Cells at inappropriate sites

Question 16

What are some challenges in flow MRD

Answer 16

Subpopulations
Changes in immunophenotype with therapy, BM regeneration and relapse
Not all blasts express aberrant populations

Question 17

Considerations with molecular MRD

Answer 17

Only a proportion of patients have a molecular marker (30% with standard PCR, 80% with NGS).
Gain/loss of mutations
CHIP - may have residual clone after therapy
Germline mutations remain at 50%