Flow

Question 1

Flow QCs

Answer 1

Factors affecting measurements on a flow cytometer includes the fluidics, optics, lasers and laser power.

These can be monitored by the use of standardised fluoro spheres.

Daily Controls should be run in the morning in the following order;
-Flow Check Pro
-Flow Set Pro
-Immunotrols

Question 2

Flow fundamentals

Answer 2

Question 3

Use of multiple flurochromes

Answer 3

• each dye must have different emission spectra
• spectral emissions minimal overlap
• a photomultiplie tube for each flurochrome
• band pass filters to detect narrow range

Question 4

Steps in stem cell collection

Answer 4

1. Collection
2. Processing for freezing & storage
3. QC testing
4. Thaw and infusion

Question 5

Pre collection requirements

Answer 5

• Target cell dose & planned collection date
• Donor suitability & eligibility
• Infectious disease testing within 30 days of collection (HIV, Hep B, Hep C, HTLV, Syphilis)
• Processing and cryopreservation requirements
• Number of infusions, planned infusion date(s)
• Consent for storage and disposal
• Patient consent for transplant of nonconforming product

Question 6

Timing and targets in autologous collections

Answer 6

• When PB WCC >1.0 x 10^9/L
• Determine CD34 count via flow cytometric methods
• Apheresis collection proceeds when PB CD34/uL > 20
• Collection 4-5 hours
• Absolute minimum target > 2 x 10^6/kg
• Preferred target dose > 5 x 10^6/kg
• ACDA as anticoagulant
• Concurrent plasma ~ 100mls

Question 7

Cryopreservation of HPC

Answer 7

-DMSO
-Remains stem cell viability & function after thawing
-Freezer mix (DMSO, NaCl, protein) + cells –> Final DMSO concentration is 10%
-Store at long term -170C for > 10 years

Question 8

Post Infusion testing in transplantation

Answer 8

Record time of ANC and plt engraftment and any reactions experience by patient at time of infusion

Question 9

CD34 flow in autologous transplantation

Answer 9

-Number of CD34 cells infused for transplantation is a clinically important marker for engraftement
-Check collected as well as post thawed CD34

In ISHAGE CD34 enumeration, Boolean gating is used to logically combine multiple gates (CD34+, CD45dim, viable, low SSC) to accurately isolate true stem/progenitor cells for transplant assessment in PB, cord blood or apheresis products.

Question 10

ISHAGE steps

Answer 10

• viability – dead cells excluded with use of a viability dye eg. 7AAD
• surface markers are used – to identify stem cells which are CD34+ and CD45dim,
• Doublet discrimination - use forward scatter to ensure single cells only counted.
• Light side scatter – stem cells will be low side scatter
• A Boolean gating strategy is used (logically combine multiple gates to accurately isolate true stem/progenitor cells for counting.)

Absolute counts – single vs dual platform. Single preferred as more accurate
- Single platform; counts CD34 AND total WCC in same flow tube with counting beads of known concentration. CD34 events per bead ratio calculate CD34+ cells. More accurate.

• Dual platform; separate haematology counter for total WBC. Measure WCC and multiple by ratio of CD34 to CD45 events. Less accurate

Recommended that a minimum of 100 CD34+ events and 750000 CD45+ events are collected

• QAP - 3 samples/year

Why ISHAGE standardisation
- Different labs can produce comparable results worldwide.
- Minimises false positives (e.g., activated lymphocytes can sometimes falsely express CD34 weakly).
- Essential for predicting engraftment success after transplantation.

Question 11

Collection measurements in autologous transplantation

Answer 11

• volume of HPC collected
• WCC of collected HPC
• CD34 counts and other cell types (t cell, b cells and NK cells)
• Sterility checks/microbial checks
• Viability
• Confirmatory ABO group check