Technical differences
When measuring such scarce cells as PIG-A mutant erythrocytes, it
is important to note that slight methodological variations can
significantly alter results. For example, using antibodies that target
the GPI-AP’s CD55 and CD59 in combination may reduce the number of false
positive mutant cells compared to targeting only one GPI-AP (see Figure
2). Alternatively, GPI anchor status can be measured using a fluorophore
conjugated variant of proaerolysin. Proaerolysin is an inactive form of
the bacterial toxin aerolysin which binds to GPI anchors, permitting
direct assessment of PIG-A status. Whilst a FITC-conjugated form
of proaerolysin is commercially available (Cedarlane, Canada), most
research groups have chosen to use antibodies that target GPI-AP’s.
Furthermore, the type and/or performance of the flow cytometer used may
impact results. Previous work carried out in our lab revealed that
variations between cytometers exists. For example, the Facs Aria I (BD
Biosciences) on average scored a higher number of mutant cells than the
Navios flow cytometer (Beckman Coulter) although there was a strong
correlation between the two machines (Haboubi et al., 2019). Flow
cytometer performance characteristics that may impact mutant cell
scoring include laser strength and alignment, flow speed, stream
characteristics and cleanliness of inner tube networks. These factors
can be mitigated with routine maintenance and quality control. Whilst it
is difficult to account for slight variations that could significantly
influence classification of PIG-A phenotype and hence falsely
classify a mutant cell, in vitro cell lines with knownPIG-A status can be used as a quality control. For example, we
have generated a PIG-A mutant L5178Y mouse lymphoma cell line by
treating wild type cells with the methyl methanesulfonate and using
magnetic bead enrichment followed by fluorescence activated cell sorting
(FACS) to create a cell line consisting of ~96%PIG-A mutant cells which is routinely used to check for flow
cytometer variations (Haboubi et al., 2019).
The gating strategy applied to categorise mutant cells is also very
important to consider. Inflating the lower fluorescence limit for mutant
classification could falsely classify erythrocytes with lower (but not
absent) GPI-AP staining as PIG-A mutant. These intermediate cells
may have less GPI anchors or GPI-AP’s due to incomplete staining,
shearing off of GPI-AP’s during sample processing or may pass through
the laser stream at an angle where fewer GPI-APs are exposed and so less
fluorophores are excited by the corresponding laser. This is
particularly important when measuring PIG-A status of biconcave
erythrocytes. The gating strategy usually involves the gating of singlet
erythrocytes using anti-CD235a (erythrocyte specific marker) followed by
the gating of PIG-A mutant erythrocytes using an unstained or
mutant mimic control (also known as an instrument calibration standard)
with this methodology being adopted by most research groups (Cao et al.,
2016; Dertinger et al., 2015; Dobrovolsky et al., 2011; Haboubi et al.,
2019; Lawrence et al., 2020). As this PIG-A erythrocyte test only
requires a finger prick volume of whole blood, re-running samples from
individuals with known mutant frequency can also act as an additional
quality control.
Although this test is quick to perform, batching samples would be most
beneficial. Not only would this save time and money, but it would also
allow the transport of samples from regional/remote locations to a
central processing laboratory. However, the stability of PIG-Astatus post blood-draw has not been measured over a prolonged period.
Within our research group we have demonstrated that erythrocytePIG-A mutant frequency is stable up to 72 hours post venepuncture
when collected in K2 EDTA coated vacutainers and stored
at room temperature (unpublished, data not shown). Dertinger and
colleagues have demonstrated PIG-A mutant stability of over a
week when the sample was kept at 4°C (Dertinger, 2015 #21). After this
time, the mutant frequency increases, potentially due to loss of
membrane integrity, GPI anchors and/or GPI-APs as cells start to die.
Cryopreservation medium for human samples similar to that supplied by
Litron Laboratories (New York, USA) for mouse and rat blood could help
to make sample batching of human blood a reality.