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.