Fluorescence Activated Cell Sorting (FACS) Followed by Fluorescence In Situ Hybridization (FISH) To Determine Clonal Origins of Cells in Myelodysplastic Syndrome (MDS) with Paroxysmal Nocturnal Hemoglobinuria (PNH).
Authors: Battiwalla Minoo , Sait Sheila , Block AnneMarie W. , Kebbewar Ibrahim , Ahluwalia Manmeet S. , Timm Earl A. , Wallace Paul K. 
Published: November 2019

Abstract
The combination of FACS and FISH has been used to identify clonal cytogenetic abnormalities in small populations of cells in diseases such as multiple myeloma. We describe our refinement and application of this technique in a patient with known MDS/PNH to dissect the clonal origins of the disorder. The subject is a 45-year old woman with MDS diagnosed five years ago when she presented with macrocytic anemia and thrombocytopenia. Bone marrow was hypocellular (20% cellularity), with 4% blasts, FAB-RA, WHO- RCMD, Intermediate-1 by IPSS, del 20q clone by FISH. Family history is significant for a familial cytopenia with two sisters developing bone marrow failure, but negative for carriage of the hTERC mutation. HLA DRB1 type was 0101;1301. There was no evidence for T-LGL by flow cytometry or by TCR gene rearrangement assay. Management has been observational only without the need for transfusions. A new PNH clone was detected for the first time by flow cytometry six months ago. Heparinized blood was processed by the laboratory on the same day as collected. WBCs were separated from RBCs by a combination of dextran centrifugation and lysis of RBCs with ammonium chloride. Normal mouse IgG (3mg/mL) was added to block Fc receptors. The cells were stained with mAb cocktails containing CD16 PE (Clone 3G8, Invitrogen), CD55 PC5 (clone IA10, BD Bioscience), and CD15 APC (clone HI98, BD Bioscience) for granulocytes; or CD14 PE (clone TUK4, Invitrogen), CD55 PC5, and CD64 (clone M22, Trillium) for monocytes. Concurrent with mAb staining proaerolysin (FLAER Ax488, Protox Biotech) was added to each tube. The cells were incubated with mAbs and FLAER reagent at saturating concentrations in the dark, on ice, for 30 minutes. They were then washed once with PBS, fixed in 0.5% methanol free formaldehyde (Polysciences, Warrington, PA) for 20 minutes and finally resuspended in PBS. Cytofluorometric analysis and sorting was performed using a FACSAria (BD BioSciences) flow cytometer. Granulocytes were defined using a Boolean combination of forward, side scatter and CD15; monocytes were defined with forward, side scatter and CD64. Granulocytes and monocytes were sorted into PNH positive and negative cells based on FLAER, CD55 and either CD16 (granulocytes) or CD14 (monocytes) expression. Data was analyzed using WinList (Verity Software House, Topsham, ME). FISH analysis for del(20q) was performed on these cells using the Vysis LSI D20S108 SpectrumOrange Probe (Abbott Molecular Inc.). Our results (table 1) show that the PNH clone (FLAER negative) is a distinct non-overlapping population from the del(20q) dysplastic population.
Conclusions: Our technique of flow-sorting followed by FISH is feasible for enriching and characterizing PNH positive and negative cellular fractions in bone marrow failure states. The PNH clone in this patient, contrary to other reports in the literature, does not overlap with the MDS clone, rather arising from normal non-dysplastic cells consistent with a putative immune evasion mechanism. This technique is a powerful clinical or research tool to elucidate clonal origins in PNH-associated bone marrow failure states.
Table 1 Pre-sort Post-sort 20q− by FISH Unsorted WBCs 13/200 Monocytes PNH− 67% 76% 34/200 PNH+ 245 81% 5/200 = negative control Granulocytes PNH− 72% 94% 34/200 PNH+ 26% 94% 5/200 = negative control