A dissection of the CD45/side scatter "blast gate"

Area of Science:

• Hematology
• Immunophenotyping
• Flow Cytometry

Background:

• CD45/side scatter (SS) gating is a standard method for isolating blast cells using flow cytometry (FC).
• This technique, often referred to as the "blast gate" (BG), can be imprecise due to contamination from other cell types, especially when blast populations are small.

Purpose of the Study:

• To evaluate the cellular composition of the blast gate (BG) in various myeloid disorders and nonneoplastic controls.
• To assess the accuracy and limitations of CD45/SS gating for blast quantification in myelodysplastic syndromes (MDSs), myeloproliferative neoplasms (MPNs), and chronic myelomonocytic leukemias (CMMLs).

Main Methods:

• Utilized 4-color flow cytometry (FC) with cluster analysis to examine the contents of the blast gate (BG).
• Analyzed samples from 21 MDSs, 14 MPNs, 7 CMMLs, and 40 nonneoplastic controls.

Main Results:

• Blasts constituted a median of 14.7%-22% of BG events across all groups, with significant contamination by granulocytes (23.3%-33.2%), monocytes (up to 24.2% in CMML), and basophils (35.4% in MPNs).
• The percentage of blasts within the BG was lower in MDSs (88.2%) and CMMLs (80.7%) compared to controls (94.2%).
• Approximately 10%-20% of blasts were found outside the traditional BG in CMMLs and MDSs.

Conclusions:

• Traditional CD45/SS gating for blast analysis is unreliable in non-acute myeloid disorders due to substantial cellular contamination within the blast gate (BG).
• The findings highlight the need for refined gating strategies to improve the accuracy of blast quantification in MDS, MPN, and CMML.
• Relying solely on the traditional blast gate can lead to underestimation of blast percentages and misdiagnosis.