869568983

two compensation matrices used per center had been established after a new compensation experiment (Table 8).

Overall, compensation matrices were shown to be similar in all seven instruments evaluated (Table 8) and their variability among instruments was similar to that observed with time within each of the laboratories for individual instruments (P>0.05, paired Student's T-test). Although compensation requirements depend on the specific PMT voltage settings, overall, high spillover was detected for the PacB into the PacO channel and for PE into the PerCPCy5.5 channel. Furthermore, intermediate spillover was found between PerCPCy5.5 and PECy7, between FUC and PE, PECy7 and APCH7, and between APC and APCH7 detectors (Table 8). Compensation experiments performed 1 month apart yielded very similar compensation values (P>0.05; paired Studenfs T-test).

CONCLUSION

Fluorescence compensation setup procedures were designed to establish fluorescence compensation matrices for every individual 8-color combination of fluorochrome-conjugated reagents in the 8-color EuroFlow panels.²⁹ The complexity of the procedurę was higher than desired due to the need for different compensation values for reagents conjugated with the PECy7 and APCH7 fluorochrome tandems. Fortunately, the frequency of compensation could be set to a time interval of 1 month, during which only minor deviations from target MFI values were recorded on well-performing instruments, as assessed by routine (daily) monitoring of the standard instrument settings (see Section 2). Notably, highly stable compensation matrices were obtained at different times among all different EuroFlow laboratories with the proposed fluorescence compensation setup SOP. This suggests that in the futurę, software Solutions for automated establishment of compensation matrices to experiments performed with adjusted PMT voltages to target MFI values may potentially be developed and implemented.

SECTION 4. SAMPLE PREPARATION AND STAINING

VHJ van der Velden\ J Flores-Montero², JG te Man/elde¹,

S Bottcher³, L Lhermitte⁴, AS Bedin⁴, J Almeida², JJ Perez⁵,

M Cullen⁶, P Lucio⁷, E Mejstrikova⁸, T Szczepański⁹, T Kalina⁸,

A Orfao² and JJM van Dongen¹

'Erasmus MC, Rotterdam, The Netherlands; ²USAL, Salamanca, Spain; ⁾UNIKIEL Kieł, Germany; "AP-HP, Paris, France; ^sHUS, Salamanca, Spain; ⁶UNIVLEEDS. Leeds, UK ⁷iP0LFG, Lisbon, Portugal; ^sDPH/0, Prague, Czech Republic and ⁹SUM, Zabrze, Poland

BACKGROUND

At present multiple protocols and reagents are available for staining leukocytes.⁵'^26,38"⁴² Most protocols include a staining

1 0”	i ^f #	**— v<% 1	i
	y	%

Ammonium    FACS łyse    OuickLysis    VersaLyse

Chloride

Figurę 3. Illustrating example of the differences in the light scatter characteristics of the major subsets of peripheral blood leukocytes observed for the distinct lysing Solutions and staining protocols. Please notę the significant reduction in the light scatter CV for the different leukocyte populations observed with FACS Lysing Solution and a SLW protocol (red square). Events shown in the upper-left corner of each dot plot correspond to PerfectCOUNT beads (Cytognos SL) introduced for the evaluation of celi loss. SLW, stain-lyse-wash; SLWF, stain-lyse-wash-fix; SLNW, stain-lyse-no wash.

FSC