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NEW NO-WASH, NO-LYSE METHOD FOR RESIDUAL LEUKOCYTE ENUMERATION USING THE ATTUNE ACOUSTIC CYTOMETER AND A CELL-MEMBRANE PERMEANT DNA DYE José-Enrique O'Connor, Alicia Martínez-Romero, Guadalupe Herrera, Laura Díaz Laboratory of Cytomics, MixResearchUnitCIPF-UVEG Amparo Pinilla, Pilar León HematologyService, “Doctor Peset” University Hospital Valencia, Spain
The need for counting residual leukocyte in blood products Allogeneic leukocytes in red blood cell units and platelet concentrates have been involved in a variety of transfusion reactions, including HLA alloimmunization, the transmission of cell-associated viruses and prions, and immunosuppressive effects. In the US, sanitary regulations require that residual leukocytes are lower than 5 x 106(~ 20 WBC/μL) per product in RBC and apheresis platelets and lower than 0.86 x 106 per product in whole blood platelet preparations (~ 2 WBC/μL). Because of this, simple and accurate routine methods for the enumeration of residual leukocytes was needed. Nageotte chamber and standard hematology analyzers have low sensitivity when considering the expected low leukocyte contamination.
FlowCytometry in residual leukocytecounting • Flow cytometry(FCM) has become a choice method to evaluate leukoreduced blood products, and such application is a typical example of rare cell detection. • FCM is used currently for accurate determination of residual leukocytes in blood components by DNA staining with more reproducibility and higher throughput than conventional counting methods. • Current residual leukocyte counting by FCM based on DNA : • Is a time-consuming, “rare-event” issue. • Requires fixation/permeabilization. • Is performed on a monoparametric basis.
FlowCytometry in residual leukocytecounting • Thus, FCM studies on leukoreduction might benefit from: • Faster acquisition rate: • To allow acquiring statistically significant numbers of leukocytes in substantially shorter periods of time. • Multiparametric /polychromatic analysis: • To allow simultaneous immunophenotypic analysis. • Use of permeable DNA-binding dyes: • To allow functional analysis. • Volume-based count calculation: • To allow determination of cell concentration without adding beads.
Attune™ Flow Cytometer: AdvantagesforRareCellCounting DirectCounting MultipleDiscriminators AcousticFocusing Violet Laser Samplerate control
Attune™ Flow Cytometer: Advantages of Violet Laser The violet laser (405 nm) has become increasingly popular in flow cytometry due to its ability to increase the multiplexing capabilities of the flow cytometer. Reagents for the violet laser include cell cycle analysis, cell viability and vitality, cell proliferation, and apoptosis.
AcousticFocusing and Violet Laser forLeukocyteCounting We have developed a no-wash no-lyse FCM method for counting residual leukocyte in leuko-reduced blood using the Attune™ Acoustic Focusing Flow Cytometerand Vybrant® DyeCycle™ Violet, a cell membrane-permeant dye for DNA analysis in living cells excited by violet laser.
No-wash no-lyseMethodfor Residual LeukocyteCounting The method involves incubation of leuko-reduced samples with 50 M VybrantDyeCycle Violet for 30 min at 37ºC without any further manipulation. • 100 μL of sample + 1 μL of 5 mMVybrant®DyeCycle™Violet • Incubatefor 20 minutes in thedark. • Make up to 3 mLwith RPMI medium. • Analyze in anAttuneflowcytometerusingviolet and bluelasers
LeukocyteIdentificationwithVybrantDyeCycleViolet VL1 (450 nm): DNA contentbyVybrantDyeCycleViolet VL1 -H (450 nm) BL2 (610 nm): Autofluorescence BL2 -H (610 nm) Cellconcentrationisdirectlyobtained in theAttuneflowcytometer
LeukocyteIdentificationwithexisting PI-BasedMethods Leucocount /BD Leucocount /FC500 Leukosure /EPICS XL • Cellconcentrationmustbecalculatedfrom a formula: • WBC/L = WBC events/Beadevents x StatedBeadspertube/Samplevolume
No-wash no-lyseMethodfor Residual LeukocyteCounting Assay performance has been validated by assessing: - Internal consistency of the Attune cytometer by running serial dilutions of fluorescent microspheres (FlowCount, Beckman-Coulter) and of whole blood in RPMI medium. - Correlation between leukocyte counts obtained with the Attune cytometer and with Neubaeror Nageottechambers. - Correlation of leukocyte counts obtained with the Attune cytometer and with a standard hydrodynamic-focusing cytometer (Cytomics FC500 MCL, Beckman-Coulter) using a commercial kit based on DNA staining with Propidium Iodide plus concentration-calibrated beads (Leucocount Kit, Becton Dickinson).
Residual LeukocyteCounting in BloodPreparations • Human wholebloodand serial dilutions in RPMI medium. • Concentration-calibratedcellularpreparationsfor residual leukocytecounting (RBC LeukoTrolcellsBeckman-Coulter): • Low control: 2 cells/μL • High control: 20 cells/ μL • Leuko-reduced RBC bags fortransfusion.
LeukocyteIdentificationwithVybrantDyeCycleViolet 18.0 WBC/ μL 18.9 WBC/ μL 500 μL/min; 500 μL 200 μL/min; 200 μL Leuko-Trol RBC HIGH: 0.66 events/μL x 30 (dilution) = 19.8 WBC/ μL Expectedcount: 20 WBC / μL. VerifiedwithNageottechamber. 18.2 WBC/ μL 1000 μL/min; 1000 μL
LeukocyteIdentificationwithVybrantDyeCycleViolet 1.5/ μL 1.8/ μL 200 μL/min; 200 μL 500 μL/min; 500 μL Leuko-Trol RBC LOW: 0.06 events/μL x 30 (dilution) = 1.8 WBC/ μL Expectedcount: 2 WBC / μL. VerifiedwithNageottechamber. 2.1/ μL 1000 μL/min; 1000 μL
LeukocyteIdentificationwithVybrantDyeCycleViolet LEUKOREDUCED SAMPLE (RBC bag): 0.01 events/ μL x 30 (dilution)= 0.3 WBC/μL
Conclusions Results show an excellent correlation of this method with other methods for residual leukocyte enumeration, as well as high internal consistency in the analysis of serial dilutions of whole blood, red blood cell bags and commercial controls with low- and high leukocyte count for quality control procedures. This is a simple method requiring less degree of sample manipulation and does not require the use of concentration-calibrated beads.