Analisi della Malattia Minima Residua nella Leucemia ... · e Laboratori di Ematologia Aziendali...
Transcript of Analisi della Malattia Minima Residua nella Leucemia ... · e Laboratori di Ematologia Aziendali...
8°UK NEQAS LI Italian Users Meeting Bologna, 7 Aprile 2016
Bruno Brando e Arianna Gatti Servizi Trasfusionalie Laboratori di Ematologia AziendaliASST Ovest MilaneseOspedale di Legnano (MI)
e-mail: [email protected]
Analisi della Malattia Minima Residua
nella Leucemia Linfatica Cronica B e nel Mieloma
Why the Study of Minimal Residual Disease is so Important:The Case of B-CLL & Multiple Myeloma
• New treatment strategies need more sensitive approaches to defineMRD at VERY LOW LEVELS (i.e. 10-4 / 10-5), to compare the efficacy of different therapeutic regimens.
• Better indicators to guide the therapy on an individual patient basisare needed, to avoid over- or under-treatment.
• The definition of clinical response (by PFS, RFS, CR, OS etc.) has remained basically the same during the last 20 years, and some indicators are now inadequate.
• Namely, the quality of Complete Remission (CR) varies largely amongdifferent regimens and fails to identify patients that may relapse.
• There is the need to define reliable Surrogate End-Point Indicatorsthat can be used in the SHORT TERM, whereas the disease course isnow typically 5-10 years long.
MRD Predice Assenza di Progressione nella LLC-B
Prog
ress
ion-
Fre
e S
urvi
val
(Interim Staging)
Marcata
differenza del
potere predittivo
di MRD <10-2
rispetto a < 10-4
Böttcher S. J Clin Oncol 2012; 30(9): 980-988
FDA POLICY ON INNOVATIVE BIOMARKERS(Applicable to the current B-CLL & MM-MRD studies)
Gormley NJ. Cytometry Part B 2016; 90B: 73-80.
Wood B. ICSH/ICCS. Cytometry Part B 2013; 84B: 315-323.
• Lower Limit of Blank (LOB):The highest signal in theabsence of the measurand.(Mean Blank + SD x 1.65).95% of negative values arebelow this limit.
• Lower Limit of Detection (LOD):(Mean Blank + SDlow x 1.65).95% of negative values areabove this limit.5% false negatives and 5% false positives are assumed
• Lower Limit of Quantitation (LOQ):The lowest level of measurandthat can be reliably quantitatedat a predefined criterion forprecision and accuracy (clinicalutility value). Never lower thanLOD.
Mean Blank
L O B
Mean Low Signal
Mean High Signal
95% NegativeValues
Such concepts derive from clinical chemistry and can be directly applied to Flow Cytometry for INTENSITYmeasurements only.
Level of theAnalyte
0
LOB= 1 event MRD Events= 156
In FCM MRD studies, the LOB / LOD / LOQconcepts must be appropriately translated:
LOB = < 10 Events in normal samplesLOD = > 30 Events detected (> 0.001% or >10-5)LOQ = > 50 Events quantitated (> 0.001% or 10-5)
[30 and 50 events derive from statistical estimations]
Consensus on MRD Detection in B-CLL and Multiple Myeloma
FCM MRD Studies: Estimated LOD & LOQ
According to the Total Number of Acquired Cells
Courtesy of Maria Arroz, 2015, modified
Total Number of
Acquired Cells(Excluding Erythroid)
LOD %≥ 30 Events
LOQ %≥ 50 Events
200,000 0.015 0.026
500,000 0.006 0.01
1,000,000 0.003 0.005
2,000,000 0.0015 0.0025
3,000,000 ~ 0.001 ~ 0.0017
5,000,000 ~ 0.0006 ~ 0.001
In each report, the LOD specific for the total amount ofacquired cells should be specified.
The acceptable level of cytometric error in the evaluation of MRD(in this case B-CLL) greatly influences the classification of the patient’s status (i.e. MRD+ or MRD-), and therefore its correlation with the clinical outcome.
Courtesy of Andy Rawstron, 2015.
B-CLL
Riassumendo 10 Anni di Evoluzione Tecnica nelloStudio Citometrico della MRD nella B-CLL
• 4 Colori sono meglio di 3 MRD 1%
• 5 Colori sono meglio di 4 MRD 0,1%
• 6 Colori sono meglio di 5 MRD 0,01%
• 6 o 8 Colori vanno bene MRD 0,001%
• Moreton P. J Clin Oncol 2005; 23: 2971-2979.• Böttcher S. J Clin Oncol 2012; 30: 980-988.• Strati P. Blood 2014; 123: 3727-3732.• Santacruz R. Haematologica 2014; 99: 873-880.• Rawstron AC. Leukemia 29 Jan 2016; doi:10.1038/leu.2015.313
L’identificazione citometrica di MRD al disopra dello 0.01% (>10-4) è unindicatore indipendente predittivo per ‘Progression-Free Survival’ e‘Overall Survival’ nei pz con B-CLL trattati con chemio/immunoterapia.
4 Tubi
2 Tubi
1 Tubo
FCM ANALYSIS OF B-CLL - MRD: Technical Options (1)
V450 FITC PE PE cy7 APC APC-H7
CD5 x CD81 CD79b x CD19 CD43 CD20
• ERIC One-Tube CORE 6 Color Panel: Good in >95% Classical B-CLL Cases
• Can be used with any analysis software, provided the HMDS-ERIC gatingstrategy is applied.
V450 BV510 FITC PE PerCP-cy5.5 PE cy7 APC APC-H7
CD5 CD22 CD81 CD79b CD3 CD19 CD43 CD20
• ERIC One-Tube 8 Colors: CD3 & CD22 added to CORE Panel to prevent stainingartifacts (but they are important if CD43 and CD81 are absent on B-CLL cells).
• Especially useful if a Pre-Treatment sample demonstrates an atypical phenotype(CD20 & CD22 to discriminate normal B Cells from B-CLL Cells).
6-Color CORE Panel and gate syntax provide sufficient internal controls to validate the analysis independently of the chose clones and fluorochromes.
Harmonized approach toB-CLL MRD Analysis
Capture gateB-CLL CD19+
B-CLLCD43+ CD81-/dim
B-CLLCD5+ CD79b-/dim
B-CLLCD5+ CD20-/dim
Besides CD5 and CD43 (‘positive’ in B-CLL), the Eric approach is more based on ‘negative’ B-CLL markers (CD22- CD79b- CD81-)
Aggiustamento dei gate nell’approccio ERIC - CORE Panel a 6 colori
B Cell Progenitors & Plasmablasts: CD5- CD20- CD43+++ CD81++
Contaminant T Cells: CD5+ CD20- CD43+++ CD79b- CD81++
6-Color Panel and gating can discriminate contaminants
B-CLL MRD Analysis by 6-Color Core Panel vs High-Throughput Sequencing (HTS - ClonoSEQ)
Rawstron AC. Leukemia 29 Jan 2016; doi:10.1038/leu.2015.313
Rawstron AC. Leukemia 29 Jan 2016; doi:10.1038/leu.2015.313
8-C
olo
r vs 6
-Co
lor
Spiking 1 Tube 8-Color
<LOD >LOD & <LOQ >LOQ
Bland-Altman
6-Color Core vs 8-Color Panel: Overlapping results in typical B-CLL MRD studies (in a Operator- and Platform-Independent fashion)
• CD3 is not required in all cases, but it is necessary when high accuracy is needed (i.e. in the 0.001 - 0.01% range and if FACSDiva is used).
• CD20 & CD22 together are redundant if ≥ 2 typical CLL markers (CD5, CD43, CD79b and CD81) are expressed and if Infinicyt is used.
The Problem With Clonality Assessment by theKappa / Lambda Ratio: Poor Specificity
B-C
LL c
ells
as
% o
f Leuc
ocyt
es
• > 40% of ‘Polyclonal’ conditions are in fact MRD Positive• > 5% of ‘Monoclonal’ conditions are in fact MRD Negative
Rawstron AC. Leukemia 2013; 27: 142-149
B-CLL
Rawstron AC. Leukemia 29 Jan 2016; doi:10.1038/leu.2015.313
B-CLL MRD Detection using the ERIC Harmonized Approach (FCM vs HTS)
• Applicable to >95% of typical B-CLL cases.
• A pre-treatment sample is not essential, if the B-CLL phenotype is typical.
• Applicable both in BM and PB samples in all therapeutic trials.
• Plenty of internal controls for a robust gating setup and adjustment.
• Easy to perform and relatively operator-independent.
• Platform-independent reagents can be used, with wider applicability.
• High-Throughput Sequencing (HTS) is more sensitive than Sanger sequencing.
• HTS shows a good concordance with FCM down to 10-4 (0.01%).
• HTS can be applied to stored DNA.
(FCR=Fludarabine,Cyclophosphamide,Rituximab)
Rawstron ACLeukemia 2013; 27: 142–149
Standard Template to evaluate Minimal Residual Disease in Classic B-CLL withone 8-color tube
CD3+ CD19+Artifacts
1-tube, 8 Colors, plusDoublet discrimination,exclusion of CD19+ CD3+ artifacts and final FSC/SSC Backgating
Minimal Residual Disease in Classic B-CLL :One tube cantell you all.
FCM ANALYSIS OF B-CLL - MRD: Technical Options (2)
V450 V500 FITC PE PerCP-cy5.5 PE cy7 APC APC-H7
CD20 CD45 CD23 CD10 CD79b CD19 CD200 CD43
• EUROFLOW Approach: LST Tube for screening and B-CLPD Tube for bettercharacterization of B-Cell lymphoproliferative disorder and MRD study.
• Merging of 2 Tubes of 2 Million cell events each + Infinicyt analysis is therecommended technique.
V450 V500 FITC PE PerCP-cy5.5 PE cy7 APC APC-H7
CD20 CD45 CD8 CD56 CD5 CD19 CD3 CD38CD4 Lambda Kappa TCR
Backbone Markers for File Merging
LST Tube
B-CLPD Tube
Reference image (blue contours) indicates the position of the B-CLL cells at onset. Red dots: MRD 0.03%, 1.5 Million cells acquired.
Reference Images and Database options establish objective and reproducible frames for comparison.
Classic B-CLLFollow-up
8-color, 1 Tube, CD43 / CD79b / CD19 / CD3 / CD5 / CD38 / CD20 / CD45B-CLL MRD (blue dots): 0.01% on 2 Million cells, single tube.Reference image (red contour) indicates the position of the B-CLL cells at onset.
Normal B Cells
Residual B-CLL
8-color, 1 Tube, CD43 / CD79b / CD19 / CD3 / CD5 / CD38 / CD20 / CD45
B-CLL MRD (blue dots): 0.01% on 2 Million cells, single tube.
APS is based on the statistical function known as ‘Principal Component Analysis’.
Use of APS - Automatic Population Separator
Reference Image
Mantle Cell Lymphoma at Onset
2nd B-CLPD Euroflow Tube: CD20/ CD45/ CD23/ CD10/ CD79b/ CD19/ CD200/ CD43
MCL phenotype is directly comparedto the expected classic B-CLL pattern obtained with the same B-CLPD tube.
Reference Images to rapidly compare a cell phenotype toan expected pattern (Mantle Cell Lymphoma vs B-CLL)
MCL
B-CLLReference
Reference Images to rapidly compare a cell phenotype toan expected pattern (Mantle Cell Lymphoma-MRD)
MCLReference
MCL MRD21 Events
NormalB Cells
21 MRD Events /500.000 TotalMRD Detected but not quantitated
Studio Citometrico della MRD nella B-CLLLe Relazioni tra Marcatori Diagnostici e Schemi Terapeutici
• L’uso di certi marcatori va armonizzato al tipo di terapia che il paziente sta seguendo (conoscere la patologia!).
• In pazienti sotto Rituximab (anti-CD20) non sono piùdimostrabili cellule B normali in circolo, per cui il CD20perde il suo potere discriminatorio tra cellule B-CLL ecellule B normali.
• In questi pazienti è quindi indifferente usare CD20 o CD22.
• Il pannello ‘Core’ a 6 colori è stato validato prospettivamentesu periferico e midollo in vari trial controllati (ADMIRE, ARCTIC,
COSMIC, FLAIR, GALACTIC) ed è applicabile anche in studi cheimpiegano Venetoclax, Ofatumumab e Ibrutinib.
• Böttcher S. Leukemia 2009; 23: 2007-2017.• Rawstron AC. Leukemia 29 Jan 2016; doi:10.1038/leu.2015.313
Fludarabine+Cyclophosphamide+Ofatumumab(COSMIC). NormalB Cells reappear afterdiscontinuation ofMoAb therapy
BM BM BM
PB PB PB
Examples of B-CLLpatients underIbrutinib monotherapy
Courtesy ofA.C. Rawstron, 2016
CD5 tends to bedown-regulatedunder Ibrutinib
0
20
40
60
80
100
0 20 40 60 80 100
Ibrutinib Therapy: Equivalent Disease Levels in PB & BM
In 56 samples after 1–6 Months of Ibrutinib, the BM MRD level is
basically the same as PB (But PB may disclose higher MRD values!)
PBC
LL %
of
leuc
ocyt
es
BM CLL % of leucocytes
Trephine biopsy in 6/19 cases
after 6 months of Ibrutinib
treatment indicate
substantially lower level of
infiltration than indicated by
the Aspirate CLL %
Courtesy of A.C. Rawstron, 2015, modified
Studio Citometrico della MRD nella B-CLLOpportunità, Problemi Aperti e Controversie
• La citometria sta consolidando un ruolo centrale nella B-CLL,sia nella diagnosi che nell’analisi della malattia residua.
• Validità dell’analisi citometrica del sangue periferico in qualsiasi regime terapeutico.
• Bulk Lysis SI (Rawstron & Stetler-Stevenson) oBulk Lysis NO (Orfao-Euroflow)?
• Più COLORI (in studio pannelli monotubo a 10 colori !) oPiù EVENTI (2x6 colori, 2 Mil. di eventi/tubo + Infinicyt)?
New Programme s
Pilot Minima l Residual Disease in CLL/ Pilot Minimal Residual Disease in AML
The Minimal Residual Disease programmes will issue up to four send outs per financial year.
Each send out will include two follow up samples of a known CLL/AML case post treatment at
different time points. The AML programme will also include a reference sample to assist with
antibody selection. Participants will be asked to quantify the level of residual disease in these
samples.
B-CLL MRD: Partenza ~ metà 2016
AML MRD: Partenza ~ fine 2016
In corso discussioni tecniche per avviareun programma di MRD per il MIELOMA
Flow Cytometric Analysis
of Minimal Residual Disease
in Multiple Myeloma
Conventional 8-color Flow-MRD
vs
Next Generation Flow-MRD
• Increasing evidence is accumulating on the power of FCM assays forMM-MRD Assessment (on sCR, OS, PFS and prognosis).
• Several studies have demonstrated the value of FCM MM-MRD assaysin individual patients, IRRESPECTIVE OF THE THERAPY RECEIVED.
• Allele-Specific Oligonucleotide PCR (ASO-PCR) is considered thevalidated golden standard for MM-MRD assessment, because it issensitive at 10-5 and is a stable indicator throughout the clinical course.
• But ASO-PCR suffers from several limitations:
CONCEPTUAL AND TECHNICAL PREREQUISITES OF MM-MRD STUDIES
Landgren O. Cytometry Part B 2016; 90B: 14-20.
• Requires patient-specific primers.• Not easily accessible.• Costly and long to develop (3-4 weeks).• Applicable to 60-70% of patients only.• Same problems related to patchy disease distribution in the BM.
BCSH Guidelines for the Diagnosis and Management of Multiple Myeloma(Bird JM. www.bcshguidelines.com/documents/myeloma_guideline_feb_2014_for_bcsh.pdf)
Table 6, page n. 78:
Immunohistochemistry forabnormal PC detection in BMis defined as UNRELIABLE
(0.26 - 1.65)
(at which level?)
78726660544842363024181260
1,0
,9
,8
,7
,6
,5
,4
,3
,2
,1
0,0
Relapse-Free Survival of MM: Impact of immunophenotyping at 3 months post-ASCT
Rela
pse
-F
ree
Sur
viva
l
— <0.01% MM-PC
— ≥ 1% MM-PC
Months from immunophenotypical analysis(3 months post-ASCT)
RFS According to % of Residual Abnormal PC
p=0.0001
40m
23m
— 0.01% to 1% MM-PC
NR
Paiva B. Blood 2008; 112: 4017- 4023
(n=200 patients)
• Landgren O. Cytometry Part B 2016; 90B: 14-20.• Rawstron AC. J Clin Oncol 2013; 31: 2540 - 2547.• Paiva B. Blood 2012; 119: 687- 691.
Cytogenetic Risk
FCM MRD Status
Progression-Free Survival (Months)
p =
MRD+ 33.7
Standard 0.014
MRD- 44.2
MRD+ 8.7
High 0.09
MRD- 15.7
Pivotal Studies on FCM MM-MRD by British and Spanish Groups
• Universal applicability and clinical significance of FCM MM-MRD.
• According to current guidelines, it is now contradictory to labela patient as in sCR when he/she is found to be FCM MRD+.
• Need for strict laboratory standardization.
Defining a Sample as UNSUITABLE for MRD Analysis
• When an excess peripheral blood contamination is observed(i.e. if CD16+ mature myeloid cells are >20%).
• When red cell precursors are overall < 10-15 %.
• When B-Lymphoid progenitors are absent or < 1-2%.
• When normal PCs are undetectable.
• Whenever a reduced representation of Bone Marrow-nativecell populations can be demonstrated, namely:
It is always advisable to classify a sample as unsuitable for MRD analysis rather than reporting an inaccurately quantitated MRD level due to peripheral blood contamination. It is however possibleto report ‘MRD Detected but not quantitated’ in selected cases.
• Aged samples (older than 48 hours).
• Visible clots are present.
• When sample deterioration has occurred:
Setting the Cutoff: Reporting a Sample as MRD - Negative
• If MRD cells are NOT DETECTED in a sufficient number,then the minimum requirement for reporting is the level ofabnormal PC collected in relation to the adopted LOD.
• Anyway it is good practice to report the Total number ofacquired events along with the number of PC events.
• LOD and LOQ levels adopted by the lab for that specificnumber of acquired events should be clearly included in the report, for comparison.
• As an example of MRD-Negative test:
Abnormal PC events collected = 15 events (positive: if>30)Total leucocyte events acquired = 5,000,000 cellsPercentage of abnormal PC = 0.000003 % (MRD-Negative)Or better: Abnormal PC = < 0.0006 % (MRD-Negative) (Reference Values: LOD ≥ 0.0006 %; LOQ ≥ 0.0001 %)
Setting the Cutoff: Reporting a Sample as MRD POSITIVE
• Sample prerequisites must be met.
• Neoplastic AND normal PCs must be detectable.
• Neoplastic PC are detected ABOVE THE LOQ %
• As an example of MRD-POSITIVE test:
Abnormal PC events collected = 1000 events Total leucocyte events acquired = 2,000,000 cellsPercentage of abnormal PC = 0.05 % (MRD Positive)
(Reference Values: LOD ≥ 0.0006 %; LOQ ≥ 0.0001 %)
Multicolor Flow Cytometry to Study MRD in MM
• The quantitative assessment of tumor load at 10-5 level has provedMORE INFORMATIVE than the clinical categorization ofpositive / negative CR.
• Highly sensitive Multicolor FCM (10-5) is likely to improve theprediction of MM patients outcome, at a level comparable tomore complex and costly molecular assays.
• Previous MM-MRD studies, defining MRD at 10-4 level, are OBSOLETE.
• Multicolor approach is mandatory for the required accuracy (≥ 8 colors)and FCM should be appropriately set to collect the necessary highcell numbers (Millions of cells!).
• Applicable to virtually 100% of patients without requiring patient-specific profiles or pre-treatment samples for comparison.
• Intra-assay QC to assess sample suitability and to evaluate thepresence of other cell types.
Backbone Markers for File Merging
FCM ANALYSIS OF MM-MRD: State-of-the-Art
H450 H500 FITC PE PerCP-cy5.5 PE cy7 APC APC-H7
CD45 CD138 CD38 CD56 β2micro CD19 cyKappa cyLambda
CD45 CD138 CD38 CD28 CD27 CD19 CD81 CD117
Two 8-Color Tubes According to EuroFlow - Version 6
1
2
• The two tubes are necessary for a complete assessment of PC features.
• For the Infinicyt File Merging option, the Fix/Perm procedure is needed also for Tube n.2, despite the absence of intracellular stainings.
• At lest 2-3 Million cell events must be collected with each tube.
• The two EuroFlow lyotubes will be commercially available in 2016.
Flores-Montero J. Leukemia 2012; 26: 1925-1929.
Backbone Markers for File Merging
H450 H500 FITC PE PerCP-cy5.5 PE cy7 APC APC-H7
CD20 CD45 cyKappa cyLambda CD138 CD56 CD38 CD19
CD28 CD45 CD81 CD117 CD138 CD27 CD38 CD19
1
2
FCM ANALYSIS OF MM-MRD: Alternative Technical Options
• Tube 1 can be used for SCREENING, whereas Tube 2 is added for follow-upand MRD studies. File Merging requires Fix/Perm of both tubes anyway.
• Tube 2 ALONE can be used for MRD studies (no need for cyLight Chains).
• β2-Micro is replaced by CD20 because the latter remains stable even afterchemotherapy and can give clues on the presence of a t(11;14) translocation.
• Addition of CD20 is also useful in the simultaneous analysis of otherlymphoproliferative disorders and Waldenström macroglobulinemia.
• Some fluorochrome choices are aimed at minimizing the number of conjugatesto be purchased.
V450 BV510 FITC PE PerCP-cy5.5 PE cy7 APC APC-H7
CD56 CD27 CD81 CD117 CD19 CD38 CD138 CD45
CD56 CD27 cyKappa cyLambda CD19 CD38 CD138 CD45
1
2
FCM ANALYSIS OF MM-MRD: Alternative Technical Options
• The HMDS - Leeds (Andy Rawstron’s) approach.
• Tube 1 can be used alone both for diagnosis and for MRD study.
• Tube 2 is optional, and the cytoplasmic Light Chains are placed at FITC/PEjust because they are the commonest conjugates.
• Emphasis is given to the internal negative control gating syntax, to clearlyassess the positive/negative judgement.
• Conventional logical gating. No β2-Micro nor Backbone markers are used.
Conventional Flow-MRD Assay: MRD Positive Case
Abnormal PC
Normal PC
CD27 CD117CD28
• MRD Study - 1 Tube, Conventional Analysis• 1 Million BM cells acquired (Erythroid removed)• LOD = 0.003% LOQ = 0.005 %
• 147 Abnormal PCs collected (0.014%)• MRD Positive
CD19 CD81
Conventional Flow-MRD Assay: MRD - Negative Case
0.026
0.00008
• MRD Study - 1 Tube, Conventional Analysis• 2.725 M BM cells acquired & erythroid removed• LOD = 0.0012%
• 2 Abnormal PCs collected (0.00008%)• MRD Negative
Consensus on a standardized,robust MM-MRD gating protocolwith conventional FCM analysis softwares is still lacking.
• Post-ABMTx BM analysis.• 2.6 Million BM cells (Erythroid removed)
• LOD = 0.0012% LOQ = 0.002 %
• 236 Abnormal PC collected (0.009%)• MRD Positive
Next Generation Flow-MRD Assay: 2 EuroFlow Tubes Merged (a)
Abnormal PC
CD81
CD81
CD27
CD56 Kappa
Lambda
CD38
CD45
Abnormal PCContourat Onset
CD81
CD19
ResidualAbnormal PCs22 Events
CD27 CD81
CD28
Normal PCs158 Events
CD138
• MRD Study (2 EuroFlow tubes merged)• 3.0 Million BM cells acquired (Erythroid removed)• LOD = 0.001% LOQ = 0.0017%
• 22 Abnormal PCs collected (0.0007%)• MRD Detected but not Quantitated
Next Generation Flow-MRD Assay: 2 EuroFlow Tubes Merged (c)
Flores-Montero J. Cytometry Part B 2016; 90B: 61-72.
Next Generation Flow MRD in MM: Infinicyt by merging the two 8-color EuroFlow tubes with 5+5 Million Bone Marrow cells: 10-5 sensitivity.
Normal Polyclonal PC
0.002% ClonalResidual PC (226 events)10 Million
BM Cells
Erythroid removed?
* Proven polyclonal by CyIg staining
% A
bno
rmal PC
inf
iltr
ation
rou
tine
(con
vent
iona
l 8-co
lor
flow
MRD)
% Abnormal PC infiltration Tube 1(Next Generation Flow MRD)
10-5
10-4
10-3
10-2
10-1
Neg
10-6
Neg 10-6 10-5 10-4 10-3 10-2 10-1
2/54 (4%)
27/54 (50%)
9/54 (17%)
16/54 (30%)
*
Next Generation Flow MRD vs Conventional 8-color Flow MRD
Courtesy ofAlberto Orfao, 2015
Conventional 8-Color Flow MRD vs Next Generation Flow MRD
CONVENTIONAL 8-Color NEXT GENERATION FCM
• Bulky cell files (1-5 Mi.) requirebulky cell preparations or multipletubes to be sequentially accruedusing the ‘append’ function
• The required bulky cell files (1-5 Mi.) are created with the‘File Merge’ function, by puttingtogether various smaller files.
• The conventional files retain thedecided MoAb composition. Moreinformation can be inferred byadding other (separate) tubes.
• The addition of multiple tubeswith the same backbone markersgenerates additional informationwithin the same merged file.
• Conventional analysis softwaressuffer very bulky files: everysmall adjustment in regionsrequires lenghty display refreshes.
• Merged files are managed in atotally different manner, so theanalysis is quick and smooth.
• Limited spectrum of statisticaloptions.
• Extended statistical andanalytical options (APS, Compass)