Flow cytometry for MRD detec1on: Focus on AML. Sindhu Cherian University of Washington, Sea6le, WA, USA

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1 Flow cytometry for MRD detec1on: Focus on AML Sindhu Cherian University of Washington, Sea6le, WA, USA

2 Residual disease in hematopoie1c malignancy Residual disease has tradi:onal been defined by morphology in hematopoie:c neoplasms Minimal residual disease (MRD) is defined as disease present at a level below the sensi:vity of morphologic detec:on Performed using a method more sensi:ve and specific than morphology Flow cytometry Molecular methods

3 Focus of this talk will be on AML Tradi:onal criteria for response in AML were created in the mid 1950s and included Morphologic criteria blast <5% Clinical criteria count recovery, no evidence of extramedullary disease We have had the ability to detect MRD using different methods for >20 years Flow cytometry Molecular methods Increasing sensi1vity 10X flow Standard PCR 100X flow Next genera:on sequencing

4 Molecular methods: MRD in AML Molecular methods: PCR for reduc:on in over expressed genes (eg WT1) Sensi:vity ~0.01% Applicable in ~50% of AML Directed PCR for a muta:on or transloca:on Sensi:vity % Highest sensi*vi*es using next genera*on sequencing methods Current targets include Muta:ons: NPM1, MLL, CEBPa Transloca:ons such as t(8;21), inv(16), t(15;17) Currently applicable ~50-70% of cases Poten:al is unknown and growing as we detect new markers with next genera:on sequencing plaaorms The role of molecular diagnos:cs for MRD detec:on in AML is likely to grow in the future

5 Flow cytometry for MRD in AML Sensi:vity ~0.01% Reported to be informa:ve in ~85-95% of cases in most studies Several variables underlie the success of flow cytometry for MRD detec:on in AML Number of events collected 500,000 to 1,000,000 More events more sensi:ve Number of colors used More informa:ve if more colors are used in the same tube Ideally 6 Experience of operator Immunophenotype of blasts and difference from normal may impact ease of detec*on Familiarity with normal an:gen expression pa6erns is cri:cal

6 Comparison of flow and PCR for MRD Detec1on in AML Flow cytometry is currently more widely applicable Flow cytometry is applicable in 85-95% of cases when using at least 6 colors Molecular methods apply in 50-70% of cases Molecular methods, when applicable are more sensi:ve Molecular (PCR for transloca:on or gene muta:on) Sensi:vity ~0.001% Next genera:on sequencing methods even more sensi:ve Flow Sensi:vity 0.01%

7 Impact of MRD on survival in AML Regardless of method used to measure MRD, the impact of MRD post therapy is clear MRD holds up in mul:variable analysis as a predictor of survival and relapse in mul:ple studies Some (including Buccisano et al and Loken et al. 2012) suggest including AML MRD in designing risk adapted therapeu:c regimens It is clear that MRD will be an important part of monitoring in pa:ents with AML post therapy and that flow cytometry has an important role in establishing MRD

8 Clinical impact of MRD in AML Author Publica1on year Clinically significant 1me point: level of MRD with clinical significance San Miguel et al End I: 0.5% End Int: 0.2% Vendif et al End C: 0.035% Sievers et al End I: 0.5% Kern et al End C: LD Al-Mawali et al End I: 0.15% Rubnitz et al End I: 1% Bucciscano et al Post C: 0.035% Loken et al Post I: Any, similarly bad if >0%/<1% or 1% Walter et al. 2011, 2012 Any MRD at CR1 and CR2 pre transplant predicted increased overall mortality and likelihood of relapse Chen et al 2015 Any MRD, end induc:on I=induc:on, Int=intensifica:on, C=consolida:on, CR-complete remission

9 245 adults in CR post induc1on for AML Cumula1ve incidence of relapse Overall survival Chen et al. JCO 2015;33(11):

10 359 consecu1ve adults with AML who underwent myeloabla1ve allogeneic HCT: MRD pre transplant was associated with poorer outcomes Overall survival Cumula1ve incidence of relapse Araki et al. J Clin Oncol Feb 1; 34(4):

11 Open ques1ons regarding MRD detec1on by flow cytometry in AML What is the best :me point to assess for MRD Controversial as to whether post induc:on or post consolida:on in most informa:ve Kern et al pose a role for early blast clearance (Day 16) Pre and post transplant MRD seems informa:ve What is the most important threshold Range of 1% to any level of MRD (no lower threshold) Most groups use WBC as the denominator while occasional groups use mononuclear cells Kern et al recommend using degree of reduc:on in abnormal blasts (log difference) rather than a percentage

12 Strategies for flow cytometric iden1fica1on of MRD Difference from normal Iden:fying a leukemia associated phenotype Best strategies combine the two

13 Difference from normal In our laboratory we focus on the difference from normal approach to define MRD. 10 color flow cytometry >500,000 events collected for MRD analysis This relies on a thorough understanding of pa6erns of an:gen expression on normal blasts. In the next few slides we will review normal an:gen expression on blasts and discuss how an:gen expression can be altered in the face of a myeloid stem cell neoplasm (in this case AML)

14 Panel used in our laboratory LSR II with 4 lasers (Becton Dickinson) Allows assessment of 10 colors

15 Iden1fying progenitors Progenitor iden:fica:on generally begins with the CD45 vs SSC defined blast gate (progenitors) can be posi:vely iden:fied using a progenitor marker (CD34 +/- CD117) Be aware: Some neoplas2c popula2ons may be overlooked if you use only this strategy AML with monocy2c differen2a2on, acute promyelocy2c leukemia Viable cells CD45 versus SSC defined blast gate Viable cells Blast gate "Blast gate" In most plots you see in this presenta:on, CD34+ blasts will be colored red

16 The CD45 vs SSC defined blast gate contains more than just progenitors When undertaking AML MRD tes:ng, it is important to recognize other cells that reside in the CD45 vs SSC defined blast gate so these are not confused for an abnormal blast popula:on Basophils, A panel for AML MRD detec:on would allow reliable differen:a:on of these popula:ons from normal progenitors and abnormal blasts Viable "Blast gate" "Blast gate" Viable cells Blast Blast gate Blast gate CD 123 PE

17 Normal progenitor development CD34 APC HLA-DR PB CD34 APC CD33 PE CD13 PE-Cy7

18

19 Progenitor abnormali1es Abnormali:es on myeloid progenitors (blasts) seen in myeloid stem cell neoplasms usually fall into one of 4 categories Abnormal intensity of an:gen expression (increased, decreased or absent). Asynchronous expression of an:gens associated with maturity with an:gens deno:ng immaturity Homogeneous expression of an an:gen usually expressed at varying levels with matura:on Expression of an:gens from other lineages. Recogni:on of such abnormali:es requires a thorough understanding of normal pa6erns of an:gen expression

20 Abnormal intensity of an1gen expression Normal AML CD33 PE CD33 PE

21 Asynchronous expression of an1gens associated with maturity and an1gens associated with immaturity Normal AML CD34+

22 Homogeneous expression of an an1gen usually expressed at varying levels through blast matura1on Normal AML CD34 APC

23 Expression of non-lineage an1gens Normal Blast gate AML CD5 PE-Cy5 CD7 PE Blast gate CD5 PE-Cy5 CD7 PE

24 Leukemia associated immunophenotype Using a different from normal strategy to iden:fy abnormal blast popula:ons in AML, leukemia associated immunophenotypes (LAIP) can be detected in most cases Most studies using 6 colors detect LAIP in 85-95% of AML For a sample list of specific LAIPS that have been described in AML in the literature see the following references Al-Mawali et al AJCP Olaru et al Cytometry Part B (Clinical Cytometry) In our laboratory (using 10 colors) we are able to define an abnormal immunophenotype in almost all cases of AML In the next few slides we will review some challenges in AML MRD detec:on by flow cytometry through a series of cases

25 Challenge 1: Blast phenotype in AML can be heterogeneous It has been long observed that abnormal blasts present in AML can have a very heterogeneous immunophenotype More than one LAIP may characterize a blast popula:on in AML At relapse or during/ater therapy, it is possible that the dominant clone will recede and that a minor clone will dominate Popula:on changes described in 31% of cases at relapse compared to diagnosis (Baer et al. 2001)

26 Example 1 Diagnosis WBCs Myeloid CD34+ CD13 PE-Cy7 CD5 PE-Cy5 Normal WBCs Myeloblasts CD13 PE-Cy7 CD5 PE

27 Myeloid CD34+.tc s. re s. WBCs in The immunophenotype of the blasts is not homogeneous CD13 PE-Cy7 CD5 PE-Cy5 Myeloid CD34+ w w w WBCs CD13 PE-Cy7 CD5 PE-Cy5

28 WBCs Abnormali1es in this case Myeloid CD34+ CD13 PE-Cy7 CD5 PE-Cy5 Abnormal intensity of an1gen expression (increased, decreased or absent). Dominant red popula:on: Increased CD34, CD33, CD13 Variably decreased CD38 Minor orange popula:on Decreased CD38 Asynchronous expression of an1gens associated with maturity with an1gens deno1ng immaturity CD15 expression on a minor subset of CD34++ blasts Homogeneous expression of an an1gen usually expressed at varying levels with matura1on Uniform CD13, CD33, and CD34 Expression of an1gens from other lineages. Minor orange popula:on CD5 expression

29 Post transplant peripheral blood, day % circula:ng CD34+ blasts WBCs Myeloid CD34+ CD13 PE-Cy7 CD5 PE-Cy5 Normal WBCs Myeloblasts CD13 PE-Cy7 CD5 PE The blasts in the peripheral blood are: A. Normal B. Abnormal

30 Post transplant peripheral blood, day 24 WBCs Myeloid CD34+ Diagnosis CD13 PE-Cy7 CD5 PE-Cy5 WBCs Myeloid CD34+ CD13 PE-Cy7 CD5 PE-Cy5 The blasts in the peripheral blood resemble: A. The dominant popula:on seen at diagnosis (red) B. The minor popula:on seen at diagnosis (orange) C. A combina:on of the two

31 Post transplant peripheral blood, day 24 WBCs Myeloid CD34+ CD13 PE-Cy7 CD5 PE-Cy5 Day 28 post transplant marrow 0.8% CD34+ blasts WBCs CD13 PE-Cy7 CD5 PE-Cy5 Normal for comparison WBCs Myeloblasts CD13 PE-Cy7 CD5 PE

32 Post transplant peripheral blood, day 24 WBCs Myeloid CD34+ CD13 PE-Cy7 CD5 PE-Cy5 Day 28 post transplant marrow 0.8% CD34+ blasts WBCs CD13 PE-Cy7 CD5 PE-Cy5 Day 28 post transplant sample with abnormal blasts highlighted in orange (0.2% of the WBC), normal background regenera:ng blasts present in red WBCs CD13 PE-Cy7 CD5 PE-Cy5

33 Myeloid CD34+ s. re s. WBCs in Diagnosis CD13 PE-Cy7 CD5 PE-Cy5.tc Relapse post transplant day 100 Myeloid CD34+ w w w WBCs CD13 PE-Cy7 CD5 PE-Cy5 The blast popula:on seen at recurrence has immunophenotypic features that overlap between the 2 dominant popula:ons present at diagnosis

34 Challenge 2: Prac1cal considera1ons How many events does it take to cons:tute and abnormal popula:on? What is the benefit of more colors?

35 Example 2 65 year old male with cytopenias Diagnos:c marrow for flow cytometry CD7 APC CD117 PE-Cy5 CD33 PE Normal for comparison CD7 APC CD117 PE-Cy5 CD33 PE

36 Abnormali1es in this case CD7 APC CD117 PE-Cy5 CD33 PE Abnormal intensity of an1gen expression (increased, decreased or absent). Decreased CD117 Increased CD34 Variably decreased CD38 Variably decreased HLA-DR Asynchronous expression of an1gens associated with maturity with an1gens deno1ng immaturity CD15 expression on CD34++ blasts Homogeneous expression of an an1gen usually expressed at varying levels with matura1on Bright homogeneous CD34 Homogeneous CD33 Expression of an1gens from other lineages. CD7 co-expression on a subset

37 Post-therapy, pre transplant specimen CD7 PE At first glance looks Normal for comparison ok but CD7 PE

38 Although, most blasts look normal and regenera1ng (red) A small abnormal blast popula1on is highlighted in purple with similar features to that seen at diagnosis CD7 PE CD117 PE-Cy5 CD33 PE Diagnosis CD7 CD7 APC APC CD15 FITC CD117 CD117 PE-Cy5 PE-Cy5 CD33 CD33 PE PE

39 Popula1on iden1fica1on Ga1ng strategy 1. CD45 vs SSC defined blast gate 2. CD34+ blasts colored red CD19 PE-CF total events collected WBC (viable only, nrbc excluded)

40 Popula1on iden1fica1on Ga1ng strategy 1. CD45 vs SSC defined blast gate 2. CD34+ blasts colored red 3. CD34 bright vs CD38 low CD19 PE-CF594 CD total events collected WBC (viable only, nrbc excluded)

41 Popula1on iden1fica1on Ga1ng strategy 1. CD45 vs SSC defined blast gate 2. CD34+ blasts colored red 3. CD34 bright vs CD38 low 4. CD34 vs CD15 demonstrates dysschronous an:gen expression 5. HLA-DR VS CD117 CD117 remains low, HLA-DR is even lower than was seen at diagnosis CD19 PE-CF594 CD34+ CD117 PE-Cy total events collected WBC (viable only, nrbc excluded) Abnormal cells are 37 total events 0.01% of the WBC

42 Popula1on iden1fica1on Ga1ng strategy 1. CD45 vs SSC defined blast gate 2. CD34+ blasts colored red 3. CD34 bright vs CD38 low 4. CD34 vs CD15 demonstrates dysschronous an:gen expression 5. HLA-DR VS CD117 CD117 remains low, HLA-DR is even lower than was seen at diagnosis CD19 PE-CF594 CD34+ CD117 PE-Cy total events collected WBC (viable only, nrbc excluded) Abnormal cells are 37 total events 0.01% of the WBC

43 How many events does it take to cons1tute an abnormal popula1on? Controversial Most studies required at least events that cluster :ghtly together with mul:ple an:body combina:ons. More colors increases your specificity as you have more an:body combina:ons to evaluate To detect an event occurring at 0.01% (1/10,000) CV of 5% if you collect 400 events/4 X 10 6 CV of 10% if you collect 100 events/1 X 10 6 CV of 20% if you collect 25 events/2.5 X 10 5 Sta*s*cal versus analy*c sensi*vity See Hedley and Keeney 2012 for a discussion of rare event analysis

44 What if you had fewer events in this case? total events viable WBC 200,000 total 100,000 total 50,000 total CD117 PE-Cy5 CD117 PE-Cy5 CD117 PE-Cy5 CD117 PE-Cy5

45 How many colors are adequate Studies evalua:ng MRD by flow have been reported to evaluate between 3 and 10 colors simultaneously In general, the more colors u:lized, the higher the sensi:vity for picking up an abnormal popula:on Olaru et al demonstrated that by increasing the number of colors form 4 to 6, a greater number of LAIPs could be detected. 6 color tube allowed detec:on of 47 different LAIPS All of the 53 AML samples studies had at least one LAIP 4 color tube allowed detec:on of only 30 LAIPS

46 What if you had fewer colors? Consider a tube with CD45, CD34, CD38 and CD15 2. CD34++/CD38 low progenitors 3. CD34++/CD15 variable abnormal progenitors 1. CD45 vs SSC defined blast gate With 4 colors, you can get partly there but cannot check expression of other markers (CD7, CD13, CD33, CD117, HLA-DR) to confirm. Albeit, 4 colors would probably work in this case with a well chosen an:body panel. We will explore this ques:on further in a later case

47 Challenge 3: Blast immunophenotype may shig Blast popula:ons may change immunophenotype with treatment failure or at relapse Reported in over half of AML cases in some studies Described in 91% of cases in one study Changes described include A shit in an:gen expression on a blast popula:on A change in the dominant popula:on seen (perhaps related to challenge 1) Markers reported to shit commonly include CD13, CD33, CD34 may be gained at relapse Loss of CD56 and CD19 has been reported at relapse The changes may prevent you from iden:fica:on of an abnormal blast popula:on if you are strictly direc:ng your search to find a LAIP seen at diagnosis Abnormal popula:ons can usually s:ll be iden:fied by using a different from normal approach. References: Baer et al. 2001, Oelschlagel et al 2000, Macedo et al. 1996

48 Diagnosis 3 months later Early MRD + sample post treatment CD38 CD15 A594 FITC HLA-DR PB HLA-DR PB Overt relapse CD38 CD15 A594 FITC HLA-DR PB

49 Challenge 4: Understanding normal in res1ng and regenera1ve seings is cri1cal Understanding immunophenotypic changes that can arise with marrow regenera:on is cri:cal in dis:nguishing MRD from marrow regenera:on in the post chemotherapy sefng Several studies have detailed common LAIPS seen in AML and dis:nguished these from changes seen in normal and regenera:ng marrow Al-Mawali et al AJCP Olaru et al Cytometry Part B (Clinical Cytometry) Also important is understanding other normal popula:ons present in the blast gate so these are not confused for abnormal blasts Remember basophils and plasmacytoid dendri:c cells? Understanding how the AML MRD panels in your lab perform with normal specimens and with regenera:ng marrow is cri:cal before undertaking AML MRD tes:ng

50 60 year old female with history of chemotherapy for breast cancer, now with cytopenias and circula:ng blasts Bone marrow flow cytometry was performed Increased cells in the blast gate (CD117+ blasts comprise 6.5% of the WBC and aberrantly lack CD34) Monocy:c cells and increased overall (68%) and include an expanded (34%, yellow) immature subset with decreased to absent CD14 and par:al CD56 Of note, concurrent molecular tes:ng demonstrates the NPM1 muta:on WBCs WBCs WBCs CD117 PE-Cy5 CD14 PE-Cy55

51 Focus on the CD117+ component Pa:ent CD33 PE HLA-DR PB Normal Abnormal intensity of an:gen expression (increased, decreased or absent). Absent CD34, bright CD33 Mildly decreased CD38 Decreased to absent HLA-DR Asynchronous expression of an:gens associated with maturity with an:gens deno:ng immaturity Decreased CD38 with absent CD34, bright CD33 CD33 PE HLA-DR PB Homogeneous expression of an an:gen usually expressed at varying levels with matura:on Homogeneous, increased CD33 Expression of an:gens from other lineages. None NPM1+

52 Day 28 post therapy Normal CD33 PE HLA-DR PB CD33 PE HLA-DR PB As compared to a normal control, progenitor matura:on remains on the expected spectrum but is shited to the right and rela:vely homogeneous

53 Day 28 post therapy No blast popula1on similar to that seen at diagnosis is iden1fied Diagnosis HLA-DR PB Blast CD33 PE HLA-DR PB HLA-DR PB CD33 PE HLA-DR PB What is the purple highlighted by blue arrows? A. Hypogranular neutrophil? B. Basophil? C. Subset of normal blasts? D. Plasmacytoid dendri:c cell? NPM1-

54 Day 50 post transplant Pa:ent Begin by evalua:ng the following plots: Normal for comparison CD34 vs CD38 CD34 vs CD15 Diagnos:c Diagnosis specimen Nothing drama1c is apparent at first glance...

55 Day 50 post transplant However, in looking at other projec1ons, a popula1on is evident that expresses bright CD33 without HLA-DR that is absent in normal specimens. basophils Normal HLA-DR PB HLA-DR PB CD33 PE CD117 PE-Cy5 HLA-DR PB? GATING STRATEGY: 1. CD45 vs SSC defined blast gate 2. CD33 bright HLA-DR nega:ve cells 3. Bright CD117 with intermediate CD38 4. Note that CD34 is now posi:ve on a subset (was absent at diagnosis total events WBC HLA-DR PB HLA-DR PB HLA-DR PB 171 abnormal blasts 0.03% abnormal blasts

56 What if you had fewer colors? Consider the 4 color tube with CD45, CD34, CD38 and CD15 we tried in case 1 WBCs Iden:fying the abnormal popula:on would not be possible with this tube in this case CD38 CD15 A594 FITC

57 Follow up-day 80 post transplant WBCs CD38 A594 Relapsed AML with 15% CD117+ blasts CD33 PE HLA-DR PB Pa:ent at diagnois CD33 PE HLA-DR PB

58 Summary MRD is useful for prognos:ca:on in many hematopoie:c neoplasms including AML In AML, MRD has been proven to provide powerful prognos:c informa:on in both adult and pediatric popula:ons As we move forward MRD may impact risk adjusted therapeu:c strategies in AML Flow cytometry is currently applicable in most cases for MRD detec:on Currently applicable in more cases than molecular tes:ng although this may be shiting in the future Strategies include a difference from normal approach combined with iden:fying a LAIP

59 Summary Several unanswered ques:ons remain in AML MRD tes:ng by flow cytometry What is the best :me point to assess for MRD Controversial: Induc:on versus Consolida:on What is the most important threshold Range of 1% to any MRD Log reduc:on in abnormal cells What do you need to do if you want to bring AML MRD tes:ng into your lab Develop panels for MRD tes:ng At minimum 6 color: more colors=greater sensi:vity and specificity Must collect >500,000 events for adequate sensi:vity Get familiar with normal At bare minimum normal and 5-10 marrow regenera:on samples

60 References Utility of flow for MRD in AML Wood. Clinical Cytometry 2016;90(1): Araki et al. J Clin Oncol Feb 1; 34(4): Chen et al. JCO 2015;33(11): Bastos-Oreiro et al. European Jorunal of Haematology. 2014;93 (3): Walter et al. Significance of MRD before myeloablative allogeneic hematopoietic cell transplantation for AML in first and second complete remission. Blood. Pre-published online July 2013 Hourigan and Karp. Minimal residual disease in acute myeloid leukemia. Nature reviews in clinical oncology 2013;10: Loken et al. Residual disease detected by multidimentional flow cytometry signifies high relapse risk in patient s with de novo AML: a report from COG. Blood. 2013;120(8): Buccisano et al. Prognostic and therapeutic implications of minimal residual disease detection in acute myeloid leukemia. Blood. 2012;119(2): Walter et al. Impact of pre-transplant minimal residual disease, as detected by MFC, on outcome of myeloablative hematopoietic cell transplant for AML. Journal of Clinical Oncology. 2011;29: Buccisano et al. Cytogenetic and molecular diagnostic characterization combined to post consolidation MRD assessment by flow cytometry improves risk stratification in adult AML. Blood. 2010;116(13): Rubinitz et al. MRD directed therapy for childhood AML: results of the AML02 multicentre trial. Lancet Oncology. 2010;11:

61 References Utility of flow for MRD in AML Al-Mawali et al. The role of multiparameter flow cytometry for detection of MRD in AML. AJCP. 2009;131: Al-Mawali et al. The use of ROC analysis for detection of MRD using 5 color multiparameter flow cytometry in AML identifies patients with high risk of relapse. Cytometry Part B (Clinical Cytometry). 2009;76B: Al-Mawali et al. Incidence, sensitivity, and specificity of leukemia associated phenotypes in AML using specific 5 color multiparameter flow cytometry. AJCP. 2008;129: Olaru et al. Multiparametric analysis of normal and postchemotherapy bone marrow: implication for the detection of leukemia-associated immunophenotypes. Cytometry part B 2008;74b: Wood BL. Myeloid malignancies: myelodysplastic syndromes, myeloproliferative disorders, and acute myeloid leukemia. Clin Lab Med. Sep 2007;27(3): , vii. Kern et al. Determination of relapse risk based on assessment of MRD during complete remission by MPFC in unselected patients with acute myeloid leukemia. Blood 2004;104: Sievers et al. Immunophenotypic evidence of leukemia after induction therapy predicts relapse: results from a prospective Children s Cancer Group study of 353 patients with AML. Blood. 2003;101(9):

62 References Cont. Rare event analysis: Hedley and Keeney. Technical issues: flow cytometry and rare event analysis. International Journal of Laboratory Hematology. 2013;35: Immunophenotypic changes Baer et al. High frequency of immunophenotypic changes in acute myeloid leukemia at relapse: implications for residual disease detection. Blood 2001;97: Oleschlagel et al. Shift of aberrant antigen expression at relapse or at treatment failure in Acute leukemia. Cytometry. 2000;42: Macedo et al. Phenotypic changes in acute myeloid leukemia: implications in the detection of minimal residual disease. Journal of Clinical Pathology. 1996;49: Flow versus molecular MRD testing Rossi et al. Comparison between muiltiparameter flow cytometry and WT1-RNA quantification in monitoring MRD in AML without specific molecular targets. 2012;36: Inaba et al. Comparative analysis of different approaches to measure treatment response in AML. Journal of Clinical Oncology. 2012;30:

63 AML MRD normal WBCs CD19 PE-CF594 CD117 PE-Cy5 CD33 PE CD117 PE-Cy5 CD33 PE

64 AML MRD Normal, CD34+ myeloid blasts WBCs CD34+ CD34+ CD34+ CD34+ CD34+ CD34+ CD19 PE-CF594 CD34+ CD117 PE-Cy5 CD33 PE CD117 PE-Cy5 CD33 PE