Flow Cytomety Immunophenotyping For

Myelodysplastic Syndromes

Sa A.Wang, MDDept. of Hematopathology

UT MD Anderson Cancer CenterHouston, TX

Myelodysplastic Syndromes

Definition:A group of heterogeneous clonal

hematopoietic stem cell diseases1)

Ineffective hematopoiesis

with peripheral cytopenia(s),

2)

Morphological dysplasia3)

Propensity for development of acute myeloid leukemia (AML)

Etiology

Primary (de novo)

Therapy-related

Clinical Scenario for MDS Bone Marrow Work-up

Patients Presented with Cytopenia(s), Cytopenia(s) often:

Unremitting ≥6 months

Hb<10

Absolute Neutrophil

count (ANC) 1.8

Plt: 100But, cytopenia can be less severe, and presents in a

shorter duration

Hematologists and primary physicians often conduct extensive clinical/laboratory work-up, or even give empirical treatment (iron, B12, folate, epo), however, when/if there is a need to rule out:

A bone marrow process

Possible Findings in Bone Marrow

As a Pathologist/hematopathologist, what we expect to find in BM that possibly explain cytopenia(s):

1. Intrinsic Bone Marrow Stem Cell Neoplasm1.

MDS or MDS/MPN or MPN (CIMF)2.

Acute leukemia (AML, ALL)

2. Bone marrow infiltrative processes1.

B cell lymphoma, especially low grade2.

T-cell neoplasm: Large granular lymphocytic leukemia3.

Plasma cell Neoplasm4.

Metastatic carcinoma

3. Bone marrow Failures (Congenital, infection, immune-

mediated, PNH, Drug/toxin, et al)1.

Aplastic

Anemia2.

Single lineage aplasia/hypoplasia

Red cell/Myeloid/Mega

Normal Bone Marrow

BM Biopsy

Normal fat/cellular distribution, megakaryocytes

are normal in number and morphology

BM Aspirate

Trilineage

Hematopoiesis, orderly maturation, and normal morphology

MDS Bone Marrow (Biopsy)

Altered bone marrow topographyAdipocyte

clustering, hematopoietic cell clustering, immature cells away from the bone paratrabeculae, increased histiocytes, stromal

cells, vasculature, and dysplastic megakaryocytes.

Morphological Dysplasia

Erythroid Dysplasia

Morphological Dysplasia

dysmegakaryopoiesis

Myeloid Dysplasia

Blasts in MDS

1.

Count 500 cells on BM aspirate smears, and 200 cells on peripheral blood smears2.

Myeloblasts: granular and agranular

type3.

The presence of Auer rods would qualify a case as RAEB-24.

MDS progress to acute leukemia, almost always AML, cases of lymphoblastic leukemia transformation are rarely reported

2008 World Health Organization Classification of Myelodysplastic Syndromes

MDS subcategories Diagnostic features

Refractory cytopenias with unilineage dysplasia (RCUD) Refractory anaemia

(RA); Refractory neutropenia

(RN); Refractory thrombocytopenia (RT)

One or two cytopenia(s) with unilineage

dysplasia<1% blasts in blood<5% blasts in bone marrow

Refractory anaemia with ring sideroblasts (RARS) Dyserythropoiesis

with ≥15% ring sideroblasts<1% blasts in blood<5% blasts in bone marrow

Refractory cytopenia with multilineage dysplasia (RCMD) Cytopenia(s) with or without Ringed- sideroblasts (RCMD-RS)

Dysplasia in ≥

two myeloid lineages<1% blasts in blood<5% blasts in bone marrow

Refractory anaemia with excess blasts-1 (RAEB- 1)

Unilineage

or multilineage

dysplasia5-9% blasts in bone marrow<5% blasts in blood

Refractory anaemia with excess blasts-2 (RAEB- 2) Cytopenia(s)

1.

Unilineage

or multilineage

dysplasia2.

10-19% blasts in bone marrow3.

5-19% blasts in blood4.

If Auer rod present

Myelodysplastic syndrome – unclassified (MDS-U) 1.

RCUD or RCMD with 1% blasts in blood2.

<10% dysplasia, with cytogenetic abnormality as presumptive evidence of MDS

3.

RCUD with pancytopenia

MDS associated with isolated del(5q) 1.

Isolated del(5q) cytogenetic abnormality2.

Normal to increased megakaryocytes

with hypolobated

nuclei3.

<5% blasts, no Auer rods4.

<1% blasts in blood

Morphological Dysplasia

The MDS categories in the red boxes

BM blasts are <5%,

more than 50% of these cases have a normal karyotype.

To diagnose a case of MDS in those categories, all depend on the presence of morphological dysplasia.

Morphological DysplasiaMorphological dysplasia can be seen in a number of

non-MDS conditions1.

Nutritional deficiency (B12, folate, copper)2.

Drug/Toxin (arsenic

and alcohol, grow factor treatment, chemotherapy)

3.

Metabolic disorders, chronic diseases4.

Infection: HIV 5.

Collagen Vascular Diseases6.

Hemolysis7.

Aplastic

anemia, treated8.

Pediatric congenital disorders9.

…

Worst of all, MDS can coexist or evolve from these conditions

Non-MDS Mimics

HIV Bone marrow

Systemic Lupus

Dysplasia in the Therapy- Related Setting

Morphological dysplasia is extremely problematic in post-therapy related setting:

We retrospectively reviewed in the past 10 years of therapy-related myeloid neoplasm with a normal Karyotype

at MDACC:

196 cases showed significant dysplasia that pathologists raised a diagnosis of t-MDS at least in the comments

Only 65 were real t-MDS after long-term follow-up (follow-up biopies, clinical/Lab data and outcome)

Suboptimal BM Specimens

Assessment of morphological dysplasia is further confounded by Suboptimal Specimens

No good spicules

for evaluation“Dried Smears”Poorly stained smears: over-

or under-

stained smearsNo smears

Normal Hematopoiesis

is characterized by highly reproducible patterns

of antigen

expression during myeloid maturation.

MDS is a stem cell neoplasm, neoplastic

stem cells may show immunophenotypic

aberrancies

It would be an objective method, the historic reliance on subjective morphologic criteria is likely to be lessened, especially when the material and morphology are suboptimal

The Utility of Flow Cytometry Immunophenotyping in MDS

MDS Flow Cytometry, Literature

The first paper actually used flow cytometry

in clinical patients was published by Stetler-Stevenson M et al (Blood. 2001 Aug 15;98(4):979-87)

The CD11b/CD16 and CD13/CD16 are still widely used combinations to assess myeloid cells.

Many publications since then, where different panels, different markers, different scoring systems have been used

Difficult for people to follow

Diagnostic Utility of FCI in Myelodysplastic Syndromes

Stetler-Stevenson M et al Blood. 2001 Aug 15;98(4):979-87

Finally, a Consensus of Flow Cytometry

in Diagnosing MDS is

made by the European group

Standardization of flow cytometry

in myelodysplastic

syndromes: report from the first

European LeukemiaNet

working conference on flow cytometry

in myelodysplastic

syndromes.

Arjan

A van de Loosdrecht, Canan

Alhan, Marie Christine Béné, Matteo

G Della, Porta, Angelika M Dräger, Jean Feuillard, Patricia Font, Ulrich Germing, Detlef, Haase, Christa H Homburg, Robin Ireland, Joop

H Jansen, Wolfgang Kern, Luca, Malcovati, Jeroen

G te

Marvelde, Gulham

J Mufti, Kiyoyuki

Ogata, Alberto,Orfao, Gert

J Ossenkoppele, Anna Porwit, Frank W Preijers, Stephen J Richards, Gerrit

Jan Schuurhuis1, Dolores Subirá, Peter Valent, Vincent HJ van der

Velden, Paresh

Vyas, August H Westra, Theo M de Witte, Denise A Wells, Michael R Loken, Theresia

M Westers

Haematologica. 2009 Aug;94(8):1124-34.

In this article, there listed Recommendations for Standardization

Acknowledging the utility of FCI in diagnosing MDS and stratifying MDS risks

Recommendation for markers and panels

Recommendation for scoring

Recommendation for interpretation: descriptive in nature, with a statement that findings could be consistent with MDS

Flow cytometry immunophenotype

of MDS

After 10 years efforts, where is the position of FCI in MDS?

The diagnostic utilityt

is acknowledged by 2008 WHO, and

Acknowledged by the MDS international working group

2008 WHO Classification

Page 92-93

Minimal Diagnostic Criteria in MDS(A) Prerequisite criteria

Constant cytopenia: Hb

<11

g

dL; ANC

<

1500

μL

or platelets <100K

Exclusion of all other hematopoietic or non-hematopoietic disorders as primary reason for cytopenia/dysplasia

(B) MDS-related (decisive) criteriaDysplasia in at least 10% of all cells in a respective lineage or >15% ringed

sideroblasts

(iron stain)

5–19% Blasts in bone marrow smears

Typical chromosomal abnormality (by conventional karyotyping

or FISH)

(C) Co-criteria (for patients fulfilling ‘A’

but not ‘B’, and otherwise show typical clinical features, e.g. macrocytic

transfusion-

dependent anemia)Abnormal phenotype of bone marrow cells clearly indicative of a monoclonal population of

erythroid

or/and myeloid cells, determined by flow cytometry

Clear molecular signs of a monoclonal cell population in HUMARA assay, gene chip profiling, or point mutation analysis (e.g. RAS mutations)

Markedly and persistently reduced colony-formation (±cluster formation) of bone marrow or/and circulating progenitor cells (CFU-assay)

Leuk

Res. 2007 Jun;31(6):727-36

Flow Cytometry Panel For Cytopenia Work-up

Include B-cell clonality

Include basic T-cell markers, especially the markers detect large gradular

lymphocytes

(CD8, CD56, CD57)

If possible, take a look at plasma cells(normal plasma cells CD38+++, CD19+, CD56-; neoplastic

plasma cells CD38+++,

CD19+, CD56-)

MDS work-up

FCI in MDS: The Analytical Approaches

Bone Marrow cell Populations

Precursors

Myeloid cells

Monocytic

cells

Erythroid

Megakaryocytes (nearly impossible to

assess by flow cytometry)

CD45 V500-A

SSC

-A

-102 -101 102 103 104 105

-400

65236

130872

196508

262144

CD45

Erythroid Lineage (not very popular)

Problems with Erythroid

Lineage assessed by Flow cytometry1.

Limited markers

are commercially available (CD71, CD235a/glycophorin, CD36)

2.

Red cell lysis

(lyse

late stage hemoglobinized

nucleated red blood cells, not the entire spectrum of nucleated RBC available for analysis)

3.

Non-specific1.

Reactive erythroid

hyperplasia, left-shifted maturation, maturation arrest of erythroid

can produce similar flow cytometry

findings as seen in MDS2.

Increased Sideroblasts

can alter the maturation pattern, but often not MDS4.

Easy to assess ring sideroblasts

by iron stain

Della Porta’s

group showed some utility in assessing erythroid

cells by flow cytometry:

Markers can be used: CD71, CD105, cytosolic

H-ferritin, cytosolic

L-ferritin

and mitochondrial ferritin

(MtF)

Changes observed:

Decreased CD71 and increased HF in MDS

Increased proerythroblasts, left-shifted

MtF

correlate with the presence of ringed sideroblastsDella Porta

MG, Leukemia. 2006 Apr;20(4):549-55.

Myeloid Lineage

Mature vs Immature myeloid cells: they have different immunophenotypes

Immature (promyelocytes, myelocytes

and early metamyelocytes)

CD10−, CD64+, CD33bright+, CD15low+, CD13heterogenous+, CD16heterogenous+, and CD11b heterogenous+

Mature (late metamyelocytes, bands and segmented neutrophils)

CD10+, CD64 dim/−, CD33dim+, CD15bright+, CD13bright+, CD16bright+, and CD11b bright+) populations.

Four Patterns of Myeloid Maturation

Stachurski

D et al. Leuk

Res. 2008 Feb;32(2):215-24

The solid circles: immature myeloid; and the dash-circles: Mature myeloid

Diagnostic pitfalls and caveats

Separate the myeloid cells into mature and immature can help to recognize a left-shifted myeloid maturation; hemodilute

specimen,

increased eosinophils…

which could be misinterpreted as MDS

Aged specimen can show alterations mimicking MDS

Aged Specimen

Increased Eosinophils

Hemodilute Specimen

Left-shifted myeloid Maturation

Maturing Myeloid Cells Assessed by FCI, our experience

Hypogranulation: Useful

However, growth factor treatment, regenerating BM can produce hypogranulation

Significant alterations in CD11b/CD16 and CD13/CD16: Useful

be aware increased eosinophils, PNH cells, aged specimens

CD56 (if high percent, and high MFI), useful

Decreased CD13, CD33, not specific

Genetic polymorphism

Synchronous left-shifted Maturation (left-shifted, not dysplasia)

Increased CD64, CD14 neutrophils,

often activation markers, not MDS

CD10 decreased alone:

not specific,

Can be seen autoimmune neutropenia, aplastic

anemia, drug induced neutropenia

Monocytes

CD13 APC-A

CD

33 P

E-C

y7-A

-102102 103 104 105

-10210

2

103

104

105

0.09%0.93%

96.67 %2.31%

CD16 FITC-A

CD

11b

PE

-A

-102102 103 104 105

-10210

2

103

104

105

0.77%6.38%

8.08%84 .77%

CD15 V450-A

CD

33 P

E-C

y7-A

-102102 103 104 105

-102102

103

104

105

0.23%0.68%

55.39%43.70%

0.23%0.68%

55.39%43.70%

CD14 V450-A

CD

64 P

E-A

-102102 103 104 105

-102102

103

104

105 8.31% 83.59%

7.61% 0.49%

8.31% 83.59%

7.61% 0.49%

CD65 FITC-A

CD

64 P

E-A

-102102 103 104 105

-10210

2

103

104

105 52 .63% 42.38 %

4.64% 0.35%

DR FITC-A

CD

123

PE-A

-102102 103 104 105

-102102

103

104

105

5.47%0.99%

90.98%2.56%

5.47%0.99%

90.98%2.56%

CD10 PE-Cy7-A

CD

184

APC

-A

-102102 103 104 105

-102102

103

104

105 95.38% 1.84%

2.69% 0.08%

95.38% 1.84%

2.69% 0.08%

CD10 PE-Cy7-A

CD

56 V

450-

A

-102102 103 104 105

-102102

103

104

105 4.99% 0.37%

92.58% 2.05%

4.99% 0.37%

92.58% 2.05%

CD117 PE-Cy7-A

CD

38 A

PC-A

-102102 103 104 105

-102102

103

104

105

0.00%0.23%

1.81%97.97%

0.00%0.23%

1.81%97.97%

CD45 APC-H7 APC-H7-A

SSC

-A

-102100102 103 104 105

-400

65236

130872

196508

262144

lymph

mono

Monocyte Changes in MDS

CD14 V450-A

CD

64 P

E-A

-102102 103 104 105

-102

103

104

105 59.89% 38.09%

2.02% 0.00%

59.89% 38.09%

2.02% 0.00%

CD2 APC-A

CD

64 P

E-A

-102102 103 104 105

-102102

103

104

105 14.78% 74.76%

5.06% 5.40%

14.78% 74.76%

5.06% 5.40%

CD14 V450-A

CD

64 P

E-A

-102102 103 104 105

-102102

103

104

10521.63% 68.68%

6.19% 0.12%

CD10 PE-Cy7-A

CD

56 V

450-

A

-102102 103 104 105

-102102

103

104

10574.36% 9.42%

14.88% 1.35%

CD13 APC-A

CD

11b

PE

-A

-102102 103 104 105

-102102

103

104

105

1.24%12.76%

43.58%42.42%

DR FITC-A

CD

123

PE

-A

-102102 103 104 105

-102102

103

104

1052.74% 89.74%

6.03% 1.49%

CD10 PE-Cy7-A

CD

123

PE

-A

-102102 103 104 105

-102102

103

104

105

0.09%0.83%

6.81%92.27%

CD10 PE-Cy7-A

CD

184

AP

C-A

-102102 103 104 105

-102102

103

104

105

1.78%72.13%

0.13%25.96%

CD10 PE-Cy7-A

CD

65 F

ITC

-A

-102102 103 104 105

-102102

103

104

105

0.07%39.57%

0.85%59.51%

0.07%39.57%

0.85%59.51%

CD45 APC-H7-A

SSC

-H

-102100102 103 104 1050

65536

131072

196608

26214478%

CD45 dim

Monocyte Changes in MDS, Our experience

Decreased CD45/SSC: useful

Decreased CD64, CD14, CD11b, CD13, CD33, CD38, HLADR, CD184 (or increased), not specific

Increased CD15, CD65, not specific

Increased CD56 (high percent and high MFI), useful

Aberrant CD2 expression, useful but very uncommon

A Prospective Study on Clinical Cytopenia

Patients by using

myelomonocytic

maturation approach by our group

We included 102 patients who presented with cytopenia

Their marrows showed either no morphological dysplasia or only mild changes insufficient to diagnose MDS.

All patients had a normal karyotype.

Group1- Myelodysplastic syndrome

Group2-Cytopenia due

to various secondary causes

Group3- Cytopenia of unknown causes

Total

Positive 9 4 9 22

Intermediate 1 5 5 11

Negative 2 52 15 69

Total 12 61 29 102

Follow-up with repeated Bone Marrow biopsy, lab work-up, hematologists’

assessment

A positive FCM result has a positive predictive value of 69% anda negative FCM result has a negative predictive value of 95%.

Truong F et al Leukemia Research, 2009

Myelomonocytic Maturation Pattern Approach

Overall: Sensitive, but not very specific

Require Expertise in Interpretation

Be aware of pitfalls and caveats

Useful in patients who have not been treated previously, and who are not acutely ill (most clinic patients)

Become less reliable in patients with other underlying medical conditions, or who are undergoing various treatment for the medical conditions

CD34+ Precursor Based FCI Approach

Blasts in MDS have an immunophenotype

of committed myeloid precursors CD34(+)CD38(+)HLA-

DR(+)CD13(+)CD33(+),

regardless of the disease subtype

Ogata K, et al: Blood. 2002 Dec 1;100(12):3887-96.

CD34-based assay is useful in low grade MDS

A high FCI score was detected in 16/27 low grade MDS regardless of karyotype

and none of the 90 controls (sensitivity 59%, specificity of 100%)

Ogata K, et al. Blood. 2006 Aug 1;108(3):1037-44.

Our Panels

We implemented CD34-based assay at MDACC, where most of the patients have received or undergone various treatment

Panels

FITC

PE

PerCP

PE-Cy7 APC

V450

V500

1

CD16

CD11b

CD34

CD33

CD13

CD15

CD45

2

CD65

CD64

CD34

CD10

CD2

CD14

CD45

3

DR

CD123

CD34

CD10

CD184

CD56

CD45

4 CD7 CD5 CD34

CD117

CD38

CD19

CD45

5

Kappa Lambda

CD19

CD20

CD5

CD45

6

CD57

CD94

CD4

CD3

CD8

CD56

CD45

First 4 tubes:

analyze three populations:CD34 blasts; myeloid and monocytes

CD10 can separate myeloblasts

and hematogones; mature and immature myeloid elements

Tube 5: B-cell tube, and Tube 6: T-cell tube

CD45 V500-A

CD

34 P

erC

P-C

y5-5

-A

-102102 103 104 105

-102102

103

104

105 0.22% 1.92%

4.70% 93.16%

0.22% 1.92%

4.70% 93.16%

FS C-H

SSC

-A

0 655 36 1 966 080

65 53 6

13 10 72

19 66 08

26 21 44

clea n blas t 0 2

0.91 %

CD34 Positive Precursors in Normal Bone Marrow versus in MDS

CD45 V500-A

CD

34 P

erC

P-C

y5-5

-A

-102102 103 104 105

-102102

103

104

105

98.07%0.39%

1.53%0.01%

98.07%0.39%

1.53%0.01%

FSC-H

SSC

-A

0 65536 1966080

65536

131072

196608

2621441.07%

CD45 V500-A

SSC

-A

-102 -101 102 103 104 105

-400

65236

130872

196508

262144

CD45 V500-A

SSC

-A

-102-101102 103 104 105

-400

65236

130872

196508

262144

CD45 V500-A

SS

C-A

-102100102 103 104 105

-400

65236

130872

196508

262144

Normal

MDS

MDS: discrete population; decreased CD45

CD10 PE-Cy7-A

CD

64 P

E-A

-102102 103 104 105

-102102

103

104

105 12.05% 0.23%

58.67% 29.05%

12.05% 0.23%

58.67% 29.05%

CD10 PE-Cy7-A

CD

34 P

erC

P-C

y5-5

-A

-10210

210

310

410

5

-102

102

103

104

105

0.00%0.00%

29.41%70.59%

CD10 PE-Cy7-A

CD

65 F

ITC

-A

-102102 103 104 105

-102102

103

104

105 12.31% 0.22%

56.81% 30.66%

12.31% 0.22%

56.81% 30.66%

CD10 PE-Cy7-A

CD

34 P

erC

P-C

y5-5

-A

-102102 103 104 105

-102102

103

104

105 100.00% 0.00%

0.00% 0.00%

100.00% 0.00%

0.00% 0.00%

CD10 PE-Cy7-A

CD

65 F

ITC

-A

-10210

210

310

410

5

-102

102

103

104

105

0.28%98.58%

0.00%1.14%

CD10 PE-Cy7-A

CD

64 P

E-A

-10210

210

310

410

5

-102

102

103

104

105

0.43%99.15%

0.00%0.43%

CD10 PE-Cy7-A

CD

65 F

ITC

-A

-102102 103 104 105

-102102

103

104

105

3.76%46.66%

0.00%49.58%

3.76%46.66%

0.00%49.58%

CD10 PE-Cy7-A

CD

64 P

E-A

-102102 103 104 105

-102

103

104

105

1.60%42.72%

1.11%54.57%

1.60%42.72%

1.11%54.57%

CD15 V450-A

CD

13 A

PC

-A

-102102 103 104 105

-102102

103

104

105

1.02%46.73%

10.8441.41%

1.02%46.73%

10.8441.41%

CD15 V450-A

CD

13 A

PC

-A

-10210

210

310

410

5

-102

102

103

104

105 97.10% 2.90%

0.00% 0.00%

CD15 V450-A

CD

13 A

PC

-A

-102102 103 104 105

-102102

103

104

105

26.06%10.61%

52.42%10.91%

26.06%10.61%

52.42%10.91%

CD34 Positive Precursors:

Loss of Diverse Differentiation in MDS or aberrant expression

CD10 PE-Cy7-A

CD

34 P

erC

P-C

y5-5

-A

-102102 103 104 105

-102

103

104

105

0.00%0.00%

1.95%97.81%

Normal

MDSLoss diverse differentiation

MDSAberrantly increased expression of CD64, CD65 and CD15

CD117 PE-Cy7-A

CD

19 V

450-

A

-10210

210

310

410

5

-102

102

103

104

105

98.65%0.40%

0.94%0.00%

MFI=13078

CD117 PE-Cy7-A

CD

19 V

450-

A

-10210

210

310

410

5

-102

102

103

104

105

MFI Ratio=35.9

MFI=6381

28.48% 0.88%

10.95% 59.69%

CD10 PE-Cy7-A

CD

123

PE

-A

-102102 103 104 105

-102102

103

104

105

19.15%13.32%

9.96%57.57%

19.15%13.32%

9.96%57.57%

CD10 PE-Cy7-A

CD

123

PE-

A

-10210

210

310

410

5

-102

102

103

104

105

94.38%0.73%

4.89% 0.00%

CD117 PE-Cy7-A

CD

38 A

PC

-A

-10210

210

310

410

5

-102

102

103

104

105 40.98% 58.39%

0.00% 0.63%

CD33 PE-Cy7-A

CD

13 A

PC-A

-102102 103 104 105

-102

103

104

105

0.51%0.76%

46.37%52.36%

CD10 PE-Cy7 PE-Cy7-A

CD

184

APC

APC

-A

-102102 103 104 105

-102

103

104

105 11.73% 46.21%

34.84% 7.22%

CD10 PE-Cy7-A

CD

184

APC

-A

-10210

210

310

410

5

-102

103

104

105 68.10% 1.17%

28.96% 1.76%

CD117 PE-Cy7-A

CD

38 A

PC-A

-102102 103 104 105

-102

103

104

105

3.59%0.01%

93.41%2.99%

3.59%0.01%

93.41%2.99%

CD33 PE-Cy7-A

CD

13 A

PC

-A

-102102 103 104 105

-102

103

104

105 4.73% 22.84%

68.52% 3.91%

4.73% 22.84%

68.52% 3.91%

Normal

MDS

CD34+ precursors:

Alterations of Levels of Expression in MDS

MDS: Increased CD117 MFI, increase CD184, decreased CD38, increased CD13

CD117 PE-Cy7-A

CD

5 P

E-A

-102102 103 104 105

-102102

103

104

105 0.00% 95.83%

0.00% 4.17%

0.00% 95.83%

0.00% 4.17%

CD10 PE-Cy7-A

CD

2 A

PC

-A

-102102 103 104 105

-102102

103

104

105

3.62%52.79%

0.00%43.59%

3.62%52.79%

0.00%43.59%

CD34 PerCP-Cy5 -5-AC

D7

FITC

-A

-102102 103 104 105

-102

103

104

105 0.00% 42.04%

0.00% 57.96%

CD10 PE-Cy7-A

CD

34 P

erC

P-C

y5-5

-A

-102102 103 104 105

-102

103

104

105

0.00%0.00%

96.00%4.00%

0.00%0.00%

96.00%4.00%

CD34+ Precursors:

Aberrant Antigenic Expression

CD10 PE-Cy7-A

CD

56 V

450-

A

-102102 103 104 105

-102

103

104

105 44.19% 3.32%

50.00% 2.49%

CD34 PerCP-Cy5-5-A

CD

5 PE

-A

-102102 103 104 105

-102

103

104

105 0.00% 28.60%

0.00% 71.40%

CD5 PE-A

CD

38 A

PC-A

-102102 103 104 105

-102

103

104

105

47.19%17.72%

3.92%31.18%

CD5 PE-A

CD

38 A

PC

-A

-102 103 104 105

-102

103

104

105 23.80% 72.78%

0.55% 2.87%

23.80% 72.78%

0.55% 2.87%

CD10 PE-Cy7-A

CD

34 P

erC

P-C

y5-5

-A

-102102 103 104 105

-102

103

104

105 77.36% 22.64%

0.00% 0.00%

77.36% 22.64%

0.00% 0.00%

A Very Hemodilute Specimen

CD45 V500-A

CD

34 P

erC

P-C

y5-5

-A

-102102 103 104 105

-102102

103

104

105

Blast 01

89.92%9.05%

1.01%0.03%

CD33 PE-Cy7-A

CD

13 A

PC

-A

-102102 103 104 105

-102102

103

104

105

2.27%0.00%

93.18%4.55%

2.27%0.00%

93.18%4.55%

CD45 V500-A

SSC

-A

-102100102 103 104 105

-400

65236

130872

196508

262144

CD10 PE-Cy7-A

CD

34 P

erC

P-C

y5-5

-A

-10210

210

310

410

5

-102

102

103

104

105

0.00%0.00%

4.88%95.12%

CD34 PerCP-Cy5-5-A

CD

56 V

450-

A

-10210

210

310

410

5

-102

102

103

104

105 0.00% 62.86%

0.00% 37.14%

CD117 PE-Cy7-A

CD

38 A

PC

-A

-10210

210

310

410

5

-102

102

103

104

105 10.87% 58.70%

2.17% 28.26%

CD117 PE-Cy7-A

CD

5 P

E-A

-10210

210

310

410

5

-102

102

103

104

105

58.70%4.35%

32.61%4.35%

The morphology of this case is inadequate; FCI shows that the CD34 cells: no hematogones; CD38dec, CD5+, CD13inc, CD56+, findings are consistent with MDS

Rule out Lymphoproliferative

Processes

CD45 APC-H7-A

SSC

-A

-102100102 103 104 105

-400

65236

130872

196508

262144

lymph B-cell

CD3 PE-Cy7-A

CD

8 A

PC

-A

-102102 103 104 105

-102

103

104

105 1.98% 64.76%

8.66% 24.60%

CD3 PE-Cy7-A

CD

4 P

erC

P-C

y5-5

-A

-102102 103 104 105

-102

103

104

105

65.34%10.30%

24.12%0.25%

CD8 APC-A

CD

57 F

ITC

-A

-102102 103 104 105

-102

103

104

105

28.15%31.00%

38.20%2.65%

LGL

CD8 APC-A

CD

56 V

450-

A

-102102 103 104 105

-102

103

104

105

65.45%33.49%

0.53%0.53%

CD7 FITC-A

CD

5 PE

-A

-102100102 103 104 105

-102100102

103

104

105

8.98%0.98%

87.10%2.93%

8.98%0.98%

87.10%2.93%

CD20 PE-Cy7-A

CD

19 P

erC

P-C

y5-5

-A

-102102 103 104 105

-102

103

104

105 1.78%

62.20% 4.65%

B-cell

1.78%

62.20% 4.65%

B-cell

CD19 PerCP-Cy5-5-A

CD

5 A

PC

-A

-102102 103 104 105

-102

103

104

105

=15.2%

49.35% 33.35%

16.55% 0.74%

=15.2%

49.35% 33.35%

16.55% 0.74%

KAPPA FITC-A

LAM

BDA

PE-A

-102102 103 104 105-10

2

103

104

105

K=0.0%

L=87.5%

Always check B cells and T cells: upper panel: a case of chronic lymphocytic leukemia; the lower panel: a case of LGL leukemia detected by MDS panel

Diagnostic Criteria (Our FCI panel)

Positive

If one aberrant lymphoid antigen expression

in Blasts If two significant alterations of level of expression

If no blast abnormality identified (very few cases)

Very significant CD13/CD16; CD11b/CD16 pattern in myeloid cells, and/or significant lymphoid antigen (CD56) expression, and significant hypogranulation.

Indeterminate (we have very few cases in this category)

If only one significant alteration of levels of expression of CD34 cells

Negative

If no blasts abnormality, only mild abnormalities in myelomonocytic

cells

MDACC Experience

First MDS or rule out MDS diagnosis at MDACC

We tested 259 patients in one year period (5/2009 to 4/2010) with follow-up information

147 MDS (62 normal karyotype, 76 abnormal, 9 not available)

6 RARS

59 RCMD

3 MDS-U

25 RAEB

5 5q-

18 MDS/MPN

35 t-MDS

112 non-MDS cytopenia

44 patients s/p

chemotherapy

17 status post stem cell transplant

6 Aplastic

anemia, treated

45 other medical cytopenia

(ITP, hemolytic anemia-CLL, liver, kidney problems, LGL, viral, low grade B cell lymphoma…)

Sensitivity and Specificity of Antigenic Alterations

Control (n=112) MDS (n=147) Sensitivity % Specificity % Accuracy %

Stage 1 Hematogone

(≤10%) 30 124 84 73 80

Plasmcytoid

dendritic

precursors (<5%) 35 104 71 69 70

Abn

CD13/CD33 19 89 61 83 70

Inc CD117 10 75 51 91 68

Inc CD123 11 73 50 90 67

Abn

CD45/SS 8 55 37 93 63

Inc CD34 2 45 31 98 60

Dec CD38 1 45 31 99 60

Inc CD184 6 39 27 95 56

CD34 (≥3%) 6 42 29 95 57

Lymphoid antigen 8 54 37 93 61

Mature myelomonocytic

antigen 8 27 18 93 50

The FCI has a sensitivity of 90.5%; specificity of 88%; PPV of 91%; NPV of 88%, and an accuracy of 90%.

Summary

FCI Assays for clinical cytopenia

without an established diagnosis should be able to:

Detect B cell clonality

Identify aberrant T cells

May be plasma cell neoplasm (CD38/CD19/CD56, gate on bright CD38 cells)

Stem cell neoplasm (MDS or related neoplasm)

Summary

MDS assays

Myelomonocytic

based assay

Sensitive, can be applied to the community setting, but less specific

Needs experience in interpretation

Recognize reactive conditions, specimen quality, and mimics

CD34+ Blast based assay

Specific, and sensitive. Sensitivity can be improved by utilizing more markers

Especially useful in patients who have been treated for hematological or non-hematological disorders

Summary

Flow Cytometry

Immunopheotyping

is very useful in Diagnosis of MDS

FCI is particularly useful in low grade MDS with a normal karyotype

and borderline dysplasia.

Either it is mild dysplasia or dysplasia is difficult to assess because of sample quality

It is very important to rule out a case not of MDS

FCI positive cases, at least require close follow-up

Some older patients may receive empirical treatment for MDS

Acknowledgement

Jeffrey L. Jorgensen, MD PhD

Marian Kersh, BS/MT

Ying Hu, BS

Guilin Tang, MD, PhD

Guillermo Garcia-Manero, MD