The Diagnosis of Myeloid Neoplasia by Flow Cytometry
Brent L. Wood MD PhDDept. of Laboratory Medicine
University of Washington, Seattle
Basic Principles
Abnormal population identification
• Normal– Antigens expressed in consistent and
reproducible patterns with maturation• Neoplastic
– Increased or decreased normal antigens– Asynchronous maturational expression– Aberrant antigen expression– Homogeneous expression
Normal B cell Maturation
Wood and Borowitz (2006) Henry’s Laboratory Medicine
Normal B cell Maturation
Wood (2004) Methods Cell Biology 75:559-576
0.1% abnormal immature B cells
ALL MRD
06-01469
Myeloid Stem Cell Disorders
• Similar immunophenotypic abnormalities– Myelodysplasia– Myeloproliferative disorders– Acute myeloid leukemia
• Differences in– Blast percentage– Extent of maturation– Basophilia– Degree of abnormality
CD45/SS
Borowitz et al (1993) AJCP 100:534-40.Steltzer et al (1993) Ann NY Acad Sci 667:265-280
CD45/SS
Wood (2004) Methods Cell Biology 75:559-576
Wood (2007) Clinics in Lab Medicine 27:551-575
Early Progenitors
Blasts• Blasts are morphologically defined
– AML is > 20% blasts in blood or bone marrow
• Morphologic “blasts” include– Stem cells– Committed progenitors (CD34+)– Promyelocytes– Promonocytes– Immature B cell precursors– Immature T cell precursors
Blasts
Blasts• Blast estimates by flow cytometry may not agree with morphology
– Specimen processing
Red blood cell lysing reagents remove variable number of erythroid precursorsVariable degree of peripheral blood dilution
• Blast estimate may be high or low depending on relative contributions– Not a problem for peripheral blood
– Blasts defined differently by morphology and flow cytometry• Include promonocytes, promyelocytes, immature B cells as appropriate
Normal Blast Maturation
Wood (2004) Methods Cell Biology 75:559-576
Normal Granulocyte Maturation
Normal Monocyte Maturation
Acute Myeloid Leukemia
Wood and Borowitz (2006) Henry’s Laboratory Medicine
Abnormal Antigen Intensity
Wood (2007) Clinics in Lab Medicine 27:551-575
Aberrant Lymphoid Antigens
Wood (2007) Clinics in Lab Medicine 27:551-575
Aberrant Maturation
Wood (2007) Clinics in Lab Medicine 27:551-575
Granulocytic Maturation
Normal Granulocytic Maturation
Wood and Borowitz (2006) Henry’s Laboratory Methods
Normal Granulocytic Maturation
Wood (2004) Methods Cell Biology 75:559-576
Wood (2007) Clinics in Lab Medicine 27:551-575
Monocytic Maturation
Normal Monocytic Maturation
Wood and Borowitz (2006) Henry’s Laboratory Methods
Normal Monocytic Maturation
Wood (2004) Methods Cell Biology 75:559-576
Monocytic Aberrancies
Wood (2007) Clinics in Lab Medicine 27:551-575
Acute Myelomonocytic Leukemia
Wood and Borowitz (2006) Henry’s Laboratory Medicine
Acute Monocytic Leukemia
Abnormal Myeloid Maturation
Normal
MDS
Myeloproliferative Disorders
Myeloproliferative disorders• CML
– Decreased CD16 expression on mature neutrophils– Decreased CD32 expression by mature neutrophils– Decreased L-selectin (CD62L) expression on the CD34-positive
cells– Aberrant expression of CD56 on the blasts and myeloid cells– Aberrant expression of lymphoid antigens such as CD2, CD5, and
CD7 on the blasts in CML blast crisis as well as CD7 expression on CD34-positive cells in chronic phase
Chronic Myeloid Leukemia
04-5708
CML in chronic phase - 2.8% blasts
Myeloproliferative disorders
• Non-CML– Increased expression of Bcl-XL in polycythemia vera– Decreased platelet GPIa/IIa, decreased GP1b and
GPIIb/IIIa, and elevated platelet P-selectin, thrombospondin, GPIV, and c-Mpl in essential thrombocytosis
– Aberrant coexpression of CD14 and CD66 on the myeloid cells in a subset of myeloproliferativedisorders
Myeloproliferative Disorders
• 76 cases referred for evaluation of MPD– All t(9;22) negative– Caveat: Diagnosis of all cases studied not known
• Evaluated by 4 color flow cytometry• Analyzed for abnormalities of blast, myeloid
and monocyte maturation• Compared with cytogenetics
Kussick and Wood (2002) Am J Clin Path 20:854-865
Essential Thrombocytosis
Kussick and Wood (2002) Am J Clin Path 20:854-865
Myeloproliferative Disorders
Population n Mean Age M:FAbnormal
Cytogenetics
Positive Flow(cases) 34 69.6 16:18 12 / 29 (41%)
Negative Flow(controls) 40 55.4 18:22 0 / 36
Kussick and Wood (2002) Am J Clin Path 20:854-865
Myeloproliferative disorders
# ofCytog.Abnls. n
Abnl.Blasts
Abnl.Myelos.
Abnl.Monos.
MeanAbnl.Myel.Ags.
MeanNon-Myel.Ags.
MeanTotalAbnl.Ags.
0 17 94% 100% 56% 3.8 0.8 4.61 8 100% 88% 50% 4.4 0.6 5.0
2 ormore 4 100% 75% 75% 5.8 0.5 6.3
TOTAL 29 97% 93% 55% 4.2 0.7 4.9
Kussick and Wood (2002) Am J Clin Path 20:854-865
MPD Conclusions• Many cases of non-CML myeloproliferative
disorders have immunophenotypicabnormalities
• Presence of immunophenotypicabnormalities correlates with cytogenetics– All cases with cytogenetic abnormalities had
flow cytometric abnormalities– Number of immunophenotypic abnormalities
paralleled increase in cytogenetic abnormalities• Abnormalities less frequent in PV and ET
Myelodysplasia
Early Studies• Many isolated abnormalities described
– loss of erythrocyte A, B, and H antigens– decreased expression of c-Mpl, GPIIb/IIIa, and GPIb on platelets in
refractory anemia– dyssynchronous expression of CD11b and CD16 in the developing
neutrophils– aberrant coexpression of CD14 and CD66 on myeloid cells– decreased CD10 on neutrophils– changes in a variety of leukocyte activation antigens, including FcR
I, FcRII, and FcR III– greater variability in the expression of CD38, CD71, CD13, and
CD33 in refractory anemia
– aberrant coexpression of CD56 on myeloid blasts
Elghetany MT. Haematologica. (1998) 83:1104-15.
Clinical utility
• Can distinguish MDS from normal and AA– Large panel of antibodies– Abnormalities in myeloid, erythroid and megs– Compared with morphology and cytogenetics– Abnormalities in subset of indeterminate cases
• Not advocated for screening
Stetler-Stevenson, et al. Blood (2001) 98:979-987.
Clinical utility• Correlation with IPSS and transplant outcome
Wells, et al. Blood (2003) 102: 394-403.
Clinical utility
Wells, et al. Blood (2003) 102: 394-403.
Clinical utility• Correlation with IPSS and transplant outcome
– Scoring system• 0 points = Normal• 1 point = Single abnormality• 2 points = 2 or 3 abnormalities OR CD34/lymphoid on myelomonocytic• 3 points = 4 abnormalities OR 1-3 abnormalities with CD34/lymphoid• 4 points = 2 to 3 abnormalities on myeloid and monocytic• Additional abnormal blasts: <5% (1), 6-10% (2), 11-20% (3), >20% (4)
• 0-1 = Normal/mild• 2-3 = Moderate• 4-9 = Severe
– Diagnosis of MDS• Score of 3 gives specificity of 100% with sensitivity of 55%; 76% correct
– Correlates with IPSS and transplant outcome
Wells, et al. Blood (2003) 102: 394-403.
Clinical utility
Wells, et al. Blood (2003) 102: 394-403.
Clinical utility
Wells, et al. Blood (2003) 102: 394-403.
Blast score in LG-MDS
Ogata, et al (2006) Blood 108:1037-1044
Erythroid Dysplasia
• Patient populations– 104 MDS– 69 Pathologic controls– 19 Normals
• Evaluated MFI– CD71– CD105– Cytosolic H&L-ferritin (HF
or LF)– Mitochondrial ferritin (MtF)
Della Porta, et al. Leukemia (2006) 20:549-555
Erythroid Dysplasia
• Linear Discriminant Analysis– MDS with erythroid dysplasia -
• 52/53 positive = 98.1% sensitivity
– MDS without erythroid dysplasia• 9/15 positive = 60%
– Normal and pathologic controls• 64/65 negative = 98.5% specificity
– MtF positive in all cases with ringed sideroblasts
Della Porta, et al. Leukemia (2006) 20:549-555
Clinical Diagnosis
• 124 cases submitted for myelodysplasia• Assessed by 4 color flow cytometry• Analyzed for maturational aberrancies
– Negative = no abnormalities– Indeterminate = mild abnormalities (< 2)– Positive = more than mild abnormalities (>= 2)
• Compared with cytogenetics and morphology– MDS = either cytogenetics and/or morphology
Kussick, et al. AJCP (2005) 124: 170-181
Distribution of cases by cytogenetic, morphologic, and flow cytometric findings.
CYTOGENETICS MORPHOLOGY FLOW
CYTOMETRY Normal Indeterm. Abnormal Normal 31 12 0
Normal* Indeterminate 12 3 7 Abnormal 3 3 20 Normal 0 0 0
Abnormal* Indeterminate 1 1 1 Abnormal 0 0 30
Myelodysplasia
N = 124
Kussick, et al. AJCP (2005) 124: 170-181
Myelodysplasia
MDS = abnormal morphology and/or cytogeneticsFlow abnormal
89% sensitivity88% specificity
Flow abnormal or indeterminate95% sensitivity67% specificity
Also true for cases with < 5% blasts84% flow abnormal were MDS
Kussick, et al. AJCP (2005) 124: 170-181
Conclusion• Flow cytometry can aid in diagnosis of MDS and MPD
– More confident diagnosis• Identify immunophenotypic abnormalities• Exclude other disorders
– Aid subclassification• More accurate blast identification and quantitation
– Assist in prognostication– Rapid
• Requires– Consistent flow cytometric technique – Knowing normal patterns of antigen expression – Knowing common abnormal patterns
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