Page 61 - Williams Hematology ( PDFDrive )

36 Part I: Clinical Evaluation of the Patient Chapter 3: Examination of the Marrow 37

specimen is the preferred method for phenotyping solid tumors and in conjunction with aberrant surface immunophenotype, can also be
is highly complementary to flow cytometry in diagnosis of lymphoid useful in establishing the clonality of plasma cell neoplasms in marrow.
infiltrates. T-cell clonality is not as easily demonstrated, because there are dozens
of Vβ specificities expressed by T cells, and antibodies exist only to iden-
COMMON FLOW CYTOMETRY APPLICATIONS tify approximately 70 percent of these. Analysis of Vβ repertoire of the
IN HEMATOLOGY αβ T-cell antigen receptor in cells with an atypical immunophenotype,
can identify clonal populations of T cells at diagnosis and posttherapy,
84
Often the diagnostic question involves characterizing an expanded blast but this methodology is less-routinely used in clinical laboratories.
population or detection and analysis of a clonal lymphoid population Presence of minimal residual disease (MRD) measured by either
(see Fig. 3–4). These determinations can be achieved by examining detection of a molecular target using PCR or aberrant immunopheno-
lineage-specific or maturation stage-specific markers. For instance, type using multicolor flow cytometry is increasingly used as a prognostic
in marrow, immature cell populations can be identified by expression marker, often in the setting of clinical trials, in a variety of hematopoietic
of antigens such as CD34 and CD117. In some instances, the stage of neoplasms, including acute leukemia, plasma cell myeloma, and CLL. In
differentiation can be determined using combinations of markers that some settings, such as chronic phase CML, MRD monitoring by molecu-
are expressed only during certain phases of differentiation (e.g., dual lar assay is standard practice. In others, both flow cytometry and molec-
expression of CD4 and CD8 in an immature T-precursor population). ular methods for MRD detection are used successfully, each having their
Stage-specific phenotypes are sometimes valuable clues to clinically own advantages and limitations. Flow cytometry assays for MRD are
85
relevant diagnoses, such as the characteristic lack of human leukocyte considerably more complex than standard diagnostic phenotyping and
antigen-D related (HLA-DR) expression in promyelocytic leukemia, need to be designed for this specific purpose, with collection of large
86
a phenotype that mimics the normal loss of this antigen in promye- numbers of events, multicolor strategies for detecting different types of
locytes. Among lymphoid leukemia/lymphomas, chronic lymphocytic subtle aberrant phenotypes, consistent data interpretation protocols, and
leukemia (CLL)/small cell lymphoma, mantle cell lymphoma, hairy confirmation of absence of “background” cells in noninvolved (but oth-
cell leukemia, and B- or T-precursor lymphoblastic leukemia, among erwise comparable; e.g., posttherapy) marrows expressing each aberrant
others, have distinctive immunophenotypes. Methodologic advances phenotype to be tested. Immunophenotypic evidence of MRD can be
such as multicolor analysis (6 or more simultaneous fluorescence col- sought by detection of specific leukemia associated immunophenotype,
ors) have allowed flow cytometers to detect and analyze diagnostically or by observation of a population in multidimensional space shifted sig-
important rare subpopulations such as Reed-Sternberg cells in classical nificantly from any corresponding normal population (“different from
Hodgkin lymphoma. Aberrant phenotype combinations suggestive of normal” approach). Immunophenotypic shifts can occur during treat-
78
malignancy, such as coexpression of high levels of CD56 or CD117 on ment, so identification of more than one aberrant phenotype to be tested
plasma cells in plasma cell myeloma, or loss of the T-cell markers CD7 is advisable when possible. Interpretation of MRD assays is dependent
or CD26 in a mature phenotype T cell in T-cell lymphoma, are diagnos- on type of neoplasia, timing of testing, treatment regimen, and standard-
tically useful. Expression intensity is a frequent clue to diagnosis; for ization of assay methods. Standardization of flow cytometry MRD assays
example, the weak surface immunoglobulin and weak CD20 expression is a challenge that will need to be resolved as these assays move into more
in CLL. In reporting flow cytometry immunophenotyping results, the routine clinical practice. In acute leukemia, where MRD testing by flow
summary immunophenotype of the relevant population(s) should be cytometry has been most intensively studied, the assays provide post-
described, rather than simply a listing of the percentage of cells positive treatment prognostic information, but the translation of this informa-
for each marker, with subpopulations noted as observed. tion into therapeutic decision making based on MRD risk stratification
Immunophenotyping of marrow by flow cytometry is a useful is further advanced in pediatric leukemia and acute promyelocytic than
87
adjunct to established morphologic and cytogenetic criteria in diag- in adult acute myelogenous leukemia (AML). MRD detection by either
79
nosing MDS, and is predictive of later development of overt MDS flow cytometry or PCR is standard clinical practice in pediatric acute
in the diagnostically challenging group of cytopenic patients with ini- lymphocytic leukemia (ALL), 85,86 where nearly all patients have leukemia
tially morphologically equivocal marrow findings. Abnormalities in associated targets suitable for either molecular or flow cytometry MRD
80
MDS marrow include an increase in the percentages of CD34+ cells assays. Detection of MRD in AML is more challenging because half of
even when blasts are not morphologically increased; decreased CD34+ patients lack a molecular target suitable for MRD testing, but the major-
B progenitors; aberrant antigen expression by myeloid progenitors, ity of AML patients have a leukemia-associated phenotype that can be
mature granulocytic cells, or monocytes; and decreased side scatter of detected at the 0.1 percent level or below by multicolor flow cytometry.
mature granulocytic cells. A simplified scoring system (so-called Ogata MRD detection in chronic lymphocytic leukemia (CLL) 88,89 and plasma
90
81
score) has been validated for use in an interlaboratory study, and inter- cell myeloma is also possible by flow cytometry and molecular tech-
national guidelines for a more extensive scoring panel have been pub- niques, and is becoming a relevant clinical issue now that therapeutic
lished by the International/European Leukemia Net Working Group for options in these malignancies are rapidly improving.
82
Flow Cytometry in MDS. Immunophenotyping may provide thera- Flow cytometry is used to enumerate CD34+ progenitors when eval-
peutically relevant prognostic information independent of existing risk uating the adequacy of blood stem cell collections (Chaps. 23 and 28),
factors incorporated in the commonly used (and recently revised) Inter- with several routine assay kits available. Flow cytometry offers the
91
national Prognosis Scoring System (IPSS) score. 83 potential to incorporate other markers defining clinically relevant pro-
92
Clonality of immunoglobulin-expressing B-cell malignancies genitor and stem cell subpopulations. Lymphocyte subset quantitation
involving marrow (e.g., CLL and lymphoplasmacytic lymphoma) can is diagnostically important in acquired and congenital immunodefi-
be determined by simultaneous assessment of surface κ and λ immuno- ciency states. Flow cytometry analysis of glycosylphosphatidylinositol
globulin light-chain expression on the surface of B cells, often in com- (GPI) linked proteins in multiple blood cell types is the gold standard for
93
bination with a characteristic neoplastic immunophenotype, such as diagnosis of paroxysmal nocturnal hemoglobinuria (Chap. 40). Flow
CD5 expression in CLL B cells. Technical considerations are important cytometry detection of paroxysmal nocturnal hemoglobinuria (PNH)
to minimize nonspecific binding of serum monoclonal immunoglobu- clones is facilitated by using FLAER (fluorescently labeled inactive vari-
lins to the surface of lymphocytes. Cytoplasmic κ and λ identification, ant of the bacterial protein aerolysin), which binds to all (GPI) linked

Kaushansky_chapter 03_p0027-0040.indd 37 17/09/15 5:38 pm