ISAC XXWORKSHOPCarleton C. StewartSigrid J. Stewart

IMMUNOPHENOTYPING ACUTE LEUKEMIAS

CD3

CD14

HLADr

CD45

This antibody combination is designed to give a differential. CD45 vs SSC is used to define lymphocytes, monocytes and granulocytes. CD14 further defines monocytes while CD3 gives T-cells and HLADr identifies B-cells and NK cells. In bone marrow, progenitor cells are CD4 dim HLADr+ and erythroid cells or platelets are CD45 negative.

CD7

CD13

CD2

CD19

This combination is used for three reasons. 1) Normal T-cells express CD2 and CD7, which are often expressed at abnormal levels by malignant T-cells. NK cell malignancy is usually CD7+CD2-. 2) CD2+CD7+CD13+ cells represent aberrant co expression of the myeloid antigen, CD13, on acute T lineage lymphocytic leukemias and lymphoproliferative disorders. 3) Co expression of CD2 or CD7 or CD13 with CD19 defines aberrant expression of these markers on B lineage malignancy. Finally, CD19 is often co-expressed on CD13 AML-FAB/M2 with a t(8,21) translocation.

CD5

Lambda

CD19

Kappa

This combination is designed to resolve B-lineage lymphoproliferative disorders. Clonal excess of kappa or lambda on CD19 positive cells or CD19CD5 positive cells are explicitly defined.

CD20

CD11c

CD22

CD25

This combination is used to further characterize B-lymphoproliferative disorders and to define the degree of maturity of acute B-lineage leukemias. In addition, hairy cell leukemia can be classified by its unique high expression of CD11c. Aberrant expression of the T-cell marker CD25 on B-cells is diagnostic for lymphoproliferative diseases when it is expressed.

CD5

CD19

CD10

CD34

This antibody combination is designed to resolve the cells within the maturation of both T and B-cell lineages. The earliest T-cells are CD5+CD10+CD34+, which differentiate into CD5+CD10+ by losing CD34 and finally into mature T-cells that express only CD5. This combination can be used to evaluate the maturity of a T lineage acute leukemia. In a like manner, the maturation of the two distinct B-cell lineages: CD19+CD5- and CD19+CD5+ can be defined. The earliest B-cells co express both CD34 and CD10. As maturation occurs, they lose CD34, then CD10 to become mature B-cells.

CD11b

CD13

CD33

CD34

This combination resolves both normal and malignant myeloid cells. The most immature myeloid cells express only CD34 and CD13. FAB M0 and M1 will generally be positive for only these two markers. As these cells differentiate, they acquire low CD33 and low CD11b expression. A dramatic increase in CD11b expression with gradual loss of CD34 expression is characteristic of differentiation into the granulocytic lineage and the malignant counterpart is FAB M2. In contrast, an increase in CD33 expression and reduction of CD34 expression is characteristic of differentiation into the monocytic lineage (FAB M4). APL has characteristics similar to both the early granulocytic and monocytic lineage leukemias suggesting they may be derived from precursors at the branch point.

CD15

CD56

CD19

CD34

This combination is used to define aberrant antibody expression on hematopoietic malignancies. CD56 is expressed early on progenitor cells (CD34+CD56+) that may also co-express CD15. We have shown that in acute leukemia, co-expression of CD15, CD56 and CD34 is associated with the t(8,21) translocation, which results in a very bad prognosis. CD15 is also expressed on granulocytes and some B-cells.

CD41

CD71

CD45

CD34

This combination is used to define erythroleukemia FAB/M6 and megakaryoblastic leukemia FAB/M7. Activated HPCs co- express CD71, the transferrin receptor, as well as CD45 and CD34. Erythroid progenitor cells can be distinguished by their very high levels of CD71 and CD34 expression. As they differentiate into precursor normoblasts they lose expression of both CD45 and CD34 and express only CD71. When HPCs differentiate into the megakaryocytic lineage they acquire CD41 and lose CD45 before losing CD34.

CD38

CD138

CD45

CD56

Myeloma cells are the only hematopoietic cells that express CD138. In addition, CD38 is expressed at very high levels and CD56 is often (~80% of the time) expressed. CD45 is expressed at low levels on immature plasma cells, but is completely absent on mature plasma cells.

ABCDEFAb1Ab1Ab1Ab1Ab1Ab1Ab2Ab2Ab2Ab2Ab2Ab2Types of Antigen Expression

MARKER EXPRESSION ON T-CELL ALLn = 54> 10% of leukemia cells express the marker

MARKER EXPRESSION ON B-CELL ALLn =284

RPCILFCBLOOD11065003PRE-B-CELLS02178081.004BONE MARROWCD10CD10CD19CD19

CD10CD10CD19CD19CD19CD20CD34CD45

FSCSSCCD45CD34SSCCD45FEATURES OF ERYTHROLEUKEMIA (FAB M6)12017021016

Phenotype of Myeloid Component of Erythroleukemia3/18 erythroleukemias did not exhibit a myeloid component4/16 erythroleukemias exhibit a megakaryocytic component

MARKER EXPRESSION ON AML CELLSn = 957> 10% of leukemia cells express the marker4% of specimens diagnosed by hematopathology as AML were found to have ALL or were normal by Flow Cytometry.

IMMUNOPHENOTYPING LYMPHOPROLIFERATIVE DISEASEmultiple myeloma T-cell lymphomas

MULTIPLE MYELOMA11296291FITC-CD38PE-CD56FITC-CD45PE-CD56FITC-CD5PC-CD19TC-CD22FITC-CD20

BONE MARROW FROM MULTIPLE MYELOMA PATIENT STAINED WITH CD38 CD56 AND CD45 FITC-CD38 PE-CD56

BONE MARROW FROM MULTIPLE MYELOMA PATIENT STAINED FOR CD38 CD56 CD45 AND DNA CONTENTGATED ON CD38+CD56+CD45- CELLS GATED ON MONONUCLEAR CELLS1030507090110140170200230DNA100200300400500600700800Number1030507090110140170200230102030405060708090100120

Typical T-cell Lymphoma or Reactive Lymph Node:

NON MALIGNANT LYMPH NODE -T CELLSTC-CD10FITC-CD5FITC-CD3PE-CD4TC-CD8PE-CD4FITC-CD3TC-CD8TC-CD2FITC-CD7

IMMUNOPHENOTYPING LYMPHOPROLIFERATIVE DISEASEB-cell/CLLRPCILFC

Aberrant Antigen Coexpression in B-cell lymphoma

Problem CaseABCDEFKappaCD19CD19CD33CD19CD19CD13KappaLambdaLambdaCD33CD13

Small Non-Cleaved(Burkitts) Lymphoma12197181FITC-CD5PE-CD19TC-CD10PE-CD19TC-CD22FITC-CD20FITC-KappaPE-LambdaFITC-IgDPE-IgMFITC-CD23PE-CD79gated on CD19+ B-cells

MANTLE CELLFITC-CD5PC-CD19TC-CD10PC-CD19TC-CD22FITC-CD19FITC-kappaPE-lambdaFITC-IgDPE-IgMgated on CD19+ B-cells07266341

ABERRANT ANTIGEN EXPRESSION ON FOLLICULAR LYMPHOMATC-CD2PE-CD19TC-CD10PE-CD19

Typical B-cell Lymphoma

There are several patterns of antigen expression. The interpretation of them is important for describing the meaning of co-expression. In A, Ab2 is uniformly expressed, while Ab1 is co-expressed by Ab2 cells in a heterogeneous manner. In B, Ab1 is uniformly expressed, while Ab2 is co-expressed by Ab1 cells in a heterogeneous manner. In C, a population of Ab1+ Ab2- cells is found, but it is the Ab2+ cells that exhibit heterogeneous expression to produce the Ab1+ Ab2+ population. Thus, Ab1 cells are not positive for Ab2. In D, there are two distinct populations: Ab2+Ab1- and Ab2+Ab1+ , while in E, the two populations are Ab1+Ab2- and Ab1+Ab+. In F, correct interpretation can be difficult because this pattern is exhibited by highly autofluorescent cells that are Ab1-Ab2- or dead cells that non specifically bind Ab1 and Ab2. If it is either of these two situations, all combinations of antibodies used will show this pattern on the same percentage of cells. If this pattern represents a true subpopulation of cells it will be unique to the one antibody combination.A B-cell leukemia illustrating co-expression of CD7 but not CD2 by CD19+ cells (A), a small cluster of CD19+CD2+ cells (red, B), co-expression of CD2+CD7+ but not CD19 on normal T-cells (blue, C) and expression of CD19 CD7 and CD2, not shown, (red, D). By requiring 10% of gated cells to co-express a marker, the population in B is not described, even though it might be a valid subpopulation because, in D, co-expression is caused by non specific staining of dead cells by all three antibodies.The next series of figures show the patterns generated by antibody combinations used for characterizing acute B-cell leukemia. In each, the top row illustrates the normal pattern and the bottom row the leukemia. In this figure, normal T-cells are yellow, B-cells are blue and the large population of leukemic T-cells express CD45 dimly and HLADr heterogeneously. It is more common to find no HLADr expression on T-leukemic cells.This combination is most important for characterizing T-ALL. Aberrant expression of one or more common T-cell markers always occurs, making the leukemic cells resolved from normal T-cells.To further define the T-cell leukemia, CD2 and CD7 are almost always expressed in an aberrant fashion. In this example, CD7 is overexpressed and CD2 is underexpressed. The normal CD2+CD7+ T-cells are clearly resolved and offer a landmark cluster for visualizing the aberrant marker expression by the leukemia.This combination is useful for determining if the T-ALL (CD5+CD19-) expresses CD10.This combination is useful for evaluating T lineage ALL. Coexpression of CD5 and CD34, but not CD19, is definitive for T-ALL. Note this combination is also used for B-ALL.1A second T-lineage ALL is shown to illustrate the varied nature of these leukemias. In this example, dim expression of CD3 is found, CD10 expression is heterogeneous and there is little expression of HLADr.B-lineage acute leukemias express CD45 in a variable way. Only 3% express CD45 at the same epitope density as normal B cells. 83% of them express CD45 at a lower level and 14% do not express CD45 at all. 19% of BALLs express CD45 heterogeneously. CD34 co expression with CD19 by B-ALL is a hallmark of pre B cells. CD19 is the best B cell marker because it is expressed 98% of the time in B-ALL. There is a very high correlation between CD34 expression and TdT expression so that it is not necessary to perform the more complex and sometimes erroneous intracellular TdT assay.CD10 coexpression with CD19 is also a hallmark of B-ALL. It is important to distinguish this from normal pre B cells, often called hematogones. In a national cooperative group study performing central morphologic and flow cytometry review, it was found that 13% of cases, that had been diagnosed at a local institution as B-ALL, were actually normal pre-B cells. CD22 is expressed on 88% of B-ALL but CD20 is expressed only 33% of the time. CD20 expression is indicative of the most mature B-ALL. CD11c is not expressed in B-ALL.One third of B-ALL express the myeloid antigen CD13. While some reports also indicate CD33 is also frequently expressed, we found expression on only 3%. Some aberrant markers may lack epitopes for some monoclonal antibodies.Pre B cells: Is this leukemia? It is important to measure several markers on any hematopoietic malignancy because aberrant expression will occur for only some markers. Here we see normal pre B cells in bone marrow, which during infection or after chemotherapy, can reach 50% of cells. The frequency in blood is less than 1/500 cells.B-lineage malignancy can be misdiagnosed by flow cytometry in individuals who have a high frequency of pre B-cells, often called hematogones. This population can be significant in patients with recovering bone marrow after intensive chemotherapy and in patients with infectious diseases. A non-malignant bone marrow with a high frequency of hematogones is shown. This very characteristic pattern is invariant among normal individuals. Because of this distinctive pattern they can be distinguished from malignancy. This combination is used for all leukemias to define their CD45 expression. Most AMLs exhibit dim expression of CD45 and are HLADr+. Acute promyelomonocytic leukemia (FABM3) is the exception because >90% of them do not express HLADr.CD13 and CD33 are the most important myeloid markers. When combined with HLADr, undifferentiated and granulocytic lineage leukemias are CD13+CD33- or CD33dim, whereas monocytic lineage malignancies are CD13+CD33+. Except for FABM3, all of the others express HLADr. In this example, the leukemic cells express CD13 (pink), but a normal monocyte population expressing CD13 and CD33 (red, green) is also present.All AMLs are positive for CD13 CD33 or both so that this combination is used to define the normal and leukemic myeloid lineage. CD11b is often expressed in an aberrant fashion by leukemic cells (green) in the bottom row.This combination is useful in the determination of CD38 expression by the leukemia. Absence of CD38 coexpression with CD34 has been shown to be a poor prognostic indicator.This combination is also useful in defining AML because CD2 or CD7 is often coexpressed with CD13. The normal T-cells (blue) also act as a landmark population. Note that in this leukemia there are more normal cells than malignant ones.This combination is useful because expression of CD56 in leukemias that exhibit 8,21 translocations have a very bad prognosis. In addition, aberrant expression of CD15 can be used to arrive at the definitive diagnosis, when present. Note the high frequency of normal NK cells (brown) that can act as a landmark population.Erythroleukemias are easy to diagnose by flow cytometry, as shown in the figure because they have a characteristic FSC vs SSC pattern that exhibits an elongated cluster ranging from very small erythroid cells to larger myeloid cells. Variants to this pattern in both scatter characteristics and phenotype are due to the myeloid component of the leukemia that is almost always associated with the disease. The erythroid component does not express any leukocyte markers. Marker expression by cells of the myeloid component is variable and summarized in the table. The most common phenotype for the myeloid component is CD4 CD11 CD13 CD33 CD34 CD38 CD45 HLADr. Markers less frequently expressed are CD11b CD15 CD16 CD32. CD41 expression on megakaryoblasts occurred in 15% of patients. A myeloid component was not found in 16% of patients diagnosed as erythroleukemia by hematopathology.CD45 gating, in which the SSC vs CD45 view of the data is used to select the gate, offers a simple means for defining the major hematopoietic lineages and their malignant counterparts. Some investigators use a linear scale (shown here) while others use a log scale for SSC. Notice how much better these populations are resolved than when FSC vs SSC is used. Since most myeloid malignancies dimly express CD45, like progenitor cells, the region is used as a gate for determining marker expression. The disadvantage of this gating strategy is that one color is always CD45, which can add to the cost of the test.The malignant population, red, is an overlaid bivariate histogram on the normal bivariate histogram. FABM0/1 are characterized by their low side scatter and dim CD45 expression. They also exhibit heterogeneous forward scatter characteristics. FABM0 cells are most often small in size than the FABM1 illustrated above.The malignant population, red, is an overlaid bivariate histogram on the normal bivariate histogram. FABM2 exhibit dim CD45 fluorescence, but they are larger cells than FABM0/1. Nevertheless, the differences are often not striking. Thus, the scatter and CD45 fluorescence characteristics are most useful in resolving leukemic from normal cells and less useful for characterizing their FAB subtypes.The malignant population, red, is an overlaid bivariate histogram on the normal bivariate histogram. FABM3 consists of two forms. The first, not shown, has a pattern like that shown for FABM2. The second type, representing the majority of APLs, are highly granular and are easily distinguished from other forms of AMLs, as shown here. Marker expression can be difficult because these cells are also highly autofluorescent. The notion that FABM3 cells do not express HLADr is wrong. In a comparative study of 62 FABM3, with several hundred AMLs, HLADr expression failed to discriminate FABM3 from other FAB classified malignancies.The malignant population, red, is an overlaid bivariate histogram on the normal bivariate histogram. FABM4/5 are malignancies of the monocytic lineage and often exhibit bright CD45 fluorescence and side scatter characteristics similar to monocytes. FABM4 always have a population exhibiting dim CD45 fluorescence (not shown). Note in this example, the high frequency of dead leukemic cells (red) to the left and above the cyan colored lymphocytes in the FSC vs SSC view.The malignant population, red, is an overlaid bivariate histogram on the normal bivariate histogram. FABM6 (and M7, not shown) are characterized by their lack of expression of CD45. In 90% of cases there is a myeloid component which exhibits dim CD45 fluorescence. FABM6 and FABM7 have similar CD45, SSC and FSC characteristics but can be distinguished from each other using CD71 or glycophorin for erythroid and CD41 or CD61 for megakaryoblastic malignancies.Myeloma cells can be found because of their unique expression of CD56 and CD38 but not any other leukocyte antigen. Occasionally dim CD20 and dim CD45 expression can be found on early differentiated myeloma. The combination of FITC-CD38, PE-CD56, TC-CD45 can be used for resolving myeloma cells.DNA content measurements can be made by staining the antibody labeled cells with Hoechst after permeabilizing them. The first step is to draw a gate around the myeloma cells.The DNA content of the MNC is shown on the left, note the aneuploid myeloma cells are a low fraction of the total. By gating on the myeloma cells, the DNA content of them alone is resolved. Patients that have myeloma with a low s-phase fraction (SPF) have a better prognosis.The hallmark of T-cell malignancy is inappropriate marker expression. This can present itself as either an abnormal epitope density or as the absence of a marker. In this example, CD4 expression in a lymphoma (green in A and B) is not different from that found on normal T-cells (green in D and E), but CD3 expression on the lymphoma cells (A) is more variable than on normal T-cells (D). The differences are the absence of CD7 and the increased density of CD2 (rust) expression in the lymphoma cells (C). Note that normal T-cells (blue in C or F) are easily distinguished from the lymphoma cells (rust in C). The CD7-CD2+ cells (rust) in F are normal NK cells.In order to evaluate malignancy in lymph nodes or solid tissue masses the normal patterns for B and T-cells must be known. This is especially important because reactive lymph nodes can have grossly altered frequencies of T and B-cells. CD3 expression is always less on lymph node T-cells than on peripheral T-cells, but CD4 and CD8 are the same. An increased frequency of the CD3+CD4+CD8+ subset is often found in reactive nodes.Sezary cell leukemias are characterized by dim CD3 expression and slightly less CD4 expression combined with CD71 expression. They may also express CD25 (not in this example) and HLADr.11LGL can be of either T-cell or NK cell origin. T-cell LGLs, shown here, express CD3 and CD57, whereas NK cell LGLs are CD3- and CD57-. CD56 and CD8 are always expressed on both. While normal LGL in high frequency might be misdiagnosed as malignant, the aberrant expression of both CD2 and CD7 proves the malignant status of this T-cell LGL.In order to evaluate malignancy in lymph nodes or solid tissue masses the normal patterns for B and T-cells must be known. This is especially important because reactive lymph nodes can have grossly altered frequencies of T and B-cells. The normal pattern for lymph node B-cells is shown, but the frequency of cells within subsets may vary markedly. In the bottom row, a CD19 gate has been used.CD2, CD7, CD10 and CD13 co-expression with CD19 in cells from lymph nodes of four patients with lymphoma is shown in A, B, C, D, respectively. Co-expression with CD19 is relatively homogeneous in the case of CD2 and CD10, but is more heterogeneous in the case of CD7 and CD13. Control B-cells from a reactive lymph node are shown in E, F, G and H for comparison. A small population of Lambda positive (violet) cells is clearly resolved from all other cells that form a continuum at a 45 degree angle in A. In B, the Lambda positive population is found to be CD19+ even though it falls on the 45 degree continuum. In C, this population is further resolved from the other cells as CD19+Kappa-. This clearly establishes the population as CD19+Lambda+ clonal B-cell malignancy. The percentages of CD19+CD13+ and CD19+CD33+ cells suggest that CD13, CD19 and CD33 are coexpressed. When one looks at the bottom row of panels, in D, there is a high proportion of CD33+CD13+ cells (red and green). In E and F, it can be seen that CD19 expression on CD13+ or CD33+ cells (green to red) is heterogeneous and the population of CD19+CD13-CD33- cells are distinct (rust). The significance of the coexpression of CD19 on CD13+CD33+ cells is unknown in this case, but may be due to non-specific antibody binding to myeloid cells.Typical expression of markers by chronic lymphocytic leukemia and small cell lymphocytic leukemia of the bone marrow is shown here. The top row shows coexpression of CD19 with CD5 but not CD10. CD5 can be higher or more heterogeneous than in this figure. CD20 and CD22 are almost always expressed in a heterogeneous fashion and it is not uncommon to find no CD22 expression. In the bottom row, light and heavy chain expression is determined by gating on CD19 which is present in all the combinations. Usually IgD is heterogeneously expressed and IgM is not expressed. A hallmark of CLL/SLL is the expression of CD79 in a heterogeneous fashion. Usually nearly all the cells will express CD23.Follicular lymphomas are characterized by their CD10 expression. They often show expression of CD2 and CD7. They may not always express light or heavy chains and may be resolved from other B-cells due to their often larger size. Virtually all follicular lymphomas exhibit a peculiar T-cell expression of CD19, as shown in the bottom row. The basis for this expression is not known, but it is diagnostic for follicular lymphomas of all types.Burkitts lymphoma is characterized by the expression of CD10 and IgM, but usually not IgD. The cells almost always express CD20, CD22, CD79, but not CD23.Mantle cell lymphoma differs from CLL/SLL in its characteristic high expression of CD20, CD22 and CD79 (not shown) and lack of expression of CD23 (not shown).Follicular lymphoma can often be distinguished from other lymphoproliferative diseases by their expression of CD10. About a quarter of them also exhibit heterogeneous expression of CD2.Chronic lymphocytic leukemias (small cell lymphocytic lymphoma) are characterized by their co expression of CD19 and CD5. Here we see four examples of aberrant expression of CD2, CD7, CD13 and CD10 on this class of lymphoproliferative disease. A correlation to clinical outcome has not yet been made.Distinguishing between lymphoma and leukemia cannot be made by flow cytometry when blood or bone marrow is evaluated because phenotypic variations can be similar in both. Thus, the diagnosis of lymphoma is made using extramedulary tissue or when it is known that lymphoma is present. Leukemia will be the diagnosis of choice when it is known no lymphoma is present. Thus, the diagnosis is based on anatomical criteria. Perhaps there is no such thing as a typical B-cell malignancy, but the most commonly found phenotype is the expression of CD19 (A), CD20 (B), CD22 (B) and one of the two light chains (C). CD5 expression is variable, typically like that shown in A. In C, kappa vs lambda is shown for cells gated on CD19 and this lymphoma is kappa light chain restricted. The corresponding bivariations for normal B-cells are shown in D, E and F.