1. IMMUNOPHENOTYPING LEUKEMIAS AND LYMPHOMAS

2. CD45 Gating Strategy NORMAL BONE MARROW LEUKEMIC (TALL) BONE MARROW A
256
512
768
1024
/6/
SSC -> 10 1 2 3 4
/6/
HLADr ->
256
512
768
1024
/7/
SSC -> 10 1 2 3 4
/7/
HLADr -> A B C D
erythrocytes and platelets
Progenitor cells from normal bone marrow express less CD45 and exhibit low side scatter so they can be easily resolved in the bivariate display of SSC vs CD45. When HLADr vs CD45 is plotted the arrows indicate the differentiation pattern of the CD45+ HLADr+ progenitor cells. Leukemias all arise from these progenitor cells so that this region can be used to resolve them from all other cells.
When phenotyping leukemia, no single combination can be used for its characterization. Rather, several antibody combinations are employed and those that follow represent our opinion on the minimum three antibody combinations that should be evaluated. Because we are products of our experience, consensus on this issue has not been achievable.
granulocytes
monocytes and B-cells

3. CD45 EXPRESSION BY ACUTE B-LINEAGE LEUKEMIA
HLADr B
CD45
HLADr C
Most acute B-lineage leukemias can be recognized because HLADr is often overexpressed (A) or CD45 epitope expression is lower (B,C) than on normal B-cells. The region identifies the position of normal B-cells. One of the most important features of leukemia is aberrant antigen expression. All antigens are not expressed in an aberrant fashion and each leukemia exhibits a different repertoire of antigens that are.
CD45
HLADr

4. 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 B-cells are colored blue. In the leukemia, most are CD45-, but a smaller subset express some CD45.

5. This combination along with the next one, where CD10 is replaced by CD34, is most important for characterizing B-lineage acute leukemia because >90% of them express CD19 and CD10 or CD34 or both.

6. This combination along with the previous one is most important for characterizing B-lineage acute leukemia because >90% of them express CD19 and CD10 or CD34 or both.

7. Nearly all acute B lineage leukemias are CD22+ and CD20- or CD20 is expressed only on a low frequency of cells in an aberrant fashion.

8. Expression of CD13 is found in 30% of B lineage leukemias and coexpression with CD19 is definitive.

9. CD19
CD20
CD10
CD22
CD45
CD34
B-lineage malignancy can be misdiagnosed by flow cytometry in individuals who have a high frequency of pre B-cells, often called hematagones. 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 invariate among normal individuals. Because of this distinctive pattern they can be distinguished from malignancy.
CD19
HLADr

10. T lineage acute leukemias are characterized by aberrant expression of at least two and usually more antigens. Using this combination, the aberrant expression of CD45 is common. While T-cell leukemias are most often HLADr-, it is positive in this example.

11. The pattern generated by this combination is so invariate in normal individuals and so often altered in TALL as to make it diagnostic for TALL. Note the normal T-cells that can be used as a landmark for describing the aberrant expression on the malignancy.

12. This combination is also useful in describing TALL
This combination is also useful in describing TALL. In this example CD7 is overexpressed and CD2 is underexpressed. Note also the normal T-cell population that can be used as a landmark.

13. This combination can be used to aid in the characterization of TALL when CD10 is positive. In this example, it is not positive. Note this combination is also used for BALL.

14. This combination is useful for evaluating T lineage ALL Coexpression of CD5 and CD34, but not CD19, is definitive for TALL. Note this combination is also used for BALL.

15. 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 th exception because >90% of them do not express HLADr.

16. 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.

17. 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.

18. 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.

19. 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.

20. Typical B-cell Lymphoma
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.
Typical B-cell Lymphoma

21. T, CD10, and Myeloid Antigen Coexpression
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.
T, CD10, and Myeloid Antigen Coexpression
in B-cell lymphoma

22. Typical T-cell Lymphoma or Reactive Lymph Node:
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.
Typical T-cell Lymphoma or Reactive Lymph Node:

23. Problem Case A B C D E F Kappa CD19 CD33 CD13 Lambda
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.
Problem Case