1. CP Unknown Heme-10/19/2011 Kumaran Mudaliar and Girish Venkataraman
Loyola University Medical Center, Pathology

2. What cells are these?- hematogones
Plasma cells are bright CD 38 with variable CD 19 expression
What cells are the purple population?
-Plasma cells

3. Additional plot of same case

4. Light chain negative cells colored green
Explain the gates in this tube containing CD45, CD19, CD20, kappa and lambda
Primary gate CD19/SSC
Light chain negative cells colored green
and forwarded to show on all plots.
Backgating and coloring the cells green

5. Hematogones Hematogones: physiologic precursors of B-cells
70 years ago, distinct ‘lymphoid appearing cells’ in the bone marrow (BM).
‘Hematogones’ (hematogonia, meaning ‘blood-maker’)
662 patients
8% pts had 5% or more of hematogones
24.6% < 16 YO had hematogones
6.3% > 16 YO had hematogones
MC in patients: lymphoma, marrow regenerative states, immuno cytopenias, AIDS
Cells of uncertain significance

6. Problem
At 5% level, they are conspicuous on marrow smear and can be confused with neoplastic lymphoblasts

7. (A) Several hematogones are illustrated in this bone marrow smear from a 3-year-old boy with immune thrombocytopenia. They bear close resemblance to the neoplastic lymphoblasts illustrated in panel B. (B) Numerous lymphoblasts are present in this bone marrow smear from a 5-year-old girl with precursor B-ALL

8. Mature hematogones

9. Figure 5. A scatter plot relating percent bone marrow hematogones to patientage for all 662 specimens. There is a significant decease in the percent hematogones with increasing age as shown by the regression line. (Two cases were off thescale of the scattergram and are not shown: a 5-month-old with 51% hematogones and a 23-year-old with 65%.)

10. Reference Ranges? No accepted reference ranges
BM examination is not performed in healthy people

11. 3 stages of Hematogone Maturation
Early
Usually comprise a small minority, but can become expanded in regenerating marrows
No expression of CD 20 /CD34+
Intermediate
Majority in most marrows
-CD34-/CD20 dim/CD10+
Late
In contrast to mature B cells, Cd34-/CD20+/maintain dim CD10 . Light chain surface+

12. Take out CD 43, CD 22, IgM, and sIg

13. As hematogones matures, they express CD 2019 20

14. 20 38

15. 19 34

16. 10 34

17. Stage 1
Hematogones
CD34+/CD10+
Gated on all CD19+ cells
Stage 2 hematogones
Dimmer CD10, CD20 het
Stg 2 (red) and stg 3 (blue)
Hematogones lack CD34
Stage 3 hematogones
-bright CD20, dim CD10
>90% of B-cells in this case are hematogones

18. Immunophenotype CD 10+ CD 19+ CD 20+ Variable expression CD 38+ CD 45+
TdT + [subset]
CD 34 + [subset]
Kappa & Lambda -

19. Differences between hematogones and lymphoblasts
Both present in CD 45 dim gate
Hematogones: low side scatter
Consistent, highly reproducible, maturational pattern
No aberrant or asynchronous antigen expression
B-ALL: relatively higher side scatter
Phenotypic abnormalities:
Myeloid Markers: 13, 33, 15
T-cell: 2, 5, 7
Concurrent expression: TdT and cytoplasmic IgM; 34 and 20 ; 34 and surface light chains
Loss of antigen expression on blasts: 45 – ; 10 – [B-ALL with MLL abnlties] ; 34 – [E2A-PBX1 fusion]

20. Even tho both may express an antigen, difference in expression patterns and levels of specific antigens exist
IF TdT: hematogones (coarsely granular / speckled) to blasts (finely granular / even distribution)
Also, hematogones have higher #’s of TdT and CD 10 molecules / cell
Leads to brighter expression on FC
Hematogones have less CD 19 molecules/ cell
Hence dimmer expression on FC

21. BM Core Biopsies Hematogones: dispersed throughout marrow
Blasts: small clusters (> 5 cells)

22. Issues Differentiation can be challenging Left Shifted Hematogones
Early stages after BM transplantation
Anti-CD20 immunotherapy
Majority of B-ALL lack CD20 expression .
-Phenotypic change
Anti CD 20 - can change the normal pattern of hematogone antigen expression by abolishing or masking the expression of CD20 in hematogones which might create problems
Phenotypic Change: (‘antigen shift or drift’) in recurrent B-ALL has been previously characterized by FC, with over 70% of cases showing loss of at least one aberrant antigen at relapse

23. Sources
McKenna RW, et al. Immunophenotypic analysis of hematogones (B-lymphocyte precursors) in 662 consecutive bone marrow specimens by 4-color flow cytometry. Blood Oct 15;98(8):