1. ICCS e-newsletter CSI Summer 2011 Jeannine T. Holden, MD Emory University School of Medicine Atlanta, GA

2. Case History The patient is a 35 year old male with a history of B lymphoblastic leukemia/lymphoma diagnosed several years prior to the present study and treated with chemotherapy. Both original diagnosis and treatment were performed at another institution. Patient is reportedly doing well clinically, and the present study is routine monitoring. His peripheral blood counts are all within normal limits. The patient does report a recent two day history of malaise and upper respiratory symptoms that are attributed to viral infection.

3. Questions… Is there any evidence of residual/recurrent B lymphoblastic leukemia/lymphoma? Is normal hematopoietic activity intact? Is there any evidence of other diseases? Are there any other problems with this case?

4. Flow cytometric immunophenotyping Bone marrow aspirate Acquired on a FACSCanto cytometer using Diva software, FCS2.0 formatted listmode data Fourteen four-color tubes Tubes #3 through #14 gated at acquisition (enriching for mononuclear cells) Files are labeled ICCS001 through ICCS014

5. Antibody panel (FITC/PE/PerCP/APC) ICCS001: isotype controls ICCS002: CD14/CD13/CD45/CD34 ICCS003: HLA-DR/CD25/CD45/CD33 ICCS004: CD3/CD4/CD45/CD8 ICCS005: CD2/CD7/CD45/CD5 ICCS006: CD10/CD19/CD45/CD34 ICCS007: CD36/CD117/CD45/CD34 ICCS008: CD15/CD11b/CD45/CD34 ICCS009: CD103/CD22/CD45/CD11c ICCS010: CD16/CD56/CD45/CD38 ICCS011: FMC-7/CD23/CD45/CD19 ICCS012: KAPPA/CD20/CD45/CD19 ICCS013: LAMBDA/CD20/CD45/CD19 ICCS014: KAPPA/LAMBDA/CD20/CD5

6. Antibody panel (FITC/PE/PerCP/APC) ICCS001: isotype controls ICCS002: CD14/CD13/CD45/CD34 ICCS003: HLA-DR/CD25/CD45/CD33 ICCS004: CD3/CD4/CD45/CD8 ICCS005: CD2/CD7/CD45/CD5 ICCS006: CD10/CD19/CD45/CD34 ICCS007: CD36/CD117/CD45/CD34 ICCS008: CD15/CD11B/CD45/CD34 ICCS009: CD103/CD22/CD45/CD11c ICCS010: CD16/CD56/CD45/CD38 ICCS011: FMC-7/CD23/CD45/CD19 ICCS012: KAPPA/CD20/CD45/CD19 ICCS013: LAMBDA/CD20/CD45/CD19 ICCS014: KAPPA/LAMBDA/CD20/CD5 Ignore high background staining with this reagent in this case!

7. Antibody panel (FITC/PE/PerCP/APC) ICCS001: isotype controls ICCS002: CD14/CD13/CD45/CD34 ICCS003: HLA-DR/CD25/CD45/CD33 ICCS004: CD3/CD4/CD45/CD8 ICCS005: CD2/CD7/CD45/CD5 ICCS006: CD10/CD19/CD45/CD34 ICCS007: CD36/CD117/CD45/CD34 ICCS008: CD15/CD11B/CD45/CD34 ICCS009: CD103/CD22/CD45/CD11c ICCS010: CD16/CD56/CD45/CD38 ICCS011: FMC-7/CD23/CD45/CD19 ICCS012: KAPPA/CD20/CD45/CD19 ICCS013: LAMBDA/CD20/CD45/CD19 ICCS014: KAPPA/LAMBDA/CD20/CD5 History of B ALL means that this tube is probably the most informative

8. So, look at the analysis pdf, scrolling to Tube ICCS006, and look for candidate malignant populations… Cells co-expressing CD19, CD34, and CD10 are readily identifiable We know that normal B-cell progenitors (“hematogones”) are likely present Can we identify any populations that are phenotypically aberrant (particularly as compared to the expected normal B- cell progenitors)?

9. One of the dotplots for this tube looks odd… B-cells and B-cell progenitors are present, but there is also an unexpected population. Can you see the unexpected population? Don’t worry if you can’t, as it’s difficult to see if you aren’t already familiar with the normal patterns as generated by another laboratory.

10. One of the dotplots for this tube looks odd… So, let’s make it easier… –The cells circled in blue are immature B-cell progenitors. They co-express CD34 and CD10. –The cells circled in green are more mature B-cell progenitors. The co- express CD10 but not CD34. –The cells circled in orange are mature B-cells. They express neither CD10 nor CD34, and do express surface immunoglobulin light chains. –The cells circled in pink are the aberrant population. If you backgate you’ll find that these cells co-express CD10 and CD34. Are these cells recurrent/residual leukemic blasts?

11. Not convinced? Well, let’s compare it to a normal hematogone pattern… –The dotplot on the bottom shows the expected normal hematogone pattern with the CD19/CD45 combination. –The dotplot on the top definitely has another population in there.

12. Not convinced? Well, let’s make it even easier… –The dotplot on the bottom shows the expected normal hematogone pattern with the CD19/CD45 combination. –Here I’ve added the colored gates, just to make it clear. Notice that the pink circle is essentially empty in the bottom dotplot.

13. But now it’s really confusing, because these dotplots are both from the same analysis in the same patient… Don’t believe me? –You can go look at the full analysis pdf and see for yourself. –And while you’re at it, look at the other CD19/CD45 combinations in tubes 12 and 13. Neither of them shows the aberrant population.

14. So, what’s going on? Residual/recurrent leukemia present, but only shows up in one tube? –Unlikely degree of sampling error. –Unlikely immunophenotype, as even if these cells do express CD19, CD10, and CD34, they also appear to express very high density CD45, and that’s not characteristic of B ALL.

15. And the answer is… “Carry over” –Tube 5 in this analysis is directed at identifying normal (and aberrant) T- cells. –Note that the CD7/CD45 combination identifies cells in both the orange and pink circles. These are normal T- cells. And of course they also express CD2 and CD7.

16. “Carry over” Already stained cells from one tube are introduced to another tube of stained cells Results in overlapping dotplots Typically identified in sequential tubes May occur as a result of… –Pipetting error (unusual) –Splashing of one sample into an another –Inefficient washing/rinsing of sipper –Machine tubing: cells from first tube remain in tubing, acquired with next tube

17. How do I know for sure that this is “carry over”? Because I deliberately contaminated tube 6 with cells from tube 5, that’s why. But here’s another example to show you what it looks like…

18. Another example of “carry over” Cervical lymph node fine needle aspirate from a 58 year old female Normal blood cell counts Cytologically benign

19. Why does this patient’s sample harbor cells that co-express CD19, CD10, and CD34? Is this an unusual early presentation of B ALL?

20. And why can’t I find a distinct CD34+ population in this tube from the same analysis in the same patient?

21. Look familiar?

23. Conclusions Exploit panel redundancy to investigate unexpected findings. Assume that errors will occur. Consider all information about a case, including the clinical history. Don’t have the clinical history? Get it.