Flow Cytometry A Guide for Data Analysis

Transcription

1 Flow Cytometry A Guide for Data Analysis Last revisions 12 Aug 2013 Ron Lee, M.D. Introduction This guide is intended to provide a basic overview of clinical flow cytometry data analysis. General Principles 1) Determine sample composition and account for all events/populations (including screening CD45-negative events/debris) using multiple antibody combinations and gating strategies 2) Characterize each population as thoroughly and accurately as possible 3) Determine if all populations are immunophenotypically normal and/or have normal maturation patterns 4) Characterize lineage/immunophenotype of any abnormal populations and level of involvement A Systematic Approach to Analyzing Flow Cytometry Data 1) Account for all lymphocytes (using CD45 v SSC and single markers v SSC ungated) a. NK cells b. CD4+ T-cells, CD4+/CD57+, look for atypical CD16 or CD56 or aberrant CD2, CD3, CD4, CD5, CD7, CD8, or CD10 c. CD8+ T-cells, CD8+/CD57+, look for atypical CD16 or CD56 or aberrant CD2, CD3, CD4, CD5, CD7, or CD10 d. B-cells, compare CD19 and CD20, gate on CD19 (all CD19, including brighter, dimmer, increased SSC) and look at CD23 and FMC7, check kappa v lambda (CD19 and CD20 gating), check CD19/20 v kappa, CD19/20 v lambda, and CD19/CD20 kappa v lambda, look for coexpression of CD5 or CD10, gate on CD19 or CD20 and plot 10 or 5 v kappa and lambda, check any bright CD19 or CD20+ events for light chains and CD103, check increased SSC or FSC 19+ or CD20+, check for plasmacytoid differentiation (CD19+, bright CD38, increased FSC) e. CD4-/CD8- T-cells (typically <1% of sample and corresponding to TCRGD+ T-cells) f. CD4+/CD8+ T-cells (typically absent to very few) 2) Account for monocytic cells (%, patterns, scatter, CD13, CD14, CD15, CD16, CD33, CD64, CD56, CD11b, CD11c, HLA DR) 1

2 3) Account for neutrophilic cells (%, patterns, scatter/hypogranularity, CD11b, HLA DR, CD13, CD16, CD33, CD10, CD14, CD56) 4) Account for eosinophils (%, CD11b+, HLA DR-/+, CD13+, CD10-, CD16-) 5) Account for basophils (%, CD13+, CD64-, CD11b+, CD22+, HLA DR-) 6) Account for mast cells (%, CD117+ bright, CD45 comparable to basophils but with increased SSC) 7) Account for blasts (%, look for atypical CD45, CD13, CD33, CD34, CD38, CD117, CD11b, CD11c, HLA DR, CD2, CD5, CD7, or CD56) 8) Account for plasma cells (%, immunophenotype, CD45, CD19, CD20, CD56, CD117 HLA DR) 9) Check CD45-negative events/debris (erythroid precursors, plasma cells, lymphoblasts, CD56+ events, non-hematolymphoid cells, etc.) 10) Account for any other events or abnormal populations (%, immunophenotype) 11) Add up all populations to account for entire sample Cellular integrity Aside from viability testing (PI, 7-AAD, trypan blue, etc.), cellular integrity can be determined by scatter properties. Samples with poor integrity show diminished scatter properties. Diminished scatter properties can also been seen in dysplastic processes. In normal samples, there should not be a significant proportion of cells with little to no forward scatter as shown below. The above histograms, from left to right, show samples with diminishing integrity and an increased proportion of degenerating cells. The forward scatter properties diminish as the integrity decreases (arrows), and the amount of events increasing in the lower right region (red circle) indicates cellular degeneration (eosinophils can also have lower FSC compared with neutrophils). 7-AAD is not needed to determine the integrity of marrow and blood samples less than 48 hours old when scatter properties are adequate. 2

3 Sample cellularity should be checked, and a comment should be made if the cellularity is low. Hemodilute marrow samples typically have an increased proportion of mature granulocytic forms and a sample composition/cellular differential similar to peripheral blood, and a comment should be made if the sample is hemodilute (or hemocontaminated in the case of tissue, fluid, and FNA samples - also consider acute inflammation and myeloid neoplasms in the case of tissue, fluid, and FNA samples). Sample Composition Accounting for all events and populations is essential. Characterizing all events should be accomplished using multiple strategies. These include, but are not limited to, analyzing cells and populations with respect to CD45 versus side scatter properties, forward versus side scatter properties, individual markers versus side scatter properties (ungated), maturation patterns, and overall immunophenotypic profiles. CD45-negative and CD45 dim positive events should always be screened and may include unlysed RBC s, erythroid precursors, platelets, non-hematolymphoid cells, B-lymphoblasts, plasma cells, and degenerated cells/ debris. The preservation of erythroid precursors is dependent on the method of lysis. Cal- Lyse (CALTAG) preserves RBC precursors better than ammonium chloride but forward scatter properties tend to be compromised. High-yield lyse (CALTAG) preserves forward scatter properties but has less preservation of RBC precursors. Ammonium chloride tends preserve the fewest erythroid precursors but is relatively inexpensive and preserves forward scatter properties. CD45 v SSC gating CD45 PC7 R2 R1 SS Log R3 R4 R5 3

4 CD45 v SSC (linear or log) is commonly used to obtain differentials and follow populations from tube to tube. In general, in the above histograms R1 (red) is the blast region, R2 (blue) is the lymphoid region, R3 (yellow) is the monocytic region, and R4 (green) is the granulocytic region (neutrophils and eosinophils). R5 is the CD45-negative region and can include unlysed RBC s, erythroid precursors, platelets, debris, degenerating cells/debris, plasma cells, lymphoblasts, and non-hematolymphoid cells (the above histograms show a relative preservation of RBC precursors with Cal-Lyse on the right versus ammonium chloride on the left). Maturing B-cell precursors (hematogones) have less FSC and SSC than myeloid blasts and are the black events to the left of R1. Of note, the regions above are intended to demonstrate where different cell populations are typically found and are not necessarily intended to demonstrate a gating strategy for analysis. If CD45 v SSC gating is the sole method used, one must be always be aware of the possibility of overlapping and hidden populations. Notable examples include plasma cells with variable expression of CD45 (typically with intermediate side scatter properties; CD45 is often negative in myeloma but can be positive; CD45 is typically positive in plasmacytic differentiation of B-cell neoplasms which have increased side scatter and often overlap with monocytes and/or basophils), monocytic precursors in the blast and granulocytic region (especially if the granulocytes have decreased side scatter), large B-cell lymphoma with increased side scatter relative to lymphocytes or in the blast region, hairy cell leukemia and other lymphoproliferative disorders in the monocytic region, pronormoblasts in the myeloid and CD45-negative regions, lymphoblasts in the CD45-negative region, and non-hematolymphoid cells as well as some lymphomas in the CD45- negative region. One should also be familiar with the locations of basophils [slightly decreased expression of CD45 and increased side scatter compared to lymphoid cells, between R1 and R2 above, typically increased in myeloproliferative disorders (especially CML)], mast cells (CD45 expression similar to basophils but higher side scatter properties, typically detected with CD117 v SSC ungated), and eosinophils (typically highest side scatter in granulocytic region and relatively bright expression of CD45, expression of CD13, and lack of CD16). Cellular differentials in marrow and blood samples should be determined and cross-checked to account for all populations. If there are no significant overlapping populations, CD45 v SSC should give a reasonable cellular differential. Populations should always be screened and cross checked using individual markers v SSC ungated. In addition to identifying abnormal populations, backgating with individual markers v SSC helps identify overlapping/hidden populations. 4

5 For example, R2 identifies the CD34+ myeloid blasts (CD34+ cells with less side scatter than the myeloid blasts are the least mature B-cell precursors/blymphoblasts), T- and NK cells (CD2+ cells), and monocytic cells (bright CD33+ cells), respectively. These percentages can be used to cross-check a cell differential using CD45 v SSC. The percentages obtained in this fashion should not differ significantly from the CD45 v SSC differential- if there are differences, this needs to be accounted for and the possibility of overlapping, hidden, and/or abnormal populations needs to be investigated (immature monocytic cells in blast region, lymphs in monocytic region, plasma cells, CD34-negative blasts, CD2- T- cells, etc.). CD45 v SSC, total leukocyte determination R1 is the total percentage of leukocytes in the sample. The percentages of individual populations for the sample composition should total ~98% in this case. 5

6 FSC v SSC In addition to utilizing CD45 v SSC, always screen FSC v SSC plots for abnormal populations. The following is a general guide to FSC v SSC. FS Lin Large lymphoid cells, plasma cells, monocytic cells, blasts Aggregates, non-hematolymphoid cells, other large cells Pronormoblasts Neutrophilic cells SS Log Small lymphoid cells Specific Cell Populations Lymphoid cells CD45 v SSC Mature lymphoid cells have bright expression of CD45 with little side scatter. R2 (blue above) identifies mature lymphoid cells with CD45 v SSC. Occasionally, mature, benign lymphoid cells will have slightly increased side scatter properties or slightly diminished CD45 expression. Abnormal lymphoid cells may have variable scatter properties or abnormal CD45 expression, and one should always backgate and compare lymphoid markers (B-cell, T-cell, and NK cell) using these 6

7 markers (CD2, CD3, CD19, CD20, etc) v SSC ungated to account for all lymphocytes. This can be especially helpful in assessing B-cells. Keep in mind, CD19+/CD20+ discordance may be seen with hematogones, lymphoblasts, plasma cells, occasional B-cell lymphoproliferative disorders (lymphoplasmacytic lymphoma, large B-cell lymphoma, etc.), and post anti-cd20 therapy. FSC v SSC Benign lymphoid cells generally have low forward and low side scatter properties (blue above). Again, backgate with CD19 v SSC and CD20 v SSC to account for all B-cells and assess where B-cells are with respect to CD45 v SSC and FSC v SSC. Markers B-cells- CD19 and CD20, as a minimum, should be used to identify B-cells (also CD22, etc.). In the absence of an explanation for CD19/CD20 discordance (anti- CD20 therapy, increased hematogones, lymphoblasts, plasma cells, etc.), these percentages should be virtually identical. T-cells- CD2, CD3, CD4, CD5, CD7, and CD8, should be used (at a minimum) to evaluate T-cells. Cross checking these percentages and intensities of expression help identify abnormal T-cell populations. Some cross-checks and general concepts to keep in mind include: 1) Total CD2 events should be very close to total CD3 events plus NK cells (minor subsets of NK cells lacking CD2 may be seen) 2) Total CD2 events should be very close to CD3/CD4 events plus CD3/CD8 events plus NK cells (normal T-cell receptor gamma delta+ T-cells are typically CD3+ bright/cd4-/cd8-/+ but there are typically very few of these cells in most normal samples) 3 The proportion of CD7-negative T-cells should be relatively low and not form a distinct, tight cluster 4) CD5-negative events should equal NK cells plus B-cells when gating on all lymphocytes 7

8 5) T-cells with diminished CD5 should not be significantly increased or form a distinct, tight cluster (T-LGL s and some T-cell subsets may have slightly diminished CD5) 6) Normal T-cells typically do not express significant amounts of CD16 or CD56 7) There should be relatively few, if any, CD4+/CD8+ T-cells NK cells- CD2, CD3, CD5, CD7, CD16, and CD56, should be used to identify and quantify NK cells. NK cells are typically CD2+ (with minor subsets lacking CD2), surface and cytoplasmic CD3-, CD16+, CD56+, CD7+, and CD5- (CD57 is typically variable). T-cells and NK cells CD45 APC-H7-A R4 SSC-A CD8 PE-Cy7-A 14.66_ _4.51 R _ _7.48 CD4 PerCP-Cy5-5-A As an example of T-cell analysis, the majority of the T-cells above are composed of CD4+ cells (RLQ) and CD8+ cells (LUQ). CD4-/CD8- T-cell receptor gamma delta+ cells are in the LLQ with B-cells and NK. The majority of peripheral blood and bone marrow samples have relatively few T-cell receptor gamma delta+ cells as well as cells which coexpress CD4 and CD8 (RUQ). The CD4:CD8 ratio in marrow and blood samples is typically ~3. CD4:CD8 ratios less than 1 should raise suspicion for viral infection (especially active EBV/infectious mononucleosis in which the CD8+ T-cells are increased and have features of activation such as expression of HLA DR and relatively bright CD38), recent treatment, chronic immunosuppression, HIV infection (typically a paucity of CD4+ T-cells), a T-cell lymphoproliferative process, etc. Tissue samples typically have a CD4:CD8 ratio ~5. Increased CD4+ T-cells in tissues with increased CD38, and otherwise no aberrant expression of T-lineage markers, may be seen in Hodgkin lymphoma (with increased CD4+/CD57+ coexpression in NLP Hodgkin), granulomatous disease/sarcoidosis, progressive transformation of germinal centers, large B-cell lymphoma where the cells are not adequately represented, and some reactive conditions. Intestinal T-cells are typically CD8>CD4 with coexpression of CD103 8

9 and diminished CD5. Apparent atypical immunophenotypic expression may be seen in celiac disease in the absence of T-cell lymphoma, e.g. loss of CD5. Lymphohistiocytic hemophagocytosis has been reported to have increased T- cells with loss of CD5. Normal T-cells are surface and cytoplasmic CD3 positive, distinguishing them from NK cells which are scd3- and ccd3- by flow cytometry. NK cells (quadrants 37, 33, and 9 above) are CD7+, CD56+, and CD5- (quadrants 7 and 36). NK cells often have partial CD8 (which is typically relatively dim). The percentage of CD3-, CD56+ cells should match the CD16+ percentage. Aberrant NK cells may have discordant CD16 and CD56 expression, or aberrant expression of T-cell markers such as CD2, CD5, or CD7. CD158 (KIR) markers may be used to assess for possible NK cell clonality, but in many cases it may be sufficient to accurately quantify and characterize the NK cells to determine if they are increased and if they have aberrant immunophenotypic expression without the use of CD158. CD56 is a marker of cytotoxic T-cells (quadrant 31), they are more commonly CD8+, and they should not be significantly increased. T-cell notes - T-cell large granular lymphocytes (T-LGL s), clonal and non-clonal, may have slightly diminished expression of CD5. Clonal T-LGL s are typically, but not always, CD57+ (often with relatively tightly clustered diminished CD5 and/or CD2) and may have CD16 and or CD56. Reliance on only on CD16 and CD56 to assess for T-LGL s is not recommended. CD94 and CD158 may be used and are typically negative on non-clonal/nonneoplastic T-LGL s. These may be helpful in cases of suspected T-LGL lymphoproliferative disorders. However, quantifiying all T-cell subpopulations, including CD4+/CD57+ and CD8+/CD57+ coexpressing cells, and looking for aberrant T-cell marker expression (often diminished CD5, CD7, and/or CD2) is usually sufficient to identify clonal T-LGL s. In general, the total of CD4+/CD57+ and CD8+/CD57+ coexpressing cells is typically less than 10% of all cells in marrow and blood samples (my double digit rule ). As a general rule, there are typically very few 9

10 CD4+/CD57+ coexpressing cells in blood and marrow samples and CD8+/CD57+ cells are typically less than 50% of total CD8+ T-cells. - Bright expression of CD38 and HLA DR expression on T-cells is a feature of T-cell activation, and infectious mononucleosis typically has a CD8+ lymphocytosis with relatively bright expression of CD38 and HLA DR. - For suspected Sezary cases, order CD7/MIG/CD3/CD45, CD7/CD26/CD3/CD45, CD7/CD26/CD4/CD45 criteria for staging MF include cutoffs of CD4+/CD7- cells at 40% and CD4+/CD26- cells at 30% (of CD4+ T-cells). - In tissues, increased CD4+ T-cells with bright CD38 without a corresponding B-cell component with relatively bright CD38 are often associated with Hodgkin lymphoma (CD4+ T-cells typically predominate in Hodgkin lymphoma, CD8 in viral infection). Increased CD4+/CD57+ T- cells raise the possibility of NLP Hodgkin. Monocytic cells CD45 v SSC Normal monocytic cells generally have bright CD45 expression with increased side scatter properties compared to lymphoid cells. R3 (yellow) identifies monocytic cells with CD45 v SSC. FSC v SSC Monocytic cells have increased forward and increased side scatter properties compared to lymphoid cells. 10

11 R2, blue, identifies monocytic cells with FSC v SSC. Markers CD11b, CD11c, HLA DR, CD13, CD33, CD14, CD15, CD16, and CD56 can be used to evaluate monocytic cells. Normal, mature monocytic cells are CD11b+, HLA DR+, CD13+, CD33+, CD14+, CD16-, and CD56-. Immature and abnormal monocytic cells can typically be identified by diminished expression of CD11b, CD13, and CD14 and/or atypical CD56 expression. Rarely, promonocytes and/or monoblasts can have an apparent mature immunophenotype. Inflammatory monocytic cells typically have bright CD45, diminished CD14 and coexpress CD16, but these are typically a very small component in blood and marrow samples but may be increased in sepsis, infection, and other reactive conditions. 11

12 Normal, mature monocytes are shown above as CD11b+ (bright), HLA DR+, CD13+ (intermediate to bright), CD33+ (bright) and CD14+. Relatively diminished expression of CD11b, CD13, and CD14 may be seen with left shifted maturation/ relative immaturity and may be seen in association with dyspoiesis. Concordant diminished expression of CD11b, CD13, and CD14 may be seen in young patients and in regenerating marrows post treatment or injury. Expression of CD56 and/or discordant or diminished expression of CD11b, CD13, and CD14 (eg. loss of CD13 or CD14 only), especially in older patients, is atypical and suggests an intrinsic marrow disorder. Loss of CD14 is also seen in PNH. 12

13 Neutrophilic Cells CD45 versus side scatter properties Neutrophilic cells have high side scatter properties and acquire CD45 as they mature. R4 above (black) identifies neutrophilic cells with CD45 v SSC. The arrow shows increasing CD45 expression corresponding to neutrophilic cell maturation. Neutrophilic cells typically have more side scatter than monocytic cells. Decreased side scatter may represent true hypogranularity or an artifact of decreased sample integrity. FSC v SSC Neutrophilic cells typically have high forward and side scatter properties. The neutrophilic cells should have higher side scatter than the monocytic cells. Decreased side scatter often represents hypogranularity. 13

14 Neutrophilic cells by FSC v SSC (R2, blue) Side scatter of neutrophils should be greater than monocytes. Decreased side scatter of mature neutrophils (Pseudo Pelger-Huet cells and hypogranular neutrophils on slide review) Markers CD11b, HLA DR, CD13, CD16, CD33, and CD10 should be used to identify neutrophilic cells and assess neutrophilic maturation. Neutrophilic cells acquire CD11b as they mature past the promyelocyte stage, typically lack HLA DR, diminish then acquire CD13, acquire CD16 as they mature ( Nike swoosh ), and 14

15 are CD33+. Bands and segs are typically CD10+. Acquisition of CD11b, CD13 v CD16, and acquisition of CD10 should be checked for the neutrophilic cells. R2 identifies granulocytic cells and their maturation is displayed with CD11b v HLA DR and CD13 v CD16. Neutrophilic cells should show a predominance of CD11b+ cells and a relatively even distribution of maturational stages with CD13 v CD16. In a sample with good integrity, an increased proportion of cells with diminished CD11b, CD13, CD16, and/or CD10 is aberrant (may represent phased maturation post recent treatment/marrow injury or an intrinsic marrow disorder). One should gate on the granulocytic cells only (blasts and monocytes excluded) and observe the proportion of CD16-negative cells (LUQ + LLQ with CD13 v CD16). Normal samples typically have 40% of gated cells or less in these two quadrants. If the total percentage of granulocytic cells in these quadrants exceeds 60%, it is indicative of left shifted maturation and is highly suggestive of a marrow disorder (increased eosinophils should be excluded, as eosinophils will increase the proportion of CD16-negative cells). In marrow disorders, there is typically a corresponding increase in the proportion of CD11b-, HLA DR- cells (LLQ in CD11b v HLA DR) and/or diminished CD10. If the blasts in such cases have aberrant CD2, CD5, or CD7 (typically greater than 20%), these findings typically represent an intrinsic marrow disorder. Cellular integrity must be checked, as cells with decreased integrity may show non-specific decreases in CD16, CD13, CD11b, and CD10. It should always be remembered, however, that abnormal maturation patterns, as well as expression of CD56, may be seen in regenerating marrows and post recent growth factors (CD14 on granulocytic cells may be due to recent growth factors as well). Additionally, absence of CD16 may be hereditary. 15

16 CD11b v HLA DR Gating on all myeloid cells (blasts, myeloid, monocytic cells) and observing the distribution of cells using CD11b v HLA DR is very helpful to determine the composition of the myeloid cells in the sample. Blasts CD45 v SSC properties Myeloid blasts typically have relatively dim CD45 expression and slightly increased side scatter properties compared to mature lymphoid cells. 16

17 R1 (red) identifies the typical myeloid blast region with CD45 v SSC. Monoblasts often have relatively decreased CD45 expression compared to mature monocytes. Aberrant B-lymphoblasts often have dim to no CD45 expression and relatively increased side scatter properties compared to mature lymphoid cells and normal maturing B-cell precursors (hematogones) but typically SSC less than myeloid cells. Pronormoblasts typically have dim to no CD45 expression and increased side scatter properties. Megakaryoblasts typically have dim CD45 expression and relatively increased side scatter properties. FSC v SSC properties Myeloid blasts typically have increased forward scatter properties and slightly increased side scatter properties compared to mature lymphoid cells. Lymphoid blasts typically have low to intermediate forward scatter properties and slightly increased side scatter properties compared to mature lymphoid cells. Monoblasts typically have increased forward scatter properties and slightly increased side scatter properties compared to mature lymphoid cells Pronormoblasts typically have increased forward and side scatter properties. Megakaryoblasts typically have increased forward scatter properties and slightly increased side scatter properties compared to mature lymphoid cells. Markers Myeloid blasts- generally CD34+, CD117+, CD11c+, CD13+, CD33+ (CD33- blasts typically in minority), CD38+, CD11b-, HLA DR+, CD2-, CD5-, CD7-, CD10-, CD19-, CD25- B-lymphoblasts- typically CD34+, CD117-, CD10+, CD19+, TdT+ T-lymphoblasts- typically CD1a+, TdT+, ccd3+ (may have ccd79a) 17

18 Monoblasts- typically CD11b+ (dim), HLA DR+, CD64+ (may have CD117) Pronormoblasts- typically GlyA+ (often heterogeneous/dimmer than more mature erythroid precursors; least mature have dim to no GlyA), CD71+, CD117+ (may have partial CD41) Megakaryoblasts- typically CD41+/cCD41+, scd62p-, GlyA-, CD11b-/+, HLA DR- Myeloid blasts should always be checked for aberrant expression of CD2, CD5, and CD7. Normal myeloid blasts typically do not have any CD2, CD5, or CD7 expression- partial expression of these markers, typically <20%, may be seen in reactive/regenerative conditions. >20% expression of these markers has been reported to be specific for myeloid neoplasia. Normal myeloid blasts CD34 PerCP-Cy5-5-A R4 SSC-A CD13 APC-A 0.00_0.00 R _ _ _0.05 CD34 PerCP-Cy5-5-A 18

19 CD11b PE-A 0.00_ _0.04 R _ _0.57 HLADR V450-A CD117 PE-Cy7-A 0.00_ _0.00 R _ _0.04 CD34 PerCP-Cy5-5-A CD11b PE-A 0.00_0.00 R _ _ _0.46 CD38 FITC-A CD34 PerCP-Cy5-5-A 12.88_ _0.00 R _ _0.00 CD38 FITC-A CD33 PE-Cy7-A 65.35_ _0.46 R _ _0.05 CD16 PerCP-Cy5-5-A 19

20 Plasma cells CD45 v SSC properties Plasma cells have variable CD45 expression and slightly increased side scatter compared to mature lymphocytes. FSC v SSC properties Plasma cells have increased side scatter properties and slightly increased side scatter compared to mature lymphocytes. Markers Normal plasma cells are typically CD45+ (minor subsets may be CD45-), CD19+/-, CD20-, CD11b-, HLA DR-, CD38+ (bright), CD56- (occasional, low level CD56+ polytypic plasma cells may be seen), CD117-, and CD138+ (may not be as good with flow compared to IHC). R2 (blue) identifies typical plasma cells with CD38 v SSC ungated. Occasionally, plasma cells may have slightly dimmer CD38 expression, but when distinctively dimmer CD38 is present on plasma cells it is a clue to a neoplastic plasma cell population. Neoplastic plasma cells typically lose CD45 and CD19 and often acquire CD56 and/or CD117. Also, FSC v SSC is helpful to identify plasma cells with dimmer CD38 expression. 20

21 Eosinophils CD45 v SSC properties Eosinophils typically have relatively bright CD45 expression compared to immature myeloid cells and have increased side scatter compared to neutrophils and neutrophil precursors SS Log FS Lin CD16 FITC 102 CD45 PC7 102 R SS Log SS Log FS Lin 103 CD16 FITC CD45 PC R1 (red, above) illustrates eosinophils in blood (CD45 v SSC, CD16 v SSC, FSC v SSC) SS Log R SS Log SS Log R1 (red, above) illustrates eosinophils in marrow (CD45 v SSC, CD16 v SSC, FSC v SSC). FSC v SSC properties Eosinophils typically have increased forward scatter and side scatter properties. Markers Eosinophils are CD11b+, CD13+, CD33+, CD10-, CD16-, and CD64-. Cases of AML M4 with abnormal eosinophils often have variable CD11b with a spectrum of maturational stages. 21

22 Basophils CD45 v SSC properties Basophils typically have slightly dimmer CD45 expression and slightly increased side scatter compared mature lymphoid cells. CD45 PC7 R1 SS Log CD11b PE 84.44_ _0.01 R _ _0.10 HLA-DR ECD CD33 PE 89.65_ _0.05 R _ _0.00 CD14 ECD CD13 PC _ _0.07 R _ _0.01 CD16 FITC R1 (red) identifies basophils (CD45 v SSC top left, CD11b v HLA DR top right, CD33 v CD14 bottom left, CD13 v CD16 bottom right). 22

23 Another example of basophils: 23

24 FSC v SSC properties Basophils typically have low to intermediate forward scatter and side scatter compared to mature lymphoid cells. Basophils have dimmer side scatter properties than eosinophils and occupy a characteristic position using CD45 versus SSC. Markers Basophils are typically CD11b+ (may be dim to intermediate), HLA DR-, CD13+, CD33+, CD16-, CD10-, and CD22+. Mast Cells CD45 v SSC properties Mast cells are typically located between basophils and eosinophils with respect to CD45 versus side scatter. CD117 PE-Cy7-A R4 SSC-A Mast cells (blue, above) with bright CD117 and CD45 v SSC (right) showing location intermediate with respect to basophils and eosinophils. 24

25 CD45 PC7 102 CD117 PC R SS Log SS Log Another example of mast cells (red, above) with bright CD117 and CD45 v SSC (right) showing location intermediate with respect to basophils and eosinophils FSC v SSC properties Mast cells typically have increased forward and side scatter compared to mature lymphoid cells and increased side scatter properties compared to blasts. Markers Mast cells typically have bright expression of CD117 and slightly increased side scatter and typically form a distinct cluster with CD117 v SSC ungated. Normal mast cells are CD117+ (bright), CD25-, and CD2-. 25

26 Non-neoplastic mast cells (CD117+ (bright), CD25-, CD2-). Maturing B-cell Precursors/Hematogones CD45 versus side scatter properties The least mature B-cell precursors have dim expression of CD45 and low side scatter properties. Intermediate stage B-cell precursors have intermediate expression of CD45 and low side scatter properties. Forward versus side scatter properties B-cell precursors typically have low forward and side scatter properties. Markers The least mature B-cell precursors are CD34+, TdT+, CD10++, CD19+, and CD20-. As they mature, they lose CD34 and TdT, become CD10+, and acquire CD20. Mature B-cells are typically CD10-, CD20+, sig+. Occasionally, hematogones may appear to have light chain restriction. In such cases, look for non-specific background movement on debris, monocytes, and/or myeloid cells. R2 (blue) includes maturing B-cell precursors (hematogones), mature lymphocytes, and the blast region. The LUQ (quadrant 6 above) shows acquisition of CD20 as precursor B-cells mature. 26

27 R2 (blue) shows the least mature B-cell precursors (CD10++, CD34+, CD19+, CD20-). These cells are also CD34+ and TdT+. R2 (blue) shows the intermediate stage maturing B-cell precursors (CD10+, CD34-, TdT-, CD19+) as they acquire CD20. Abnormal B-lymphoblasts in B-ALL typically deviate from the normal precursor B-cell maturation patterns. Erythroid cells CD45 v SSC properties Erythroid precursors originate in the blast region and typically lose CD45 and SSC as they mature. 27

28 FSC v SSC properties Immature erythroid cells have increased forward and side scatter properties. More mature erythroid cells have low scatter properties. Markers Maturing erythroid cells are GlyA+ (bright) and have intermediate to bright CD71. As erythroid cells mature, CD71 diminishes. Vitamin B12/folate deficiency Erythroid cells are relatively increased in B12/folate deficiency. DNA S-phase can be very helpful in this setting. Normal, non-hemodilute bone marrows typically have S-phases ~11% (5.2-17%, 2SD range). Increased S-phases (>17%) can be seen in hyperproliferative marrows (myeloid and/or erythroid). S- phases 30% or greater in marrows are almost always related to B12 and/or folate deficiency. Erythroid cell case examples 71 FITC-A R4 SSC-A glya PE-A 0.00_0.00 R _ _ _ FITC-A CD45 B APC-H7-A SSC-A 28

29 Post treatment marrow (CLL/SLL) with decreased neutrophils and relatively increased erythroid precursors 71 FITC-A R4 SSC-A CD45 B APC-H7-A SSC-A glya PE-A 0.00_0.00 R _ _ _ FITC-A Increased erythroid precursors, myelodysplasia with 46,XY,del(5)(q22q33)[14]/46,XY[6] The above histograms show an expansion of erythroid precursors. Intermediate stage precursors typically have bright GlyA and CD71. The least mature erythroid precursors are CD71+ with variable GlyA (arrow). These cells often correspond to CD117+ erythroid blasts, but are typically underrepresented due to sample lysis. Deviations from the above pattern and/or increased CD117+ erythroid blasts are atypical. 29

30 CD45 PC7 SS Log R1 CD71 FITC 4.34_ _8.73 R _ _2.95 GlyA PE MDS/RARS R1 (red) includes erythroid precursors and shows GlyA+ (bright) with variable CD71 (RUQ)(Cal-Lyse). Note neutrophilic cells with decreased SSC. glya PE-A 67.83_ _0.00 R _ _ FITC-A Peripheral blood, sickle cell patient, unlysed RBC s (LUQ), few nucleated RBC s (RUQ) 30

31 Non-hematolymphoid cells CD45 versus side scatter properties Non-hematolymphoid cells are CD45-negative and typically have increased scatter properties (typically in area highlighted by red circle). Forward versus side scatter properties Non-hematolymphoid cells typically have increased scatter properties. Markers CD56, CD99, Ber-EP4, and cytokeratin can helpful in evaluating nonhematolymphoid cells. Pronormoblasts can have dim Ber-EP4 reactivity, so GlyA may be needed to distinguish pronormoblasts from non-hematolymphoid cells. CD45-, CD56+, CD117+ events may also represent aberrant plasma cells. Myelodysplastic/Myeloproliferative Processes Myelodysplasia Potential findings in cases of myelodysplasia: Myeloid blasts- aberrant expression of CD2, CD5, CD7, or CD56; aberrant loss of CD34, CD117, CD13, CD33, CD11c, and/or HLA DR; aberrant clustering with CD45 v SSC, CD34 v CD38; aberrant CD13 and/or CD33; aberrant ctdt. Neutrophilic cells- decreased CD11b, CD16, and/or CD10; expression of CD56; decreased side scatter Monocytic cells- decreased CD11b, HLA DR, CD13, and/or CD14; expression of CD56 Myeloproliferative Neoplasms (MPN) 31

32 Myeloproliferative neoplasms often have some abnormal flow cytometry finding. Increased basophils, aberrant blast findings (particularly expression of CD5 and/or CD7), and CD56 on the neutrophilic and/or monocytic cells are clues to myeloproliferative processes. Some findings associated with MPN s: Myeloid Blasts: 1) Expression of CD5, CD7, CD36, and CD56 (>20% compared to control set at 2%) 2) Variable expression of HLA DR 3) Loss of CD13 4) Underexpression of CD45 and HLA DR (1/4 log shift) 5) Partial loss of CD13, CD33, and CD117 Myeloid cells: 1) CD56 expression 2) Increased basophils 3) +/- increased eosinophils Cytogenetic, FISH, and or molecular testing may be helpful in cases suspected of being myeloproliferative disorders. JAK2 testing may be helpful for cases of suspected myeloproliferative disorders (especially PV, IMF, or ET). Acute Leukemia Acute leukemia is generally defined as greater than 20% blasts (morphologic criteria) and is classified by the lineage of the blasts, the degree of differentiation, and cytogenetic and/or molecular abnormalities. The percentage of blasts may be may be artificially increased by flow cytometry due to red cell lysis. Lysis methods which preserve more red cells may have blast percentages which more closely resemble morphologic counts. Lysis methods which do not preserve red cell precursors may show significantly higher blast percentages compared to morphologic criteria. Blasts may also be underrepresented due to hemodilution, and the cellular differential and background myeloid maturation patterns help determine the degree of hemodilution. The blast percentage, as determined by flow cytometry, is not recommended to be determinative for diagnosing acute leukemia- morphology is the gold standard for diagnosis of acute leukemia. Treatment decisions are based on the subtype of acute leukemia and, in selected cases, whether or not the blasts express particular markers. Thus, accurate characterization of the blasts is very important. The first critical decision is whether the leukemia could be acute promyelocytic leukemia (APL). The immature cells in APL are typically CD34-, HLA DR-, CD11b-, CD11c-, CD117+, CD64+/- (dim), CD13+, CD33+, and MPO+. Some cases of APL have partial expression of CD34, HLA DR, and/or CD11b, and these often correspond to the 32

33 microgranular variant of APL. The aberrant promyelocytes in cases of microgranular variant of APL also typically have partial expression of CD2. Some non-apl acute myeloid leukemias can mimic APL, but the most common mimickers typically have more HLA DR, CD34, CD11b, and/or CD11c than APL and often have relatively less forward scatter and side scatter properties than APL (well preserved APL s have intermediate to high forward scatter properties compared to mimics which often have intermediate/relatively lower FSC properties). These mimics are also typically CD11c+. If APL cannot be definitively excluded based on the flow data, FISH (PML/RARA) analysis should be requested. Very rare cases of AML M3 are cytogenetic and FISH negative but molecular positive, but these typically have the characteristic acute promyelocytic morphology. Lymphoproliferative Disorders B-cell Lymphoproliferative Disorders B-cell lymphoproliferative disorders are identified by abnormal light chain expression and/or abnormal immunophenotypic expression. Normal B-cells are typically CD10- and CD5- and have polytypic/polyclonal expression of kappa and lambda light chains, typically at a ratio of 1-2:1. There should be equivalent intensity of CD19 and CD20 for the kappa and lambda positive cells (different intensities may be seen in biclonal processes). Occasionally, polytypic/polyclonal B-cells may have partial expression of CD10 (follicular hyperplasia) or CD5 (typically younger patients). Follicular hyperplasia in lymph nodes typically has variable proportions of CD10+/CD20+ bright polytypic/polyclonal B-cells. General immunophenotypic profiles (variations exist) Classic B-CLL/SLL: CD5+, CD10-, CD19+, CD20+ (dim), dim sig, CD11c typically partially CD11c+, CD23+, FMC7-, CD38-/+ B-PLL: CD5v, CD10-, CD19+, CD20+, sig+, CD23v, FMC7v, CD38v Splenic B-cell marginal zone lymphoma: CD5-, CD10-, CD19+, CD20+, intermediate to bright sig, CD23-, FMC7v, CD11c+/-, CD103 typically negative but may be partially positive Hairy cell leukemia (typically found near or in monocytic region): CD5- (very rarely positive), CD10- (~10% are CD10+, no clinical difference compared to CD10- cases), CD19+ (bright), CD20+ (bright), intermediate to bright sig, CD23-, FMC7+, CD123+, CD103+, CD11c+ (bright), CD25+ (very rare cases reportedly CD103- or CD25-) Splenic B-cell lymphoma/leukemia, unclassifiable (includes splenic diffuse red pulp small B-cell lymphoma and vairy cell variant: CD5-, CD10-, CD19+, CD20+, intermediate to bright sig, CD23-, FMC7+, CD123-, CD103-/+, CD11c+, CD25-33

34 Lymphoplasmacytic lymphoma: CD5-, CD10-, CD19+, CD20+, intermediate to bright sig with brighter cig, CD23-, FMC7-, CD38 variable (often can prove B-cell/ plasmacytoid/cd38+ (bright) monoclonality) Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma): CD5- (rarely positive), CD10-, CD19+, CD20+, sig+, CD23- Nodal marginal zone lymphoma: CD5-, CD10-, CD19+, CD20+, sig+, CD23- Follicular lymphoma: CD5-, CD10+ (occasionally negative), CD19+ (often dim), CD20+, intermediate to bright sig, CD23v, FMC7+, CD38+ Pediatric follicular lymphoma (increased proportion of BCL2-): CD5-, CD10+ (occasionally negative), CD19+, CD20+, intermediate to bright sig, CD23v, FMC7+, CD38+ (be wary of CD10+ clonal populations which may be seen in younger patients and may not actually represent lymphoma) Primary intestinal follicular lymphoma (most with localized, indolent disease): CD5-, CD10+ (occasionally negative), CD19+, CD20+, intermediate to bright sig, CD23v, FMC7+, CD38+ Primary cutaneous follicle centre cell lymphoma (often BCL2-): CD5-, CD10+/- (typically + in cases with follicular pattern and in cases with diffuse pattern), CD19+, CD20+, usually sig-, CD23v, FMC7+, CD38+ Mantle cell lymphoma: CD5+, CD19+, CD20+, intermediate to bright sig, CD23-, FMC7+, CD38+, CD11c-/+ Large B-cell lymphoma (general): Mature B-cell lineage typically with increased FSC/SSC and increased Ki-67 or DNA S-phase Subtypes: T-cell/histiocyte-rich large B-cell lymphoma Primary DLBCL of the CNS Primary cutaneous DLBCL, leg type EBV+ DLBCL of the elderly Primary mediastinal (thymic) large B-cell lymphoma: CD5-, CD10-/+, CD19+, CD20+, sig-, CD30+/-, bcl2+/- ALK+ large B-cell lymphoma: CD45-/+, ALK+, CD30-, CD138+, CD3-, CD20-, CD4-/+, CD57-/+ Plasmablastic lymphoma: plasma cell phenotype (including CD38 and CD138), CD45-/+, CD20-/+, PAX5-/+, ccd79a+/-, cig+/-, CD56-/+, CD30+/- (EBV+, HHV8-) Large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease: HHV8+, increased plasmablastic cells Primary effusion lymphoma: usually a very atypical immunophenotype, possible positive markers include CD45, CD19, CD20, ccd79a, sig/cig-, HLA DR, CD30, CD38, and CD138 Burkitt lymphoma: CD5-, CD10+, CD19+, CD20+, intermediate to bright sig, CD38+, very high S-phase by DNA analysis (typically 30% or higher), Ki-67 typically >90% by flow B-cell lymphoma, unclassifiable, with features intermediate between, diffuse large B-cell lymphoma and Burkitt lymphoma: CD5-, CD10+, CD19+, 34

35 CD20+, intermediate to bright sig, CD38+, very high S-phase by DNA analysis (typically ~30% or higher), Ki-67 typically ~90% by flow Variations: CD5-, CD10-, CD103- B-cells: CD10-negative follicular lymphoma, CD5- negative B-CLL/SLL, CD5-negative mantle cell lymphoma, lymphoplasmacytic lymphoma, LBCL CD5+, coexpression of or variable expression of CD23 and FMC7: variant B- CLL/SLL or mantle cell lymphoma CD10+/CD5+: DDx includes all CD10+ and CD5+ lymphoproliferative disorders (as well as floral variant of follicular lymphoma and intravascular large B-cell lymphoma) T-cell Lymphoproliferative Disorders The classification of T-cell lymphoproliferative disorders incorporates morphologic, immunophenotypic, genetic, and clinical data (leukemic/bm, nodal, extranodal, cutaneous). By flow cytometry, abnormal T-cell populations are typically detected by identifying aberrant immunophenotypic expression of markers or by a significant increase in a specific subset of T-cells. Crosschecking percentages of T-cell subsets in multiple tubes helps identify loss of antigenic expression. Normal T-cells are surface and cytoplasmic CD3 positive, distinguishing them from NK cells (scd3-, ccd3-). The majority of peripheral blood and bone marrow samples have a CD4:CD8 ratio between 0.7 and 4.0. Anything outside of this range may indicate viral infection or a T-cell lymphoproliferative disorder. Absolute levels for atypical T-cells in peripheral blood samples should always be determined. Normally, LGL s account for 10-15% of circulating lymphoid cells with absolute values less than 400/uL. Counts greater than 2,000/uL usually represent a lymphoproliferative process, but lower counts do not exclude an LGL lymphoproliferative disorder (some use >500/uL). General immunophenotypic profiles (variations exist) T-PLL (T-CLL) CD4+, CD8+, TdT-, CD1- (may be CD4+/CD8- or CD4-/CD8+) T-LGL processes CD8+, CD7+, CD57+ (typically), CD16v, CD56v, dim CD5 Variant T-LGL processes CD4+, CD7+, CD57+; CD4+, CD8+, CD57+; CD4-, CD8-, TGD+, CD57+ EBV+ T-cell lymphoproliferative disorders of childhood: CD3+, CD2+, CD8+ in acute primary EBV infection, CD4+ in severe chronic active EBV infection, rarely CD4+/CD8+, CD56-, TIA1+ (CD4+ Adult T-cell leukemia/lymphoma (HTLV-1 associated) CD4+, CD7-, CD25+ (few CD4-/CD8+ or CD4+/CD8+) Enteropathy-associated T-cell lymphoma 35

36 CD4-, CD8-/+, CD7+, CD103+, CD5- Enteropathy-associated T-cell lymphoma, monomorphic variant CD4-, CD8+, CD7+, CD103+, CD56+, CD5-/+ Hepatosplenic T-cell lymphoma CD4-, CD8-/+, CD5-, TCRGD+, CD56+/- Subcutaneous panniculitis-like T-cell lymphoma CD4-, CD8+, CD56-, TCRGD- Mycosis fungoides/sezary syndrome CD4+, CD7-, CD25- ALCL, Primary cutaneous T-cell lymphoma, Lymphomatoid papulosis CD30+, variable CD3, CD2 and CD4 Primary cutaneous gamma-delta+ T-cell lymphoma CD4-, CD8-/+, CD7+/-, CD5-, CD56+, TCRGD+ Peripheral T-cell lymphoma, unspecified Non-specific aberrancies Angioimmunoblastic T-cell lymphoma CD4+ > CD8+, CD10+ NK cell Lymphoproliferative Disorders NK cells are typically scd3-, ccd3- (most cases by flow), CD16+, CD56+, CD57+ (partial/heterogeneous), CD2+, CD7+, CD5-, CD4-, and CD8-/+ (partial/heterogeneous). NK cells usually are less than 25% of lymphoid cells. A diagnosis of NK cell lymphoproliferative disorders can be supported by increased levels and/or aberrant immunophenotypic expression, including CD158 subsets. General Profiles (variations exist) Chronic lymphoproliferative disorders of NK cells: scd3-, ccd3-, CD16+/-, CD56+/-, CD4-, CD8-/+, CD57+ Aggressive NK cell leukemia: CD2+, s/ccd3-, CD56+, CD16+/-, CD57- Extranodal NK/T-cell lymphoma, nasal type: CD2+, CD3-, CD4-, CD5-, CD7- /+, CD8-, CD16-, CD56+, CD57- Plasma Cell Disorders Plasma cell disorders are classified by clinical criteria and the level of plasma cells by morphologic assessment. Always quantify and characterize all plasma cells- if there is a mixture of monoclonal cells and polyclonal cells, describe both populations since low levels of monoclonal plasma cells and polyclonal plasma cells are typically seen in cases of MGUS (but may also be seen post treatment for myeloma). Plasma cells are typically underrepresented by flow cytometry due to variable/patchy marrow involvement, hemodilution, processing, and/or cell preservation. Normal plasma cells are CD56-negative and typically are less than 1% of all events in bone marrow samples. Increased percentages, CD45-36

37 negativity, CD117 positivity, and CD56 positivity are clues to neoplastic plasma cells. CD45 v SSC and FSC v SSC can be used to immunophenotype plasma cells. Bright intensity of CD38 may give a false impression of CD45 positivity in that tube, so CD45 assessment in multiple tubes is recommended to determine if there is true CD45 positivity. Peripheral blood samples may be submitted for flow cytometric evaluation in cases of MGUS or myeloma. In these cases, the level of plasma cells should be reported, regardless of clonality, along with the percentage of events and number of events (e.g. 0.1% at 50,000 events). If clonality can be assessed, report it, but the most important factor for some is the level of circulating plasma cells. Hodgkin Lymphoma Flow cytometry may show some fairly characteristic findings in cases of classic Hodgkin lymphoma. Reed-Sternberg cells are not typically detected in routine clinical flow cytometry, but some T-cell findings are often good clues to Hodgkin lymphoma. These include a relatively increased CD4:CD8 ratio (often but not always), relatively decreased polytypic B-cells, and, importantly, relatively bright expression of CD38 on the T-cells (majority CD4+) with no significant corresponding bright CD38 B-cells (gate on all lymphocytes and plot CD3 v CD38, HL typically has an upside down exclamation mark appearance). NLP Hodgkin typically has increased CD4+/CD57+ coexpressing cells (but these may be seen in large B-cell lymphoma as well). There is typically minimal CD57 coexpression in normal lymph nodes. The changes seen in HL may also be seen in cases of large B-cell lymphoma and some non-neoplastic conditions (granulomatous disease, sarcoidosis, etc.). In non-neoplastic conditions, the changes are often borderline and not as pronounced as neoplastic cases. Reed-Sternberg cells have been reported to be detected with collection of 200K- 500K events, the use of specific markers, and patterns are used to identify RS cells when to isolate relatively few events. If some CD30 events are detected, the following may be useful for HRS cells: 30/64/15/45 30/64/95/45 40/64/95/45 30/20/5/45 Ancillary testing to consider: FISH considerations CLL panel for CLL/SLL IGH/BCL2 for follicular lymphoma 37

38 MALT1 for extranodal marginal zone lymphoma IGH/CCND1 for mantle cell lymphoma C-MYC, IGH/BCL2, BCL6 for Burkitt/Burkitt-like/aggressive LBCL IGH/BCL2, IGH/BCL1, MALT for B-cell NOS (CD5-, CD10-) Myeloma panel for myeloma ALL panel for B-lymphoblastic leukemia MDS for hemodilute or low cellularity marrow samples Chromosome 7 probe for hepatosplenic T-cell lymphoma (isochromosome 7) Eosinophilia panel ALK for ALCL Cyclin D1 immunohistochemistry on paraffin sections for mantle cell lymphoma Molecular testing to assess for clonal TCR gene rearrangements and IGH gene rearrangements in equivocal cases or cases where flow is discordant with morphology Molecular testing for BCL1 or BCL2 in selected cases where FISH or IHC not possible (higher false negative rate, however, for molecular compared to FISH) Chromosomes Chromosomes, FISH and molecular testing for AML Considerations/Pitfalls/Limitations Artifacts Dead cells, debris, platelets, unlysed RBC s, and nucleated RBC s all can interfere with accurate analysis. These need to be recognized to fully interpret the data. Variable Involvement, etc. Lymphoproliferative disorders, non-hematolymphoid processes, plasma cells, etc. may have patchy involvement, may be fragile, may not make it through transport and processing, and, depending on the degree of hemodilution in BM samples, may not be adequately represented. Thus, negative flow does not exclude anything. Variability (tube-to-tube, laboratory, fluorochrome intensities, etc.) Preset gates may shift due to tube-to-tube variation (software can correct for this, but it should still be checked). FITC tends to have dimmer intensity compared to PE. Tandem dyes may show relatively increased reactivity (CD64 is typically positive on granulocytic cells but is dimmer than monocytic cells). If expression is questionable, run the marker of interest in PE with another marker of intermediate intensity to isolate the cells and avoid false pull up. Post treatment (regeneration, growth factors, chemo, anti-cd20, anti-cd33) 38

39 CD56 Post treatment marrows can have partial expression of CD56 on the myeloid and monocytic cells as apparent new findings. Monocytes may also be relatively increased post treatment. Recent growth factors can cause a transient increase in blasts and left shifted myeloid maturation. Relatively bright expression of CD13 is seen with growth factors (especially Neupogen) and CD14 may be expressed by myeloid cells post recent growth factor administration. Sepsis CD64 may be seen on neutrophils in cases of sepsis. We have also seen CD64 on neutrophils in cases of recent growth factors with no clinical evidence of infection as well as in cases of myeloid neoplasia. CD64 is positive on immature myeloid cells (dimmer than monocytic cells) and CD64 may also be positive on myeloid cells as a conjugate dye. Additional tubes/markers Additional tubes/markers should be ordered and designed to answer specific questions/discrepancies. Critical markers should probably be ordered in PE and should be ordered with a control with an anchor marker for the cells in question (try to avoid an anchor marker with very bright reactivity). Ex: 19/MIg/45 19/5/45 Not: MIg/MIg/45 19/5/45 Ex: MIg/19/45 TdT/19/45 Not: MIg/MIg/45 TdT/19/45 Acute leukemia markers/blasts MPO, TdT, CD64, ccd3, CD1a, ccd79a, ccd22, GlyA, CD71, CD62p, CD25, CD41 or ccd41 preferably with an anchor marker (CD117, CD34, etc.) 39