Flow Cytometry User’s Guide | NeoGenomics Laboratories

Flow Cytometry
User’s Guide
Updated March 13, 2023

Rev03132023 Page 1 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Table of Contents
NeoGenomics Flow Medical Guidance ......................................................................................................... 3
NeoGenomics Gating Strategy .................................................................................................................. 3
Gates ..................................................................................................................................................... 5
B-cell Gating .......................................................................................................................................... 5
T-cell Gating .......................................................................................................................................... 6
Plasma Cell Gating................................................................................................................................. 8
Flow Cytometry Panels ........................................................................................................................... 14
Flow Cytometry Automatic Add-on Criteria ........................................................................................... 17
IT Guidance ................................................................................................................................................. 18
Reporting Cases ...................................................................................................................................... 18
Personal Macros ..................................................................................................................................... 18
Regating .................................................................................................................................................. 18
Flow Regating Software (FCS Express Reader) Minimum Requirements ........................................... 18
Working with Single or Multiple Workbooks .......................................................................................... 19
Features of Professional Input Screen .................................................................................................... 20

Version 03.13.2023 – Updates to Flow User’s Guide:

• Page 13 and 16: Updated additional info and list of antibodies (Table 3) for B-ALL MRD Panel regarding
hemodilution evaluation in bone marrow specimens.

Rev03132023 Page 2 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

NeoGenomics Flow Medical Guidance

Flow cytometry is a method to evaluate the expression of surface and cytoplasmic antigens on individual cells using fluorescently
labeled antibodies. NeoGenomics Laboratories uses a 10-color antibody panel for leukemia/lymphoma flow cytometry testing.
10-color analysis maximizes the amount of information that can be obtained from short samples as well as increasing the
combinations of markers that can be evaluated on every case.

NeoGenomics Gating Strategy

In order to ensure the highest quality flow cytometry data, NeoGenomics performs a multi-fold gating strategy to reduce
artifacts. This involves gating on time, single cells, and forward versus side scatter prior to looking at antibody staining patterns.

Time/Stable Flow Gate is a plot of time vs a scatter plot to determine how even the flow was during the run (Figure 1). Areas
where there was poor flow can be excluded from areas of good flow by time gating, which will ensure a higher quality of data.
Causes of poor flow include clogs, back pressure, air bubbles and tubes that run dry.

Figure 1: Time gate

Proper flow cytometry data analysis requires single cells (singlets) (Figure 2). When cell clumps pass through the laser intercept,
they will take longer than single cells. This in turn affects the area of the signal. Using a pulse geometry gate (such as FSC-H x FSC-
A), doublets can be easily eliminated. This reduces the possibility of false co-expression of antigens because two cells passed
through the flow cytometer at the same time.

Rev03132023 Page 3 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Figure 2: Singlet gate

Forward (FS) is a measure of cell size and side scatter (SS) is a measure of cell granularity. Degenerating cells and debris have
lower forward and side scatter than viable cells. Therefore gating on forward versus side scatter allows debris to be removed
from analysis.

Most of the flow cytometry data analysis is based on linear side scatter (SS) vs. CD45 gating. We have CD45 in every tube,
thereby making back gating much more informative. All gates have their own particular color, making them easy to follow
throughout the analysis (Figure 3). In some plots, total CD19 or CD3 gating is applied.

Figure 3: Gating strategy

Population analysis: Population analysis is the analysis of combined immunophenotypic and optical properties of each
population of lymphocytes, monocytes, CD45-dim cells, CD45-neg cells and granulocytes as well as other cellular populations,
if necessary.

Rev03132023 Page 4 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Gates
1. Lymphocytes: These events have a very intense CD45 expression and the least amount of SS. They are depicted in
dark blue.
2. Monocytes: These events have an intense CD45 expression and slightly more SS than Lymphocytes. They are depicted
in lavender.
3. CD45 Dim (Blast pocket): These events have a dim CD45 expression and varying SS properties. These events include
normal myeloid & lymphoid precursors and basophils as well as blasts. They are depicted in red.
4. CD45 Neg: These events have a dim to negative CD45 expression and varying SS properties. These events include
erythroid precursors, cell debris and few non-hematopoietic cells in normal samples as well as abnormal plasma cells,
lymphoid blasts, and metastatic tumor cells in abnormal samples. They are depicted in light blue.
5. Granulocytes: These events have intermediate CD45 expression and the greatest SS of all events. They are depicted
in green.
6. Mononuclear: The mononuclear gate consists of lymphocytes, monocytes, CD45 -dim and CD45-negative events.
The benefits of the mononuclear gate include: assessing true size of abnormal populations that are not confined to a
single region based on CD45 versus side scatter gating and the ability to use internal negative control populations for
assessing true dim staining versus increased background staining. In addition, the mononuclear gate also allows
simultaneously assessment of multiple populations at once due to display of color back-gates for each CD45 versus side
scatter region.
7. Plasma cells: The plasma cells have variable CD45/SS properties and therefore cannot be easily detected by the
CD45/SS gating strategy; therefore CD45/CD38 gating strategy is used. CD45-positive plasma cells are depicted in
orange and CD45-negative plasma cells are depicted in green. Initial screening with CD38/CD56/CD19/CD45 in the
standard panel allows for detection of increased or abnormal plasma cells. An add-on tube is available to assess
clonality and includes CD138 and cytoplasmic kappa and lambda. Normal plasma cells are usually positive for CD19 and
CD45, and negative for CD56, while abnormal plasma cells are often positive for CD56 and negative for CD19 and CD45.
Plasma cells express bright CD38 and variable CD138 with rare exceptions. Note: Myeloma patients who have been
treated with anti-CD38 monoclonal antibody therapy may not have detectable CD38 expression by flow cytometry, but
these cells usually retain CD138 expression.

B-cell Gating
Several different gating strategies are employed to evaluate B-cells (Figure 4).

1. B-cells gate: Highlighted in brown, based on the lymphocyte gate, encompasses lymphocytes expressing CD19 and/or
CD20.
2. CD19-positive B-cells gate: Highlighted in pink, based on the B-cells gate, encompasses all B-cells expressing CD19.
3. Additional B-cells gate: Highlighted in green, based on the B-cells gate, used to highlight secondary B-cell populations
falling within the B-cell gate.
4. CD5-positive B-cells gate: Highlighted in orange, based on the lymphocyte gate, encompasses lymphocytes co-
expressing CD5 and CD19.
5. CD10-positive B-cells gate: Highlighted in dark purple, based on the lymphocyte gate, encompasses lymphocytes co-
expressing CD10 and CD19.
6. CD11c-positive B-cells gate: Highlighted in pink, based on the mononuclear gate, encompasses all mononuclear events
co-expressing CD11c and CD19.
7. CD10/CD19-positive Gate: Highlighted in red, based on the mononuclear gate, encompasses all mononuclear events
co-expressing CD10 and CD19.
8. CD19-positive cells are gated based on cell size (forward scatter) and the kappa/lambda ratio for small and large CD19-
positive cells is displayed.

Rev03132023 Page 5 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Figure 4: B-cell gating strategies

T-cell Gating

Several gating strategies are employed to evaluate T-cells. First, total CD3-positive cells are gated on cell size (forward scatter)
and the CD4:CD8 ratio is displayed on both small and large cells. Second, each quadrant of the CD4 vs CD8 plot showing total
CD3-positive cells is highlighted in a different color. These colors are displayed on the plots showing patterns of expression for
other T-cell markers (Figure 5). This allows simultaneous evaluation of T-cell markers on each subset of CD3+ cells (CD4+, CD8+,
CD4/CD8 double positive and CD4/CD8 double negative). The T-cell markers are displayed on gates of total lymphocytes so
that abnormal T-cells, which may lose CD3, will not be missed.

Third, an additional page is added to show markers expressed on NK-cells (CD56+CD3-) and CD56+ T-cells (Figure 6). T-cell large
granular lymphocytes (T-LGLs) usually have variable expression of CD56 and CD57. Absence of CD56 does not exclude the
presence of T-LGLs. The TRBC1/LGL Add-On is available for further characterization of T-LGLs and NK-cells.

Rev03132023 Page 6 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Figure 5: T-cell gating strategies

Figure 6: Sub-gating for CD56+CD3+ T-cells and NK-cells (CD56+CD3-)

Rev03132023 Page 7 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Plasma Cell Gating

CD45/CD38 gating strategy is used to gate on plasma cells in the main panels (Figure 7). In the plasma cell add on tube, CD138
gating is also employed.

Figure 7: Plasma cell gating strategy

Lymphocytes
1. A relative increase in lymphocytes raises concern for a lymphoproliferative disorder. Conversely, a relative decrease by
itself has unknown clinical significance.

2. There are usually more T-cells than B-cells, otherwise a B-cell disorder is suspected.

3. A polyclonal kappa/lambda pattern makes a B-cell leukemia/lymphoma unlikely. However, small monoclonal
populations in a polyclonal background may exist.

4. Features suggestive of a B-cell lymphoma/leukemia are:

a. Kappa/lambda ratio > 3.0 or < 0.3. NOTE: Some normal reactive germinal center cells may have a
kappa/lambda ratio as high as 14.4 or as low as 0.13 (rare reports in the literature).
b. Abnormal Pan-B-cell marker expression, such as CD19+, CD20-neg/dim or CD19-/CD20+.
c. Aberrant expression of CD5 or CD10 or bright CD11c. NOTE: CD23 expression can be seen in normal B-cells.
CD10 expression can be seen in normal germinal center cells.
d. Mature B-cells (CD45+++, CD20++) without (or with very dim) surface light chain expression.
e. Large B-cells (FS higher than normal lymphocytes in the same sample).
f. If the total of B-cells, T-cells and NK-cells does not account for the majority of the cells within the lymphocyte
gate, then one should carefully review the flow data for a possible abnormal population, such as CD3-
negative T-cells, CD19-negative B-cells, myeloid cells, or other population. Lymph Sum token in Lymphocyte

Rev03132023 Page 8 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Differential section of the flow workbook will turn red if the total of CD19+ B-cells, CD3+ T-cells and
CD56+CD3- NK-cells is less than or equal to 95%.

5. The possibility of a NK-cell lymphoma/leukemia may be raised by relative increase in NK-cells (sCD3-, CD7+, CD56+
lymphocytes) to >20% of all lymphocytes, but it cannot be confirmed by clinical flow cytometry at this time.

6. Add-on tests:
a. Intracytoplasmic B-cell tube (ICB) can be requested when B-cells lack detectable surface light chain
expression: cKappa, cLambda, CD23, CD19, CD11c, CD10, CD5, CD34, CD20, CD45.
b. Hairy cell leukemia markers: CD22, CD25 and CD103 with Kappa, lambda, CD19, CD11c, CD20.
c. CLL/Mantle Cell Companion tube: CD3, CD5, CD19, CD22, CD36, CD43, CD45, CD52, CD200; can be useful in
discrimination of chronic lymphocytic leukemia/small lymphocytic lymphoma and mantle cell
leukemia/lymphoma. Positivity for CD52 expression on lymphocytes indicates possible response to therapy
with alemtuzumab, a recombinant anti‐CD52 antibody. CD200 is a sensitive marker for CLL and is usually
highly expressed, however it is not specific for CLL. Most mantle cell lymphomas have negative or weak
staining for CD200.
d. TRBC1/LGL Add-On: CD3, CD4, CD7, CD8, CD16, CD45, CD56, CD57, TCR-gamma/delta, TCR-alpha/beta.
e. TRBC1/T-Cell Lymphoma Companion Panel: CD3, CD4, CD7, CD8, CD25, CD26, CD30, CD52, CD279 (PD-1),
CD45. This panel assesses T-cells for the presence of targetable antigens to guide therapeutic decisions.
f. CLL MRD assay is available for monitoring of minimal residual disease in patients with CLL. It follows the
standardized protocol developed by the European Research Initiative in CLL (ERIC) and can detect MRD at the
0.01% level.

Granulocytes
1. Most mature granulocytes are CD10-positive. The CD10-positive/CD10-negative granulocyte ratio is the greatest in
peripheral blood samples (Figure 8). The vast majority of peripheral granulocytes are CD10+. A relative increase in
CD10- peripheral granulocytes suggests left shift. The CD10-positive/CD10-negative ratio is around 0.2 (20%) for
normal bone marrow samples (Figure 9). A higher ratio in a bone marrow sample suggests hemodilution. A lower ratio
suggests left-shifted myeloid maturation.

2. CD13 is a myeloid maker with biphasic expression. One peak is in immature granulocytes that are CD11b and CD16
negative or dim. Another is in mature granulocytes that are CD11b+ and CD16+.

3. Eosinophils are also CD16- granulocytes and have bright CD45 with high side scatter. Loss of CD16 can be seen in PNH
cells, since CD16 is a GPI-anchored antigen on neutrophils (it is a transmembrane molecule on NK-cells).

4. The CD13 vs CD16 plot of granulocytes shows “√”-shaped distribution. Dyssynchronous maturation leads to a distorted
“√” shape often seen in MDS or MPN.

5. Aberrant CD56 expression is seen in MDS or MPN as well as some reactive conditions.

Rev03132023 Page 9 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Figure 8: Granulocyte pattern recognition in normal peripheral blood

Rev03132023 Page 10 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Figure 9: Granulocyte pattern recognition in normal bone marrow

Monocytes
1. Peripheral blood monocytes account for approximately 10% of all circulating leukocytes and are traditionally divided
into three phenotypically and functionally distinct populations based upon differences in expression of CD14 and
CD16 encoding for the lipopolysaccharide receptor and the low affinity FC gamma receptor (FCGR3), respectively.
2. Classical (CD14hiCD16neg) monocytes account for 80–90% of human blood monocytes, intermediate (CD14hiCD16hi)
monocytes comprise ~2–5% and the non-classical (CD14lowCD16hi) monocytes account for the remaining 2–10%
(PMID: 28596372).
3. Multiple diseases including chronic myelomonocytic leukemia (CMML), infections, autoimmunity, and chronic
inflammation are associated with changes in monocyte subsets.
a. An increase in the fraction of classical monocytes to >94.0% of total monocytes has been reported to help
distinguish CMML from other causes of monocytosis (PMID: 25852055).
b. Intermediate monocytes are more abundant in bacterial sepsis, dengue fever, Crohn's disease,
cardiovascular disease and rheumatoid arthritis (PMID: 28596372).
c. Non-classical monocytes are more prevalent in periodontitis and reduced in stroke (PMID: 28596372).
4. Normal monocytes also express CD64 and will show variably decreased CD14 expression with left-shifted maturation.
5. Monoblasts may be negative for CD14 and are often negative for CD34.
6. Aberrant CD56 expression can be seen in MDS or MPN, but also in reactive conditions (e.g. post chemotherapy).
7. When monocytes are gated based on CD45 vs SSC pattern, other cells may also be present in the gate, including, but
not limited to, abnormal lymphocytes (e.g. hairy cell leukemia), dendritic cells and granulocytes with low side scatter.

Rev03132023 Page 11 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

a. Before evaluating the proportions of various monocyte subsets, the CD14/CD16 double negative cells are
excluded, yielding what is named the “Clean Monos” gate in the workbook (Figure 10).
b. Classical, intermediate and non-classical monocytes are referred to as Mo 1, Mo 2 and Mo 3, respectively, in
the workbook (Figure 10).

Figure 10. Example of Normal Peripheral Blood Monocytes

8.

Basophils

1. Basophils express slightly less CD45 than lymphocytes, but more than blasts. Basophils can be separated from
monocytes in the mononuclear gate based on expression of CD33 without HLA-DR.
2. Basophils express CD9, CD11b, CD13, CD33, CD36, CD38 (bright), CD123 (bright) and are negative for CD19, CD34,
CD64, CD117, and HLA-DR.

Eosinophils

1. Eosinophils have high side scatter and express brighter CD45 than most granulocytes.
2. Eosinophils are distinguished from the rest of the granulocytes by lack of CD16 and CD10. They are positive for CD13
and CD33.

CD45 Dim Cells

1. Relative increase raises concern for acute leukemia or excess blasts.

2. Acute leukemia is usually straightforward and tentative morphologic and immunophenotypic classification can be
achieved in the vast majority of cases. However, acute promyelocytic leukemia (APL) cannot be completely excluded.
Not all APL cases have the typical immunophenotype characterized by lack of expression of both CD34 and HLA-DR.
Stat FISH for t(15;17) is recommended on any case of suspected APL. Proper WHO classification requires completion of
cytogenetics, FISH and/or molecular studies as well as morphological confirmation of blast count (the current gold
standard).

3. Excess blasts may be seen in a regenerating marrow or in patients receiving growth factors. Abnormal myeloid blasts
may be detected by their maturation pattern of HLA-DR /CD33 /CD34 /CD117 /CD45.

4. Excess precursor B-cells (CD19+, CD10+, CD45 dim), or so called hematogones, may be seen in young patients and
other non-neoplastic conditions. The differentiation of hematogones from lymphoblasts can be challenging.

Rev03132023 Page 12 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

CD10/CD20 plot may be helpful because hematogones show gradual gains of CD20 with decreasing CD10, while
lymphoblasts form a tight cluster or have aberrant expression of myeloid markers, for example.
5. Add-on tests:
a. B-ALL tube: nTdT, cMPO, cCD3, CD10, CD19, cCD22, CD34, CD45, cCD79a
b. T-ALL tube: nTdT, cMPO, CD1a, cCD3, CD7, CD11b, CD19, CD43, CD45
c. AML tube: nTdT, cMPO, cCD3, CD11b, cCD22, CD34, CD45, cCD79a, CD117, CD123
d. Erythroid/Megakaryocyte tube: CD13, CD34, cCD41, CD45, cCD61, CD71, CD117, CD235a
e. CD123 is now available in the AML-add-on tube and can be helpful in diagnosis of blastic plasmacytoid
dendritic cell neoplasm, but can be seen in ALL and AML.
f. CD1a is now available in the T-ALL tube for further characterization of T-lymphoblasts.
g. B-ALL MRD assay is designed for monitoring of minimal residual disease and follows a standardized protocol
with a sensitivity of 0.01%. It is useful to run at initial diagnosis to have a baseline phenotype for correlation
with MRD results. Tube #2 includes antibodies intended for the purpose of evaluating the extent of
hemodilution in bone marrow specimens.

CD45 Neg Cells

1. Relative increase raises concern for suboptimal sample processing (unlysed erythroids), true erythrocytosis, metastatic
cancer cells, plasma cell myeloma and acute leukemia (ALL, erythroid leukemia).

2. Dysplastic erythroid precursors may show loss of CD71 (transferrin receptor). Normal mature erythrocytes in the
peripheral blood are negative for CD71.

3. Add-on tests:
a. Erythroid/Megakaryocyte tube: CD13, CD34, cCD41, CD45, cCD61, CD71, CD117, CD235a

Plasma Cells
1. Plasma cells express bright CD38 and variable CD138 with rare exceptions.

2. Plasmacytosis (no numeric reference range for flow cytometry) raises concern for plasma cell dyscrasia, plasma cell
myeloma (multiple myeloma) or monoclonal gammopathy of undetermined significance (MGUS).

3. Normal plasma cells are usually CD19+, CD45+, and CD56-, while abnormal plasma cells are usually CD19-, CD45-, and
often CD56+. If an abnormal plasma cell population is predominant (>95%), a plasma cell neoplasm (multiple myeloma
or plasmacytoma) is likely. If the abnormal plasma cell population is between 50% and 95% of all plasma cells, the
diagnostic entity is either MGUS or a previously treated multiple myeloma in the appropriate clinical setting.

4. When variable plasma cell populations are present, a diagnosis of multiple myeloma is unlikely.

5. In any case suspicious for plasma cell dyscrasia, an add-on test for intracytoplasmic light chain markers is warranted.
Again, a clonal population should be at least 95% of all plasma cells for a phenotypical diagnosis of plasma cell
dyscrasia. NOTE: a discrepancy between flow cytometry and morphological examination may be encountered due to
uneven distribution of sample cells between biopsy and aspirate or fragility of plasma cells during flow cytometry.

6. Add-on test:
a. Plasma Cell Tube: cKappa, cLambda, CD20, CD38, CD56, CD138, CD19, CD117, CD45

Rev03132023 Page 13 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Flow Cytometry Panels

NeoGenomics Laboratories offers several panel options to suit our clients’ needs (see Table 1). Our Standard Panel is
recommended for all specimen types (PB, BM, tissues and fluids) and consists of 24 markers covering B-cell, T/NK-cell, plasma
cell and myeloid neoplasms (Tubes 1-3, see Table 1). The Extended Panel (tubes 1-4 in Table 1) consists of 31 markers and is
primarily recommended for BM or PB specimens as it includes markers for erythroid and megakaryocytic differentiation. The
T&B Tissue Panel (tubes 1-2, see Table 1) consists of 17 markers for workup of T/NK- and B-cell lymphomas in tissues where
there is no suspicion for myeloid sarcoma. For specimens with limited cellularity, the laboratory will perform a targeted
antibody panel based on the reason for referral, supporting documents and any prior history in our LIS.
We also offer smaller follow-up flow panels, which are recommended only for patients with a previous diagnostic flow
specimen performed at NeoGenomics (see Table 2). The Follow-up Panels include: AML (Myeloid tube + AML tube), B-ALL (B-
cell tube + B-ALL tube), B-cell lymphoma (B-cell F/U tube), HCL (HCL tube), Plasma Cell (PC tube), T-ALL (T-cell tube + T-ALL
tube), and T-cell lymphoma (T-cell tube). High sensitivity minimal residual disease flow panels are available for monitoring of B-
ALL, CLL, and MM (see Table 3).

Rev03132023 Page 14 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Table 1. Flow cytometry tube configuration

Antibodies

Tube PB/
Tube # APC- APC- V450/
Name FITC PE ECD PC5.5 PC7 APC KrO
A700 A750 BV421/
SNv428
1 T-Cell CD38 CD56 CD3 CD5 CD7 CD2 CD19 CD4 CD8 CD45

2 B-Cell Kappa Lambda CD23 CD19 CD11c CD10 CD5 CD34 CD20 CD45

3 Myeloid CD16 CD13 CD64 CD117 CD33 CD34 CD19 CD14 HLA-DR CD45

4 Extended FMC-7 CD235a CD41 CD138 CD11b CD34 CD19 CD71 CD15 CD45
Short
T&B Tissue Kappa Lambda CD3 CD19 CD10 CD34 CD5 CD4 CD8 CD45
Sample
AML nTdT cMPO CD34 CD117 cCD22 cCD79a cCD3 CD11b CD123 CD45

B-ALL nTdT cMPO CD19 cCD22 CD10 cCD79a CD34 cCD3 none CD45

T-ALL nTdT CD1a cCD3 cMPO CD7 none CD19 CD43 CD11b CD45
CLL/MCL
CD52 CD200 CD3 CD22 CD5 CD36 CD19 CD43 none CD45
Companion
Erythroid-
CD71 CD13 cCD41 CD117 cCD61 none CD34 CD235a none CD45
Mega
Add-On Hairy Cell Kappa Lambda CD19 CD22 CD11c CD103 CD25 CD20 none CD45
Tubes
ICB cKappa cLambda CD23 CD19 CD11c CD10 CD5 CD34 CD20 CD45

Mast Cell none none CD34 CD117 none CD2 CD25 none none CD45

Plasma Cell cKappa cLambda CD20 CD38 CD56 CD138 CD19 CD117 CD45 none
TRBC1/T-
Cell TRBC1 CD26 CD3 CD279 CD7 CD30 CD25 CD4 CD8 CD45
Lymphoma
Companion
TRBC1/
TRBC1 TCR γ/δ CD16 CD3 CD56 CD7 CD8 CD4 CD57 CD45
LGL

WBC FLAER CD64 none none CD14 CD24 none none CD15 CD45
PNH
RBC CD235a CD59 none none none none none none none none
Qualitative
BAL 4/8 none none CD3 none none none none CD4 CD8 CD45
4/8 Ratio

Note: This table is a complete itemization of our current tube configuration. Combinations are subject to change as the NeoGenomics
Medical Director deems it necessary. BAL 4/8 (CD4/CD8 for BAL) does not have a professional component. It is imperative on the
submitted requisition that the patient’s clinical history and the diagnosis under consideration are as accurate and precise as possible.
Providing such information will give us a better idea whether to run a comprehensive panel or a sub-panel in a more targeted
approach.

Rev03132023 Page 15 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Table 2. Flow cytometry follow-up panels tube configuration

Antibodies

Panel Tube PB/

Name APCA APCA V450/
(follow-up) FITC PE ECD PC5.5 PC7 APC KrO
700 750 BV421/
SNv428
Myeloid
CD16 CD13 CD64 CD117 CD33 CD34 CD19 CD14 HLA-DR CD45
Tube #3
AML
AML nTdT cMPO CD34 CD117 cCD22 cCD79a cCD3 CD11b CD123 CD45

B-Cell
Kappa Lambda CD23 CD19 CD11c CD10 CD5 CD34 CD20 CD45
Tube #2
B-ALL
B-ALL nTdT cMPO CD19 cCD22 CD10 cCD79a CD34 cCD3 none CD45

Hairy Cell Hairy Cell Kappa Lambda CD19 CD22 CD11c CD103 CD25 CD20 none CD45

Plasma Cell Plasma Cell cKappa cLambda CD20 CD38 CD56 CD138 CD19 CD117 CD45 none

T-Cell
CD38 CD56 CD3 CD5 CD7 CD2 CD19 CD4 CD8 CD45
Tube #1
T-ALL
T-ALL nTdT CD1a cCD3 cMPO CD7 none CD19 CD43 CD11b CD45

Table 3. Flow cytometry minimal residual disease (MRD) panels tube configuration

Antibodies
Panel
(MRD) Tube PerCP- PE- APC-
FITC PE APC APC-H7 V450 V500-C BV605 BV711 BV786
Name Cy5.5 Ccy7 R700
CD269
MM MRD MM VS38c CD117 CD56 cKappa CD27 cLambda CD138 CD45 CD20 CD81 CD19
(BCMA)
CD66c/ CD304/ CD13/
#1 7-AAD CD34 CD10 CD19 CD20 CD45 CD38 CD58 CD22
CD123 CD73 CD33
B-ALL MRD FITC PE APC-750 Pac Blue KrO
#2
(BM only) CD16 CD13 CD14 HLA-DR CD45

Antibodies
Panel
(MRD) Tube PerCP-
FITC PE PE-Cy7 APC APC-H7 V450 V500
Name Cy5.5
CLL MRD CLL CD81 CD79b CD22 CD19 CD43 CD20 CD5 CD3

Rev03132023 Page 16 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Flow Cytometry Automatic Add-on Criteria

Add Plasma Cell tube:
• ≥1% possible phenotype PCs (CD56-CD19+)
• Distinct clustering of suspicious possible plasma cells with any % , CD19- and /or CD56+, and /or CD117+56-19-

Add AML tube:

• ≥20% suspicious of Blast population
o CD34+,
o And/or CD34-, CD117+/- blast with or without any other myeloid markers
o Prior NeoGenomics history of AML of >6 months

Add B-ALL tube:

• ≥10% suspicious of Blast population
o CD34+,
o And/or CD10+/CD19+
o Prior NeoGenomics history of B-ALL of >6 months

Add HCL tube:

• B-cell population at least 5% of total cells that have the following phenotype: CD5-, CD10-, CD11c+(mod-bright)

Add Mast Cell tube:

• A Mast Cell/Mastocytosis diagnosis or history AND > 0.1% mast cells

Repeat B-cell tube after warm wash:

• B-cell population at least 5% of total cells
o Poor separation of Kappa/Lambda pattern
o Unclear Kappa/Lambda pattern that could be cleaned up

Add B-cell tube with intracytoplasmic kappa/lambda:

• B-cell population at least 5% of total cells that has negative, very dim or unclear light chain (Kappa & Lambda)
expression

Note: All other add-ons will be requested by the client or the pathologist signing out the case. Tech-Only clients may opt out of
these add-on criteria by marking the appropriate box on the requisition.

Rev03132023 Page 17 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

IT Guidance
Reporting Cases
The NeoGenomics Laboratory information system is called NeoLINK (APvX). This system is used to send data files to clients and
can be used by clients to report their cases. Through NeoLINK (APvX), clients will receive a .pdf file of the flow histograms (flow
workbook), an image of the CD45 versus side scatter plot, the cell differential and percentages for each of the gated regions.

Personal Macros
Personal macros can now be created in NeoLINK (APvX) for Flow cytometry. To access macro database, click on Flow Macros
button. For detailed instructions on how to create personal or practice specific macros, please refer to the NeoUniversity
online on demand training video under NeoLINK Training at www.neogenomics.com.

Regating
If regating of the Flow cytometry data is needed for any reason, NeoGenomics provides several regating options based on
desired level of interaction.
• Regate requests may be made via the Test Add On or Regate Request buttons,
• By contacting Client Services at 866-776-5907, Option 3, and/or
• If a client prefers to do his or her own regating, then publishing of flow cytometry data can be arranged by contacting
his/her sales representative. Published flow data is accessed via the Self Regate File button. FCS Express Reader
software is required to perform self-regating and is available for free download.

More information on regating is available in the NeoUniversity online on demand training video under NeoLINK Training at
www.neogenomics.com.

Flow Regating Software (FCS Express Reader) Minimum Requirements

• Windows 7 or higher. FCS is not compatible with Windows Vista.
• Pentium III –class PC, 500 MHz
• 1 GB RAM
• 400 MB hard drive space
• Recommended system: 4 GB RAM, multi-core computer
• The manufacturer recommends downloading the version of the reader that corresponds to the version of the software
used to publish the workbook. NeoGenomics is currently using FCS Express version 6.0.
• Visit the manufacturer’s website for free download of FCS Express Reader:
http://www.denovosoftware.com/site/Reader.shtml

Rev03132023 Page 18 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Figure 10: Worklist

Click on Accession, Case

Number, or Patient Name
columns to open case

• (Figure 10) A sample NeoGenomics Laboratories NeoLINK (APvX) Worklist. The worklist includes all cases within a
specific account and can be filtered or sorted as desired.
• Click on the accession number, patient name or case number to open the Flow case that is ready to be analyzed and
completed.
• After clicking on the case to analyze, the Flow Input screen opens as shown in (Figures 11-14).
• The Patient Summary tab displays the patient demographics at the top of the page, while the Prior/Concurrent Cases
tab displays concurrent cases and any associated previous to which access is available (Figure 11).

Working with Single or Multiple Workbooks

• From the worklist, navigate and review the .pdf scatter plots (by selecting the One Workbook Available/Multi
Workbooks Available button) and preview / sign the final report (Figures 12, 15).
• If there is only one workbook associated with a case, then it will open directly by clicking the One Workbook Available
button.
• If add-on tubes are performed, then they will be uploaded in a separate workbook(s). The original workbook will be
named with the case number (e.g. FLT20-012345) and add-on tubes will be named with the case number followed by a
descriptor (e.g. FLT20-012345AML).
• If more than one workbook is available, then after clicking on the Multi Workbooks Available button, a list of the
workbooks will appear in a new window.
• Click on the name of the workbook to open. More than one workbook can be opened at a time by holding down the
CTRL key while clicking to highlight the file names.
• If a workbook is re-gated, amended or corrected, then the file name and top of the workbook .pdf will be labeled as
such.
• The workbook can be minimized while the Flow input screen is open so that the scatter plots are available while
completing the final report.

NOTE: Multiple workbooks within the same case number will be incorporated into the same report.

Rev03132023 Page 19 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Features of Professional Input Screen

Figure 11: Report input screen: patient info versus prior/concurrent case view

View concurrent cases (under the same accession,

Patient Info tab contains patient
if any) and prior cases for the patient, which were
demographics. Toggle between Patient
accessioned for any clients to which you have
Info and Prior/Concurrent Cases.
access. Toggle between this tab and “Patient
Interpretation screen will always remain
Info” tab while remaining in the same case.
under these two sections.

Previous history for this

Previous report versions patient
that were signed out for
this case

Rev03132023 Page 20 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Figure 12: Report input screen Create macros.

Refer to training
video in NeoU
Click to open pdf of flow
data. Name of button will
change from No to One to If publishing of flow data
has been requested, then Easily add
Multi Workbooks Available additional markers
depending on how many access self regating here
Cancel current case and or test and request
workbooks are available. convert to Global for regating
NeoGenomics to make electronically.
Go back to Quick links to this document interpretation
Worklist and tube configurations

Choose font color

Add hospital or practice name to for diagnosis
Choose among normal,
abnormal, positive, negative the report. This will display in
etc. The diagnosis type is not large font in the upper left
required and will not show on corner of the final report.
report, but it can be used to
generate metrics, if desired,
and changes font color of text
in diagnosis field.

Rev03132023 Page 21 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Figure 13: Report input screen continued

Diagnosis macros automatically populate all fields of the report and insert tokens for percentages when applicable. Some
macros contain leading verbiage and as such, have to be reviewed and edited (direct entry via cut-and-paste, etc.)

Checking QNS box automatically adds QNS

macro, unselects all fields in Population
Analysis and deselects flow tables.

Uncheck box to remove

population analysis field
from the report.

Rev03132023 Page 22 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Figure 14: Report input screen continued

Click Add Coexpression to add

free text markers with
percentage to any gate table If check box is selected, then gate tables will
show on report unless there are no
percentages in that table. Click ‘Deselect All’
if no gate tables are desired on the report.

If boxes are left blank, then these fields

will not display on report.

• After adding interpretation to the Flow input screen, click on the “Preview Report” button.
• By clicking this button, changes will be saved and the report preview will display.
• At this point, options are to go back to make more changes and create new preview or sign the report to complete the
case (Figure 15).

Rev03132023 Page 23 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Figure 15: Preview and sign report

Sign out report

Markers performed autopopulates the report based

on markers accessioned.

• After a case is signed out, the Flow workbook containing the data plots and the report is available from the work list in
the column labeled “Report” (Figure 16).
• If more than one Flow workbook was created, such as when add on markers are requested, the flow workbook (FWB)
icon will contain a ‘+’ sign . Click the ‘+’ sign to display a list of available workbooks in a small pop-up window. Click
on the title of a workbook to open it.
• Alternatively, click on the button in the Status / Test Add column and select from the choices in the Reports box to
view all of the workbooks after a case is complete.
o In this screen, both the flow workbooks and reports will appear as a .pdf icon. Hover over the FWB icon to
see the name of the workbook file. Signed reports may also have a Word file associated with them
depending on client preferences (Figure 17).

Rev03132023 Page 24 of 25
 Flow Cytometry User’s Guide | NeoGenomics Laboratories

Figure 16: View flow workbook and report after case is complete via worklist

Click FWB icon to view

flow workbook, or click .pdf
icon to view report after a
case is signed out.

Figure 17: Alternative method for viewing flow workbook and report after case is complete via case
status screen

Click Revise Case to pull case back to Client Click Launch Virtual MicroScope to view cytospin
Interp Step and make any need revisions. image before or after a case is reported.

Click here to view flow workbook(s) or report. If more

than one workbook was created, then this is the only
way to see all workbooks after case is complete.
For further assistance, please contact:
NeoGenomics Client Services at 866.776.5907 and select option #3

Rev03132023 Page 25 of 25