FLOW CYTOMETRY –

CLINICAL APPLICATIONS
Everything you need to know about flow cytometry at Ziv
Medical Center

2010
THE FLOW CYTOMETRY UNIT AT ZIV
MEDICAL CENTER

Beckman Coulter Epics XL-MCL:

488nm laser, 4 FL channels, manual or automated
carousel sample loading
WHAT IS FLOW CYTOMETRY?
 Cyto = Cell metry = measure
Flow = fluid stream
 “Cytometry is a process in which physical and/or
chemical characteristics of single cells … are measured.
In flow cytometry, the measurements are made as the
cells or particles pass through the measuring
apparatus, a flow cytometer, in a fluid stream.” –
Howard Shapiro, Practical Flow Cytometry, available
online free at www.coulterflow.com .
ADVANTAGES OF FLOW CYTOMETRY
 Flow cytometer can make measurements on tens of
thousands of cells in minutes.
 Multiple parameters can be measured at the same time.

 Relatively simple sample preparation.

 Use of monoclonal antibodies allows for measuring

specific immunophenotypes, such as different kinds of
lymphocytes.
INSTRUMENT BASICS – TEST AT THE
END

chamber
TYPICAL FLOW CHAMBER – NO TEST
AFTERWARDS, YOU’LL NEVER SEE IT
FRONT SCATTER RELATES TO SIZE
SIDE SCATTER (90O) RELATES TO CELL
COMPLEXITY
WITH THESE ALONE, LEUKOCYTE
GROUPING CAN BE SEEN

Granulocytes

Monocytes

Lymphocytes
 FLUORESCENCE BASICS – TEST ONLY
FOR THOSE WHO WANT EXTRA CREDIT

http://biology.berkeley.edu/crl/flow_images/fluorescence_detector.gif

•Common Fluorophores excited by a 488nm laser: FITC, PE, PI,

PE-TR, PE-Cy5
•Overlap of emission spectra can cause signal in a non-specific
channel.
•This overlap is corrected electronically by “compensation”, or
subtraction of the electronic signal caused by the wrong dye.
 MONOCLONAL ANTIBODIES – START
PAYING ATTENTION SOON
 Monoclonal antibodies allow specific antigens on the
membrane or inside a cell to be “tagged” with a
fluorescent dye.
 This yields a wealth of data that is clinically useful, such
as the percent of lymphocytes which are B-cells, T-cells,
or NK-cells. Abnormal percentages can indicate
suspicious cells.
 Aberrance of antigen expression and light scatter
properties can indicate abnormal or suspicious cells.
EXAMPLES OF LINEAGE SPECIFIC MARKERS
– EXTRA CREDIT ONLY
 T-cells: CD3, CD5, CD7, CD2…
 B-cells: CD19, CD20

 Monocytes: CD14 bright

 Granulocytes: combinations of CD13, CD33, CD15,

CD16, CD11b
 Platelets: CD41
 Fluorchrome (FITC)

Antibody (anti-CD45)
cell
Membrane Antigen (CD45)
 LEUKOCYTES DIFFER IN CD45
EXPRESSION AND SIDE SCATTER

Granulocytes

Monocytes

Lymphocytes
 DATA DISPLAY –
TECHNICAL STUFF YOU PROBABLY WON’T NEED TO
KNOW, BUT YOU CAN SAY YOU HEARD ABOUT IT

 The electronic signals are now processed by the

computer. For each cell we have now gathered data
which can include SSC (side scatter), FSC (front scatter),
and four FL channels.
 Most common displays are the histogram and the dot
plot.
 The histogram is a count of each cell which had data in a
particular “channel”, or value.
 The dot plot is a plot of dots! Each dot represents a cell
and the value of the parameter chose.
SAMPLE DATA DISPLAY
A Histogram of the PE
channel data for a
monoclonal Lambda
sample.

A dot plot of the PE vs.

FITC channels for the
same sample.
SAMPLE CD45 PC5/CD19 FITC /CD5 PE
ABNORMAL SAMPLE 45-19-5
 TACHLIS –WHAT SAMPLES CAN YOU SEND?
REQUIRED KNOWLEDGE TO PASS TEST
 Peripheral Blood – purple or green caps (EDTA or
heparin, citrate also possible)
 Bone Marrow -- purple or green caps (EDTA or
heparin)
 Lymph Nodes (via pathology)

 Body fluids (peritoneal, plural, etc..) -- purple or green

caps (EDTA or heparin)

 Please consult with Dr. Dali first to confirm that flow

cytometry is recommended.
 CLOTTED SAMPLES CANNOT BE ANALYSED.
Lymphoma
PNH - PAROXYSMAL NOCTURNAL
HEMOGLOBINURIA
Symptoms
 PNH is a complex disease with signs and symptoms that are
nonspecific, unpredictable, and often similar to those of other
diseases. While symptoms can vary widely in severity among
patients with PNH, they share a common cause of chronic
hemolsyis. Some more common symptoms include: 6,8
 Debilitating pain, especially in the stomach

 Difficulty swallowing

 Severe anemia

 Shortness of breath

 Dark-colored urine (hemoglobinuria)

 Disabling fatigue

 Erectile dysfunction
 FLOW CYTOMETRY FOR
HEMATOLOGICAL DISORDERS
 Lymphomas
 B-cell
 T-cell
 NK-cell

 Leukemias
 Acute
 Lymphoid
 Myeloid

 Chronic
 Lymphoid
 Myeloid

 PNH - Paroxysmal nocturnal hemoglobinuria

 MDS – Myelodysplastic Syndromes

 Hereditary Spherocytosis
 WHAT ELSE DO YOU NEED TO KNOW?
TAKE NOTES NOW, THIS IS THE INTERESTING PART

 Samples are accepted Sunday morning – Thursday noon. Urgent

samples can be processed on weekends with approval of Dr. Dali
only.
 CBC samples from the same day must be submitted to the
hematology laboratory separately.
 Requisition form must be filled out including:
 Patient information sticker
 Referring doctor
 Reason for immunophenotyping request (susp LY, susp blasts,
etc..)
 Relevant medical history (known illnesses, drug treatments,
immune system problems)
 Type of sample (peripheral blood, bone marrow, fluid)
 Date of sample
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CD19+dim/CD20+/CD22+/CD79b+/CD23+dim/CD38+/CD71+/CD10+dim/CD25+dim/CD43-/FMC7-/CD11c-/LAMDA+

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Lympho:11.8% Mono:2% Gran: 73.6%

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4.8% of CD19+ KAPPA £› Ÿ£¡ CD20 9% S-phase, possible DNA
aneuploidy observed
©£±´ CD43 £› Ÿ£¡ CD10 83% of CD19+ LAMBDA
©£±´ CD11c ©£±´ FMC7 £› Ÿ£¡ CD23
£› Ÿ£¡ CD71 £› Ÿ£¡ CD22 £› Ÿ£¡ CD25
£› Ÿ£¡ CD38 £› Ÿ£¡ CD79b

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 ‫‪Thank You‬‬
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