RUMAH

SAKIT KANKER
"DHARMAIS"
( PUSAT KANKER NASIONAL )
FLOWCYTOMETRY
DNA ANALYSIS IN
CANCER
Instalasi Patologi Klinik
RS Kanker Dharmais

OUTLINES
What Is FCM ?
Principle :
FCM Fluidic, optic, electronic
Ploidi
Cell cycle analysis
DNA How its measured by FCM ?
Hematology Malignancy
APPLICA
Solid tumor
TION

What
is
FCM ?

FCM: examining physical and

chemical properties of cells,
beads, or particles as they pass in a
fluid stream through a measuring
apparatus (flow cytometer).
Measurements are made on a per-cell
basis atroutine ratesof500 to 4000
cells per second

Princi
ples

Fluidic : To introduce and focus the cells

for interrogation
Optic : To generate and collect the light
signals
Electronic : To convert the optical signals
to proportionate electronic signals and
digitize them for further analysis on the
computer

FluidicsHydrodynamic
Focusing
Low Sample Pressure

High Sample Pressure

sheath

sheath
sample

sheath

sheath
sample

Flowcytometry

 Ploidy is a measure of the

number of chromosomes in a cell.
Chromosomes contain the genetic
material known as DNA
Diploid : normal chromosom in
healthy cells (the sets of 23 each).
Diploid cancer cells tend to be
slower-growing, less aggressive
cancers.
Aneuploid: When cancer cells
are rapidly dividing, mistakes in
the distribution of chromosomes
can happen, resulting in some
cells having too many
chromosomes (hyperploid) and
others too few (hypoploid) An
aneuploid cancer may be more
aggressive than a diploid cancer.

M phase kinase

M phase

CELL CYCLE

G2 phase

Go phase
Mitosis

Daughter
cells

Interphase
S phase

End stage

Death
G1 phase

S phase promoting factor

CELL CYCLE ANALYSIS PROCEDURE

Sample
preparation
: Tissue

single cell
suspension
Trypsin buffer
Trypsin inhibitor
RNAse
nuclei
susp

Analyzed by FCM
DNA
Staining :
Nuclei
stabilizer
PI staining

CEN for daily

calibration CTN for
validation/CV
CV < 8% is consider
valid
MODFIT mode
software for data
acquisition and data
analysis

The Voltage
Pulse
As a cell passes through the laser,
more and more fluorescent light is
emitted until the cell is in the center
of the laser maxima)
As the cell leaves the laser, less and
less fluorescent light is emitted

And since emitted photons are

converted
to photoelectrons in the PMT, this
creates

The Pulse

Created by Ryan
Duggan

Time

FL-2 Height
detector

Above
threshold

Measurement of a Doublet
pulse

FL-2 Height
detector

Time

Threshold

Voltage Intensity

Measurements of the Pulse

Pulse Height

Pulse Area

Pulse Width=
time of flight

Time
Created by Ryan
Duggan

Laser

Pulse
Area

Pulse Height

Pulse Area

Pulse Width

G2/M

Doublets

S
G1

Pulse Width

The beginners guide to

pulse processing

Removal of cell clumps

(DoubletsDiscriminationModul)

Ungated

R1

Doublets

Gated

DNA stained with propidium iodide

S Phase

G1

G2/M

DNA Content
Note linear scale

DNA analysis in tumours: Aneuploid Rhabdosarcoma

Aneuploid G1
Diploid G1

DNA Index: 1.34

Diploid G2
Aneuploid G2

DNA index =
G0-G1 sample/
G0-G1 reference
Diploid :
DNA Index 0,95-1,05

DNA analysis in tumours: Near diploid fibrosarcoma

Aneuploid G1
Diploid G1

Aneuploid G2

Diploid G2
DNA Index: 1.08

HISTOGRAM OF CELL CYCLE ANALYSIS

HISTOGRAM OF CELL CYCLE ANALYSIS

CELL CYCLE ANALYSIS OF 2 PATIENTS WITH LUNG

CANCER

Patient 1. G1 : 56.2 %
S : 34.6 %
G2+M :
9.2 %
DI :
1.04

Patient 2. G1 : 79.5 %
S
: 17.1 %
G2+M : 3.4 %
DI : 1.70

Asian J. Exp. Sci., Vol. 23, No. 1, 2009; 33-38

DNA-Ploidy - A Prognostic Factor of Acute Lymphoblastic

Leukemia (ALL) in Childhood
O. Basu , F. Zlzer, P. Uma Devi, and C. Streffer
Abstract :.
The recurrence-free survival of patients with hyperploid and euploid tumours was analyzed
according to Kaplan and Meier, and the results were compared with those for other possible
prognostic factors. Data analysis, incl. multivariate
analysis, showed that DNA ploidy was indeed of significant prognostic value, with a
risk ratio similar the initial leukocyte count. The appropriate cut-off point between
diploid/near-diploid and hyperdiploid cases seems to be a DNA index of 1.10 rather than the
most widely used 1.16. Overall, we are of the opinion that future studies should continue to
include the DNA ploidy as a possible prognostic factor for acute lymphoblastic leukaemia in
childhood.
Key words : Childhood leukaemia, Predictive assay, Risk factor, Ploidy, Flow cytometry.

DNA ploidy analysis as an adjunct for the detection of relapse in

B-lineage acute lymphoblastic leukemia
Barton Kenney1, Arthur Zieske1, Henry Rinder1 and Brian Smith1
Leukemia & Lymphoma 2008, Vol. 49, No. 1 , Pages 42-48

We hypothesized that flow cytometric DNA ploidy analysis may detect relapse in aneuploid ALL
cases that might be missed by flow immunophenotyping. We retrospectively studied ALL cases at
our institution between 1991 and 2003 (n=114). Aneuploid populations were present at
diagnosis in 32% of all patients. Sixty-five percent of all patients had normal leukemic
immunophenotypes, defined as being similar to normal precursor B-cells, while 35% had
aberrant immunophenotypes with myeloid or T antigen co-expression. In ALL cases that were
originally aneuploid, follow-up ploidy-analysis detected relapsed disease in all cases which were
also detected by flow immunophenotyping, suggesting that ploidy analysis is highly
sensitive for detecting ALL relapse. However, in 5 cases in which the diagnosis of relapse
could not be reliably made by flow immunophenotyping, ploidy analysis successfully detected
aneuploid cells, i.e., relapse, in all five; these included 3 patients with normal and 2 with aberrant
original immunophenotypes. These results suggest that it may be beneficial to perform
ploidy analysis as an adjunct to flow immunophenotyping in following patients with Blineage
ALL
who
demonstrate
aneuploidy
at
diagnosis.

Consensus Review of the Clinical Utility of DNA FCM in Colorectal

Cancer'
Kenneth D. Bauer: C. Bruce Bagwell, Walter Giaretti, et all
Cytometry 14:486-491 (1993)

Cell DNA content analysis (DNA ploidy and PA) may be helpful in
choosing for adjuvant therapy patients with Dukes stage B cancers .
Although DNA ploidy and/or PA for stage C patients, it does not assist in clinical
decision making because all of these patients are currently routinely treated with
adjuvant therapy. However, this information may be useful for counseling patients
as to their prognosis. DNA ploidy and PA may be useful in predicting response to
chemotherapy. A recent preliminary report suggests that among patients
with Dukes C disease treated with adjuvant chemotherapy, patients with
tumors having high PA preferentially benefitted from adjuvant 5fluorouracil therapy. This reemphasizes the need to incorporate cell DNA
content analysis into the eligibility requirements for patients entering cooperative
group trials.

DHARMAIS CANCER HOSPITAL EXPERIENCE

DNA & SPF ANALYSIS IN CANCER (th 97-2000)
To obtain
baseline data
and informations
that might
provide a clearer
perspective on
the profile of
DNA ploidy
status and
proliferative
activity of
cancers in
Indonesia

To evaluate
their possible
clinical
applications
as markers of
prognosis

SPF Categories :
Low : 10%
Intermediate :
10% SPF 20%
High : 20%
DNA Categories :
Diploid
Aneuploid

PROFILE OF PLOIDY STATUS AND

PROLIFERATIVE ACTIVITY OF SEVERAL CANCERS
Ploidy status

Proliferative activity

Type

DI

Dipl

Aneupl

SPF

Low Interm High

Breast

57

1.18

26

31

22.7

10

12

35

Lung

43

1.01

35

38.4

34

Orbital

20

1.10

19

18.7

Larynx

30

1.03

23

28.16

23

Rectal

20

1.13

17

20.3

NPC

25

1.32

22

18.4

11

Total

195

48

147

36

41

118

DISTRIBUTION OF PLOIDY STATUS AND SPF VALUES

OF BREAST CANCER IN RELATION TO STAGE
PLOIDY STATUS

PROLIFERATIVE ACTIVITY

STAGE

DIPL

ANEUPL

LOW

INTERM

HIGH

II

36

15

21

22

III

19

10

13

IV

DISTRIBUTION OF PROLIFERATIVE ACTIVITY AND ITS

RELATION TO PLOIDY STATUS IN ORBITAL CANCER
(n=20)

DI = 0.7 - 1.79 Mean 1.1

SPF = 3.7% - 47.2%

Mean 18.7%

DISTRIBUTION OF PROLIFERATIVE ACTIVITY AND ITS

RELATION TO PLOIDY STATUS IN LARYNGEAL CANCER

DI = 0.5 - 1.54 Mean 1.03

SPF = 8.4 - 59.1

Mean 28.16

SURVIVAL CURVE ACCORDING TO SPF

IN LARYNGEAL CA

PROPORTION OF
RADIO-RESPONSIVENESS OF NPC IN RELATION TO SPF AND PLOIDY

PLOIDY AND SPF IN RELATION TO SURVIVALS

AND DEATHS IN RECTAL CANCER

Summary
Flow cytometry is a technology that rapidly
measures multiple characteristics of a cell or
particle.
The assessment of DNA ploidy status and
proliferative activity is an objective and
reproducible component of diagnostic
procedures
Aneuploidy as evidence of aberrancy is
anticipated to correlate with deviation from
normal behaviour and hence clinical
aggressiveness
High SPF is associated with greater malignancy

Summary
Cellular proliferative activity and ploidy
status may be used as markers of
prognosis for cancer
High SPF and an-euploidicity are
associated with poor prognosis
Risk for recurrence is higher in tumors with high
SPF values and aneuploid tumors

Thank You