USE OF FLOW CYTOMETRY

IMMUNOPHENOTYPING FOR DIAGNOSIS OF

LEUKEMIA AT MOI TEACHING AND REFERRAL
HOSPITAL

Dr. Lotodo T.L.C , MBchB (MoiUni),

Mmed Path (UoN)

Back ground
World Health Organization classification of

tumours of haematopoietic and lymphoid

tissues is a collaborative project of the European
Association of Haematopathology and the Society
for Haematopathology.
The project began in 1995 and brought together
over 50 pathologists and oncologists to revise the
classification of hemopoeitic malignancies based
on the REAL
The 2008 revision of the World Health Organization (WHO) classification
of myeloid neoplasms and acute leukemia: rationale and important
changes, Blood. 2009;114(5):937.

Backg.
REAL classification of lymphoid was based

upon clinical features, morphology,

immunophenotype, and genetics
In 2001 3rd edition was made and in 2008
4th edition was made.
WHO classification broadly classifies the
,malignancies as
myeloid,lymphoid,histiocytic/dendritic and
mast cell disorders

Introduction
Flow cytometry is a technology that

simultaneously measures and then analyzes

multiple physical characteristics of single
particles, usually cells, as they flow in a fluid
stream through a beam of light.
The properties measured include a particles
relative size, relative granularity or internal
complexity, and relative fluorescence intensity.
These characteristics are determined using an
optical-to-electronic coupling system

Specimen processing
Specimens commonly analyzed are bone

marrow aspirate, peripheral blood and

lymphoid tissues FNA material and body
cavity fluids
Blood and BMA is processed within 24-48hrs of
collection stored at room temp
They are collected into Heparin or EDTA tube
Tissues like lymphnodes and trephines, trephine
are submitted in culture media e.g RPMI to
maintain viability
The tissues are mechanically dissociated with a
scalpel to yield a cell suspension

Spec
A cell count is obtained before flow is run-

100,000 events or more required.

Red cells are lysed to obtain a population
of nucleated cells before staining
The sample is then stained with a cocktail
of fluorochrome conjugated
monoclonal antibodies
Analysis of intracytoplasmic markers
require an additional fixation and
permeabilization step to allow antibodies to
pass through the cell membrane
A predetemined panel of antibodies may be

Analysis
A measurement for the diffraction of

light in a flat angle is the forward

scatter (FSC), which depends on
the volume of the cell.
A measurement for the diffraction
of light in a right angle is the so
called sidewards scatter (SSC). It
depends on the granularity

The process of collecting data from

samples using the flow cytometer is

termed 'acquisition

Data

Acute promyelocytic
leukemia

CD34 and HLADR are

characteristically negative in APL.

CD13 and CD117 are usually

positive. CD33 and CD13 are

expressed in APL.

Acute monocytic leukemia

Study
Objective: The main objective of the study was to

compare morphological and flow cytometric diagnosis

in patients previously diagnosed with leukemia
Materials and Methods: Retrospective study was
carried out at Moi Teaching and Referral Hospital
between the period July 2013 and June 2014 After
Institutional Research Ethics approval was granted
Consecutive sampling was done and information was
extracted from patients files that were previously
diagnosed with leukemia
The data was collected using a data collection form
where socio-demographic data, morphological and
flow cytometry results were recorded.

Morphological diagnosis
The original classification scheme proposed

by the French-American-British (FAB)

Cooperative Group divides AML into 8
subtypes (M0 to M8)
ALL into 3 subtypes (L1 to L3)

Flow cytometry
Equipment: Four colour computerized BD FACS.
The fluorochromes FITC, PE, Per CP, and APC

were applied
Panel of monoclonal antibodies used include:
-Pan Leukocyte antigen: CD 45 is a pan leukocyte
marker
- B- cell CD10, CD19, CD20, cyt CD79a
-T cell; CD3, CD4, CD7, CD8, cyt CD3
-Myeloid; CD33, CD117, MPO.
-Immature cell antigens; CD34, and HLADR
-Erythroid marker: CD71

Results
The total number of samples run between

July 2013 and June 2014 were 101,

diagnosed as:
i)AML-11,
ii)ALL-32
iii)Negative-44
iv)Inclonclusive-14
The findings for this study were based on
33 results for patients who had both flow
cytometry and morphological diagnosis

Results
Morphology

Flow +ve
-ve

+ve

-ve

Total

12

18

Total 15

11

14

17

32

The probability of a person having

the ALL (morphology)and Cytometry

test showing positive results is
66.7%

Results
Morphology
+ve

-ve

Total

10

13

23

Total 17

15

32

Flow +ve
-ve

The probability of a person having

the AML(morphology) and

Cytometry test showing positive
results is 77.8%

Discrepancies of flow and

morphology
Serial
no

Morpholog Flow
y

Comment

003

AML M5

CD45(dim)-95%,
CD33(95%), CD38(86%),
HLADR(92%),
CytCD3(95%), MPO-4%
-Reported as T-ALL

Biphenotypic

006

AML M5

CD 45(dim)-21%,
cytCD7a-21%-Reported
as B-ALL

Few events70,000

007

Inconclusive CD45(dim)-66%,
CD38(51%),CD33(50%)
HLADR(43%)-Reported
as AML

Inadequate for
morphological
evaluation

016

AML M2

CD45(bright)-30%,
CD5(31%)

Few events15,000

018

ALL

CD45dim)-73%,
CD5(61%), cyt

Biphenotypic

Discrepancies of flow and

morphology
Serial
no

Morpholog Flow
y

Comment

019

AML M1

CD45(dim)-82%,
CD33(74%),
HLADR(73%),
cytCD3(93%)-Reported
as T-ALL

Biphenotypic

024

ALL

CD45 (bright)-28%,
CD3(20%)-Inconclusive
results

Few events10,000

025

AML M1

CD45(bright)54%,CD45(dim)-22%
CD19(24%), CD3(54%),
CD5(52%),HLADR-35%,
CD79a(23%)-Reported
as T-ALL

Few events10,000

WHO-2008:
MWWHOHixeWenotype Acute
LeukemiaWHOW
B cell lineage

T cell lineage

Myeloid lineage

Strong CD19
with :
cCD22, CD10 or
cCD79a

CD3 (c or s)

cMPO

CD19 with at least

two of:
cCD22, CD10 or
cCD79a

2 or more of:
CD11c, CD14,
CD36, CD64

Vardiman et al., Blood 2009,

Pit falls in morphology and flow

diagno.
Morphology
Differentiating ALL
and AML M0 /M1
Aspicular/hemodilu
te BMA
Viral infections
involving marrowlarge abnormal
cells that look like
blasts

Flow cytometry
Few events<100,000
Poor sample collection
Abberant expression of some

markers
Few flow cytometrists
Consultation services

Conclusions
Flow cytometry is an important diagnostic

tool for diagnosis of acute leukemia and

should be available for patients in public
hospitals.
It should be used together with adequate
clinical info and morphology for
comprehesive diagnosis
Future-cytogenetics can be developed
through proposal writing for grants

Acknowledgments
Moi University /MTRH-Kirtika Patel (PI),

Festus Njuguna, Wilfred Emonyi, Simeon

Mining, Nathan Buziba, Nicholas Kigen
Indiana University-Magdeline Czader,

Asirwa Chite, Terry Vik, Jodi Skiles

V U University medical center,

Natherlands- Marissa Westers, Valerie de

Haas, Floor Abink, Gertjan Kaspars