Kesho Conference 2014

1. 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)

2. 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.

3. 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

4. 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 particle’s
relative size, relative granularity or internal
complexity, and relative fluorescence intensity.
 These characteristics are determined using an
optical-to-electronic coupling system

5. 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

6. 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 used

7. 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‘

8. Data

9. Acute promyelocytic leukemia
 CD34 and HLADR
are characteristically
negative in APL.
 CD13 and CD117 are
usually
positive. CD33 and
CD13 are expressed
in APL.

10. Acute monocytic leukemia

11. 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.

12. 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)

13. 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

14. 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

17. Results
Morphology
+ve -ve Total
Flow +ve 12 6 18
-ve 3 11 14
Total 15 17 32
 The probability of a
person having the ALL
(morphology)and
Cytometry test
showing positive
results is 66.7%

18. Results
Morphology
+ve -ve Total
Flow +ve 7 2 9
-ve 10 13 23
Total 17 15 32
 The probability of a
person having the
AML(morphology) and
Cytometry test
showing positive
results is 77.8%

19. Discrepancies of flow and
morphology
Serial
no
Morphology Flow 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 events-
70,000
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 events-
15,000
018 ALL CD45dim)-73%, CD5(61%),
cyt CD3(34%),
MPO(41%),CD71(74%)-
Biphenotypic

20. Discrepancies of flow and
morphology
Serial
no
Morphology Flow 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 events-
10,000
025 AML M1 CD45(bright)-
54%,CD45(dim)-22%
CD19(24%), CD3(54%),
CD5(52%),HLADR-35%,
CD79a(23%)-Reported as
T-ALL
Few events-
10,000

21. 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, 114:937-51

22. Pit falls in morphology and flow
diagno.
Morphology
 Differentiating ALL
and AML M0 /M1
 Aspicular/hemodilute
BMA
 Viral infections
involving marrow-large
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

23. 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

24. 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