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

Path (UoN)

USE OF FLOW CYTOMETRY IMMUNOPHENOTYPING FOR DIAGNOSIS OF LEUKEMIA AT MOI TEACHING AND REFERRAL

HOSPITAL

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 particle’s

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 used

Analysis A measurement for the

diffraction of light in a flat angle is the forward scatter (FSC), which depends on the volumeof the cell.

A measurement for the diffraction of light in a right angle is the so called sidewardsscatter (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

ResultsMorphology

+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%

ResultsMorphology

+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%

Discrepancies of flow and

morphologySerial

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%)-

Reported as T-ALL

Biphenotypic

Discrepancies of flow and

morphologySerial

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

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

WHO-2008:

MWWHOHixeWenotype Acute

LeukemiaWHOW

Vardiman et al., Blood 2009, 114:937-51

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

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