From www.bloodjournal.org by on October 8, 2010. For personal use only.

1986 68: 1355-1362

Classifying acute leukemia by immunophenotyping: a combined FABimmunologic classification of AML

PB Neame, P Soamboonsrup, GP Browman, RM Meyer, A Benger, WE Wilson, IR Walker, N Saeed and JA McBride

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From www.bloodjournal.org by on October 8, 2010. For personal use only.

Classifying

Acute

Leukemia

by Immunophenotyping:
Classification of AML
M. Meyer, A. McBride
to of the that with we the

A Combined

FAB-Immunologic
By Peter B. Neame, Praniti Soamboonsrup, Irwin
A and type panel five 1 38 was of commercially of acute available were leukemia with obtained lymphoblastic distinguished the on acute by of the AMI. used

George

P. Browman, Saeed,

Ralph and John

Ann

Benger,

W.

Edwin

C. Wilson,

R. Walker,

Niloufer
antibodies and sub-

monoclonal to (AL). the distinguish The cases.

immunophenotype M5 was possible classification ogy/cytochemistry recommended least try. of and myand the At in all cases present. for typing

FAB than devised

subtypes 80% for

Ml of cases. and AML

M2. FAB

M4.

and

heteroantisera cases compared discriminated from acute not separate (one criteria case). to

in greater was

A combined morpholIt is at for be done cytochemisstandardS

immunophenoThe (ALL) of immunoleukemia and cases). and panel of recog(22%

immunophenotype immunophenotyping negative suggest that until

type
(FAB)

French-American-British myelogenous leukemia (13% immunologic correspondence cytochemistry lymphoblasts

subtyping. should

classification

phenotype (AMI) nized cases

or inconclusive a gold

identifying
method of morphology,
S

leukemic
acute

subtypes
leukemia and

is

developed.
immunophenotyping.

the
a combination

best

cases),
cases eloblasts derived

mixed
with

lineage

phenotypes
populations Using

is by using

cytochemistry

1986 by Grune

& Stratton, Inc.

subtype

9HE

FRENCH-AMERICAN-BRITISH fication acute of acute leukemia the morphologic and leukemia

institutions

(FAB)

classi-

MATERIAL

AND and

METHODS Immunological Studies (122 over tested

2/2

.1
of

standardize

was proposed in 1976 cytochemical classification could

trials.

to

Morphologic.

Cytochemical

so
and

that

comparisons
various

be

made

The
adults, years,

peripheral
16 children) between

blood
with December

and bone
acute 1 983 and

marrows
(AL) June

of 138 patients
diagnosed were 1 986,

between

between

Roman-

leukemia

owsky included

morphology

and

cytochemical (MPO) and and

stains Sudan

were

used, which B (SBB) esterase subtypes and

+
4])3.4

for

myeloperoxidase

Black

membrane, classified by the

Wright-stained

surface

cytoplasmic, and FAB Cooperative

and cytochemical

nuclear Group
stains.

antigens and were classification using

Cases of chronic

for granulocytic differentiation (NSE) for monocytic differentiation.2

were identified: six myeloid

nonspecific Initially nine M6)

+ 2

smears

myelogenous
this study.

leukemia
The

(CML)

in blast
stains

crisis
used (CAE)

were
included

not included
periodic acid phosphatase

in
acid

(Ml but from M7

through a further [Ml

three was

cytochemical

lymphoid
subtype

(LI M7

through (as distinct

L3)

megakaryoblastic microsvarious used to

Schiff (PAS), SBB, MPO, and in some cases chloracetate

(AP).26 with by using ies were According to the the monoclonal selected
79I,827

nonspecific
esterase

esterase-butyrate
and

(NSE),

added in 1985, though methods in addition to light copy were used for identification.56 In the interim heteroantisera and monoclonal antibodies had been classify
criteria

number

antibodies
for testing

of cells available, and heteroantisera

microscopy. The basis of previously

testing was done shown in Table I

antibodreported type

immunofluorescence

monoclonal

acute
had

leukemia.74
also become

Difficulties
apparent.34

with

FAB

diagnostic

on the

endeavored revisions remain fication different and

to

improve

their

The classification

FAB investigators by introducing

FMCIO,
available.

Leu

M3,

Some antibodies PIMI, and PLTI)

(AML2.23, PMN6, PMN29, were added as they became

in 198I and in interpretation.7 for comparative laboratories

in l9856; however, some difficulties Instituting an immunologic classipurposes used various Also, classification reagents. how and and
study was

had

potential monoclonal has not

problems antibodies been

as

FAB

Classification Group
peripheral

The FAB Cooperative

cation was used on the

classification25
blood and bone

with minor
marrow

modifismears.

heteroantisera.74824 to the FAB immunological we describe

a myeloid

subclassification well-

corresponding defined using

In available this

The myeloid
criteria: Ml:

leukemias

were subclassified ofmyeloblasts

according with <10%

to the following granulocytic dif-

paper monoclonal

a panel subtyping
first

of commercially were leukemic

readily

Predominance

ferentiation.

antibodies
ofour

five heteroantisera acute

from to select

evaluated
cells. The

for

distinguishing

purpose

From

the and

Leukemia McMaster March

Management University. accepted Hamilton Dr Peter Oxon, to Dr General L8L ofthis article article must with Inc. Peter 2X2. by the to 3. 1986;

Group. Hamilton. July Civic B.

Hamilton Ontario. 5. 1986. Hospitals Neame, 189

Regional Canada. Research Wilsdon of East. by page marked solely to

available antibodies that ries using

immunologic reagents a useful and heteroantisera to classify could reagents.

diagnosis. whether the an

panel of monoclonal acute leukemia so various case with we were

pattern

Hospitals Submitted P.B.N.

comparisons similar
FAB

be made Secondly, obtained

between on each

laboratoto compare its comein

come-

is supported correspondence Paddock, reprint Ontario, payment. this fact. by Grune Hamilton

we wished

Fund.
Address
Way,

the immunophenotype sponding

determining

In particular
immunophenotypic

interested

Lyne

Kidlington. requests Canada, costs This

England B. Neame, Barton Department Street in part 1734

Address Hematology, Hamilton, charge indicate

sponding with from the panel was to derive,

classification

FAB myeloid subtypes could be selected of monoclonal antibodies used. Our third aim if possible, a combined FAB-immunological acute leukemia. to the diagnostic In another paper we of evaluation of the contribution of the immuconcordance

Hospital.

The publication advertisement

I 986

were defrayed therefore 18 U.S.C.

be hereby

to subtype a formal
subtypes.25

in accordance
& Stratton,

present
leukemic

nophenotypic

classification

0006-4971/86/6806-0027$3.00/0
1986: pp 1355-1362 1355

Blood.

Vol 68,

No 6 (December),

From www.bloodjournal.org by on October 8, 2010. For personal use only.

1356

NEAME

ET AL

Table
Cluster Designation (CD Number)5a7123

1 . Antibodies

Molecular
(kilodaltons,

Weight

Antibody

kd)sa7173

specificity

Source Becton Coulter Dickinson Immunology 27 28 28 12 29 Immunology Dickinson Dickinson Immunology Immunology

Ref.

Leu 1 T3, T4, T6, T8, Ti 1

5 3, 4, 1, 8, 2 NA
20,21, 19

67 19-29, 90
35, 140,95

T lineage 55, 45/12, 32. 50 Tlineage T lineage

Blineage

OKT9
B1,B2,B4

Orthodiagnostic
Coulterlmmunology

BA1, J5

BA2 (Ia)

24. 9 10 NA NA Mol w13, w14, NA NA 1) 29 NA NA, NA

15

45/55/65.
100

24

B lineage CaIla B lineage, myeloblast Granulocytic,

cytic

Hybritech Coulter monocytic, monomonoBecton Becton Coulter Coulter monoHybritech Becton monoUCLA

7, 12 30 31 10, 32, 10, 33 9 37

HLA-DR Leu M 1 My7. My4, AML

29-34 NA (x-hapten) NA. w14 11 160, 68-74, 155/94

My9, Mo2 2.23

Granulocytic,
cytic

55, 55 NA NA NA NA, NA
NA

Monocytic Granulocytic,
cytic

Leu M3 D5D6(CAML PMN 6, PMN

Monocytic Granulocytic, cytic Granulocytic

Granulocytic

Dickinson Tissue Typing Lab

11 18 9
34

Hybritech
AMD-Cedarlane

FMC 10 PLM 1 PLT1 FVIIIR EMcAb TdTt

Antihuman

NA NA NA NA NA NA
and

NA NA NA NA NA NA

p1. GP lIb/Illa
karyocytic

mega-

AMD-Cedarlane Coulter Cappel B.S. Clarke McMaster University Pharmacia Tago Immunology

35, 37

36

Megakaryocytic Megakaryocytic Erythroid/Lymphoid Lymphoid B lineage

36, 38

39 40, 41

Immunoglobulint,
chain, kappa

lambda

light

chain
antibody-not commercially available.

#{149}Eyhroid monoclonal

PoIycIonal. NA, Not available.

than 30% myeloblasts with >10% differentiating NSE <20%. M3. (a) Hypergranular promyelocytes with numerous Auer rods on Wright-stain or CAE. (b) A variant form showing cells with bibbed, multilobed or reniform nuclei (NSE-negative) and relative scarcity of hypergranular promyelocytes or of primitive cells with multiple Auer rods. M4: Monocytic cells with >20% but <80% NSE-butyrate posiM2: granulocytes, tivity.

More

immunoglobulin stage fluorescein

was used as the second antibody and for the third conjugated avidin was added. After three further washes, cytospin preparations were made and examined under the fluorescent microscope. Heteroantisera Surface immunoglobulin. Cells were incubated with scein-conjugated antihuman immunoglobulin for 30 minutes. three washes, cytospin preparations were made and examined a fluorescent microscope.#{176}
Cytoplasmic immunoglobulin. After fixing for five

cells with >80% NSE positivity. (a) Monoblas(b) Monocytic, differentiated. M6: More than 50% erythroblasts with >30% myeloblasts excluding the erythroid cells. The criteria used for the myeloid subclassification were similar to the revised FAB classification recently published6 and were introduced in our laboratory in 1983 as a result of the FAB reports in l98l and 198242 and from examination ofour data.
M5: tic, poorly differentiated.

Monocytic

fluoroAfter using
in After

minutes

acetone,
with

cytospin

preparations the preparations

ofcells
antihuman

were incubated using

for 30 minutes a fluorescent immunotrans-

fluorescein-conjugated

immunoglobulin.

three washes, microscope.4

Terminal

were examined
transferase.

deoxynucleotidyl

fluorescent
ferase

assay

using
was

rabbit
performed

anti-terminal
as previously

An indirect deoxynucleotidyl
described.2639

(anti-TdT)

Cell

Preparation

and Immunofluorescence

Monoclonal antibodies. This has been described in detail in a previous paper.2 In brief: after Ficoll-Hypaque gradient separation, cells were incubated for 30 minutes with the monoclonal antibody (McAb). After three washes the cells were then incubated for 30 minutes with fluoroscein-conjugated antimouse immunoglobulin. To increase the reactivity of some monoclonal antibodies (My9, My7, My4, Leu Ml, PMN 6/29, and B4) biotin-conjugated antimouse

Criterion mononuclear
positive.

for all methods. Cases where more than 25% of cells were reactive with antibody were considered

Comparison FAB

ofthe

Derived

Immunophenotype

with

the

Classification observers
as described

Two experienced FAB classification

independently
above),

classified
randomly

(using
ordered,

the
and

From www.bloodjournal.org by on October 8, 2010. For personal use only.

IMMUNOPHENOTYPING

ACUTE

LEUKEMIA

1357

Table

2.

Reactivity

Patte

rn of Acute
Reactivity

Myeloid
pattern:

Leukemia

with

Monoclonal

Antibodies
positive) AML 2.23

No. positive!
My4

No. analyzed (percent

PMN 6/29

FAB

(No.)

My9

My7

Leu Ml

Ia

M1(18) M2(17) M3(8) M4(15) M5(14) M6(2)t M7(1)* Total (Ml, Total Total
ALL

14/17(82) 12/15 6/6 1 1/13 1/2 1/1 2, 3, 6) mono AML (M4, (75) 5) 34/41 26/28 60/69 6/50 variant. (83) (93) (87) (12) Non-mono (80) (100) (85)

11/17(65) 13/15 6/6 6/13 1/1 1/1 32/40 19/28 51/68 7/50 (80) (68) (75) (14) 13/15(87) (46) (87) (100)

0/18(0) 15/17 3/6 13/14

1/1

1/18(5) (88) 1/16 0/7 14/14

-

0/10(0) (6) 2/10 4/6 (20) (67)

1/6(17) 1/5 (20) 4/4 3/4

-

9/15(60) 7/1 1 (64) 1/8 14/15 13/13

-

(50) (93)

(0) (100)

(100) (75)

(13) (93) (100)

15/15(100)

13/15(87)

12/15(80)

7/13(54) 1/9 (1 1)
-

6/7(86)

0/1 19/43 26/29 45/72 5/47 (44) (90) (63) (1 1)

0/1 2/42 26/29 28/71 1/49 (5) (90) (39) (2)

0/1 6/27 8/22 14/49 0/19 (22) (36) (29) (0)

0/1 6/16 15/27 2/8 (38) (56) (25)

0/1 17/35 27/28 44/63 34/44 (49) (96) (70) (77)

9/1 1 (82)

Promyelocytic tEMcAb
PLT1
>

25%
>

andPLMl

25%

coded Wright-stained smears together with their cytochemical preparations. The results of the formal evaluation of the initial I 05 cases are presented in another paper.25 The concordant FAB results were used for comparison with the results of the immunophenotype. Certain patterns for FAB M I , M2, M4, and MS using monoclonal
antibodies against myeloid differentiation antigens were recognized

My7
positive

and
for

My9
both

The

remaining
(My7,

55
My9).

cases

(73%) My9, My7,

were
per-

antibodies

Ninety-six

cent both.32

of the Three

cases

of AML 75 cases

expressed that

either failed

or by

of the

to be identified

from our initial data. the immunophenotype

diagnosis and then

These patterns were formally tested by reading of coded cases without knowledge of the FAB
comparing the results with the FAB diagnosis

My7 and My9 were later myeloid in origin. In two were considered to be M5 both was (>80%) and were and MPO-positive, used(My7,My9,

recognized by other of the patients, the by morphology and My4-positive. negative with

criteria to be bone marrows NSE reaction SBBantibodies

that had been previously above.

recorded

by the two observers

as described

A further all myeloid

case,

My4,andLeuMl).

RESULTS Comparison Observers ofthe FAB Classification by the Two

From surveying our initial results we recognized that both My7 and My9 antibodies would be useful as an initial screen for acute myeloid leukemia. Pooling My7 and My9 antibodies was first undertaken when insufficient cells had been obtained by marrow cases
the the

aspirate

or on peripheral

blood.

In 20

of
diagnosis I 7%

the

1 38 of 83% cases),
[4],

cases, of the

there cases

was

agreement

in In the

the

FAB

further
with Using

(I I 3 cases).

remaining

pooled My7/My9 antibodies results when the reagents were

were compared used individually.

(23

(Ml/M2

1 5 cases showed FAB subtype differences Ml/M5 [I], M2/M4 [2], M4/M5 [2J or unclassifiable
cytochemical

reaction,
observed

pooled reagents, an increase and sometimes in the number

in the majority of cases. In

in the intensity of the of positive cells, was

a similar manner, we

Ll/L2 [6]) while eight cases were 30 cases (22%), there was negative The two observers were concordant 20 of these cases. and Of two the remaining minor undifferentiated initial The Table
reactivity, ration or

(AUL).
staining. diagnosis

In
in

now pool Leu Table guish between

1 and FAB

TIl (see that the Ml

Table and M2.

4). can of the distinNone 18 cases

in their subtype

FAB

2 shows

immunophenotyping

10 cases,

eight

were

were

differences.

classified as FAB Leu MI-positive, cases (88%). The reactivity been

Table

M I by morphological whereas FAB M2 were of the reagent AML 2.23

examination were positive in 15 of 17 has not previously (FAB M3).9

Selection reagents I . Some

infrequent

of Antibodies used reagents to type showed response

positivity in

acute

leukemia a lack
AML

are

shown or matu-

in poor

of specificity,
and were

reported
3.

in acute
ity Patt

promyelocytic
em of A IL with J5
0/6 Ti 1 0/6

leukemia
Monoclo
Leu 1 0/6

an irregular our panel. are shown

to differentiation,

Reactiv (No.)

nal Antibodies
TdT 6/6

dropped
Type NuIlt (6)

from notype

The reagents in Tables

ultimately 2, 3 and 4.

used to immunophe-

B4
6/6

a
6/6

Common

(26)

26/26 0/13
6/6

26/26 3/13
3/5

0/24 13/13
0/6

0/23 12/13
0/6

25/25 13/13
0/6

20/2 3/13
4/4

Relationship FAB The clonal shown identified were My7

Between

Jmmunophenotype in AML

and

T(13)
B (6)

Classification reactivity antibodies in Table and

Total

Non-T

(38)

38/38 0/13 0/79

29/37 3/13 0/75

0/36 13/13 3/76

pattern against 2. It will and My9;

of 75 cases myeloid be noted 75% five My7

of AML

with

mono-

TotalT(13) AML Heteroantiserum. tcALLa

Slg

0/35 12/13 0/76

31/37 13/13 2/86

30/3 3/13 43/63

differentiation antigens is that 87% of the cases were 75 cases, and three 12 and My9;

by My9

by My7.#{176} Of these

negative

Non-T,

Non B-ALL.

From www.bloodjournal.org by on October 8, 2010. For personal use only.

1358

NEAME

ET AL

Table Acute
First Line

4.

Monoclonal Leukemia

Antibodi

es for Classifying

A cute Acute

Leukemia Lymphoid B4 Leukemia Leul/Ti Slg 1t

Myeloid
My 7/9

TdT

2nd Lines

Leu M 1 , My4,
EMcAb, PMN Ia, 6/29.

Ia

J5, cz

T3. T6. T9

AML 3rd Lines FVIIIR,

2.23 PLT1 Kappa Lambda

B1,B2

T4, T8

Gpllb/lIla
. Heteroantiserum.

tAn

Second

addition would be Leu 9, (CD7. and third line subsequently erythroid monoclonal

40 kd).73 performed

according

to lineage available.

established

by the first

line.

EMcAb,

antibody;

not commercially

We

found

that

all four

cases of FAB reaction

the variant

M3

including

a variant

Relationship

Between

Immunophenotype

and

form43 gave a positive cases were My4 but the hypergranular

1a,

with

AML form was

.

2.23. All of these Ia in contrast to combination in FAB positively of MI, in the It gave
classified

FAB As tion FAB

Classification previously between L3 showed Cz, the

in ALL demonstrated, FAB no SIg LI, L2 there was a lack and

case one

of correlathe ALL as (B4, classified

form and

which My4

was 1a was

The

classification and typed

AML M4,or
As

2.23k, M5.

not observed reacted M4 and

(5%)

immunophenotype.235
J5+

Furthermore,

M2,

positivity results reported with

as cALL

previously

reported,#{176} My4 of cases in only of two FAB

SIg).#{176}5 with acute lymphoid leuin the literature.82295253 antibodies of 51

majority positive

(90%) reaction

MS.

a
as

of 42 cases

Our immunophenotypic kemia correspond to those The

patients

nonmonocytic by the FAB classification reaction. In seven cases NSE-butyrate were not concordant.
Eighty-six

and and

a negative NSE My4 reactivity were of the and tested cases with was

reactivity
classified

pattern
as

monoclonal

specific
tested,

for non-T

ALL are shown in Table 3. B4 was lineage ALL being positive in all patients in
six

cases and and be two sheep a

which cases case of

typed were

as AML One

anti-TdT also Tllered to

positive. mixed

positive SIg.

and negative in all except Because BA2

1 3 cases
cases of

of T-ALL.
B-ALL out

Anti-TdT
showing

was positive cases of BAI ALL, cases

red cell receptor-positive acute cases diagnosed myeloid markers

was considleukemia

was positive cases than

in four

of nine

lineage

(AMLL).267 Two further FAB classification showed lymphoid 3). By observing certain marker (TIl)

as AML by the plus a single (See Table

AML and two out of three was considered less reliable both antibodies were (24%) diagnosed lymphoid markers typing.

of T-ALL, and B4 for subtyping our panel.54

because Null Twelve

dropped

from

on immunophenotyping patterns using monoclonal

as ALL by the and a myeloid

FAB classification showed marker on immunopheno-

antibodies

against myeloid differentiation antigens, a myeloid immunophenotype corresponding to the FAB myeloid subclassification was recognized for M I , M2, M4, and M5 (Table Figure 1 shows that by the immunophenotypic criteria, of cases diagnosed as M I , 93% as M2, 8 1% as M4 and as MS diagnosis. AML draw and 2.23k, a firm corresponded The and to their My4, but
appropriate

5).
85%

Cases With Disagreement Between Diagnosis and the Immunophenotype Table disagreement immunophenotype. to tiated originally by cytochemical unclassifiable 6 shows the results the of

the Morphological

100%

I 4 cases cases Eight Ml, of or L2)

where diagnosis were the

there and undifferencases

was the were

FAB appeared are

morphologic to be 1a, too small of M649

between

morphologic of the

hypergranular conclusion. Criteria

FAB our

M3 numbers for the

Thirteen (AUL,

staining.

diagnosis

by morphology.

M73536 have

been published

in the

literature.
Ml

Ininuncphenotype M2 0 14 M4 1 1 MS 0 0

Table
M 1 M2

5.

Myeloid

Immunophenotypic

Subclassiflcation
M1 17
2

My7/9-positive, My7/9-positive,

Leu M 1 - and My4-negative Leu M 1-positive. My4-negative

M3a M4

la-negative, (a)
(b) Grey positive

AML
zone My4

2.23-positive,
(between

My4-negative
45-55%) and PMN 6/29-

:
PMN 6/29-

My4-positive

but less than 45%

1
0 20 85%

1
0 15

13
1 16

0
13 13 100%

MS

(a) (b)

My4-positive Grey zone My4

weater

than

55% 45-55%) and

(between

93%

81%

negative
Too few cases to draw a firm conclusion.

Fig myeloid

1. Comparison subclassiflcation

of results of the immunophenotypic and the FAB-cytochemical classification.

From www.bloodjournal.org by on October 8, 2010. For personal use only.

IMMUNOPHENOTYPING

ACUTE

LEUKEMIA

139

Table

6.

Laboratory

Data

of I 4 Patients

with

Disagreement
Positive Immunologic Markers

Between

F AB Diagnosis

and lmmunophenotype
Final Diagnosis AML

No.
1

FAB Ml

Cytochemistry Myeloid Undifferentiated

Immunophenotype UD

2 3 4 5 6 7

M 1 AULt AUL L2 M 1 Ml

UD UD UD UD UD UD

TdT. TdT, TdT, My9,

B4. J5 B4, J5, B 1 , BA2 64, My7, BA2, My4 Lambda Leu Ml, Double labelpopula-

Common Common AML-M4 B ALL M x L My7

ALL ALL

ALL ALL ALL AML ALL M x L

Null ALL (plus My7)

B4, B 1 , B2, SIgA, TdT(36%).

My9, ling My7, showed

B4(20%).
My4 separate

tions
cells

of lymphoid

and myeloid Common Null ALL Ia Neg) AML ALL (plus My7) ALL ALL AML
ALL ALL

8 9
10

M2 AUL
AUL AUL
AUL AUL

UD UD
UD UD
UD UD

TdT, TdT,
My7, TdT,
TdT, TdT,

B4, J5, My7 BA1

B4, B4.
B4, B4,
,

BA2
(TdT

Neg,
Ia

(plus B4) ALL ALL

1 1
12 13

JS.
J5, J5,

Common

Ia Ia

Common Common
AML (Ml)

ALL
ALL AML

14

AUL

UD

My7/My9

UD,
tAUL,

undifferentiated. acute undifferentiated leukemia x L, dual population of myeloblasts (M1/L2). and lymphoblasts.

In the

remaining

six cases

there

had

been

FAB

concordance all

but the result was type. One of these monoclonal and strong
notype

in disagreement cases showed

with negative FAB SBB.

the immunophenoreactivity with M 1 morphology The immunophe-

myeloid and lymphoid first two panels are

includes antibodies

leukemia. investigated that recognize

Cases not defined by a third panel megakaryocyte/platelet

by the which

antibodies used despite positivity with MPO and

differentiation or further The immunophenotype lymphoid

tiated

discriminate subtypes of ALL. will discriminate cases of acute leukemia (22% which remain and thereby noted In addition, of cases)264757 and
It seems

and
nosis

included three cases with a mixed a single case with a dual population
(biclonal, biphenotypic).264755

lineage phenotype26 of lymphoblasts and The cases

observed

from

myeloid

undifferenclarifies between cases and, myeloid

the

by cytochemistry

of cases)

myeloblasts

final

diag-

the majority
observers

of the disagreements
using

(M I v L2)

for

therapeutic phenotype
to the

purposes was considered

of the
lineage

with ALL

a mixed or AML

the lineage dual

FAB

classification.

lineage according

as either immunologic

with
less cells

a mixed
commonly, (<1% of

phenotype populations
can be

( I 3%
be regarded

predominant

on combined assessment.

of lymphoid
identified.

morphologic,

cytochemical,

and
DISCUSSION

cases)265558

immunophenotype The FAB classification256 provides a common language diffiThe

should

as a reliable

method

for comparing and treating acute culties with some of the diagnostic
major difficulties include distinguishing

leukemia; however, criteria are evident.7

of classifying acute leukemia. the same cluster differentiation the First and Second Leukocyte
possibly be used to replace some

Monoclonal antibodies with (CD) number as defined by Antigen Workshop597 could

of the monoclonal

antibodies the same of My4 by

where M234 recent From

cytochemical though revised our their FAB initial

staining distinction

M 1 from L2 in cases is negative356 and Ml from has been improved in the

utilized in our panel; however, despite their having CD number, we found differences in the reactivity and MO2.72 The subclassification is of acute lymphoblastic

classification.6 investigation, a panel of commercially

leukemia

immunophenotyping

oc2329.5153

To date

available mia was 4). tively Since on

antibodies to discriminate and subtype acute leukedevised and is used in the Hamilton region (Table its 58 initiation, AL cases. the It panel was has been developed tested to prospecobtain the

all our cases of non-T-ALL ALL was identified in the

have shown B4 reactivity. Null panel by reactivity with anti-TdT observed presumed B4 . As previously did not correspond there subthe
FAB

and B4 only, but Anderson et al2 have cases of non-T ALL that were TdT and
reported the ALL immunophenotype

maximum In addition, most mens useful which

of information the panel information contain

using the smallest number of tests. is performed in sequence so that the an is likely to be derived insufficient number from speciof cells for

with has

the FAB

classification.2#{176}5 using different antibodies between the FAB myeloid in AML923#{176};however,

have correlated with the

In most previous reports been little correlation antigenic

of certain

complete analysis. The first line screen is used to distinguish acute myeloid from acute lymphoid leukemia and to separate B and AMLL.
recognized

types and expression of

criteria

phenotype
antigens

T lineage The
by the

ALL. second
first

It will panel
panel

also
and

identify
further

some a rare
subtypes

cases case
acute

classification.#{176}6
to subtype

Using AML, FAB

our

immunologic MI M2,

panel M4,

and and

derived MS was

discriminates

not

correspondence
,

of the immunophe-

notype

to the

subtypes

From www.bloodjournal.org by on October 8, 2010. For personal use only.

1360

NEAME

ET

AL

Table

7.

Criteria

for FAB

Myeloid

Subclassiflcation and Immunophenotype

Using

recognized come outcome progress), with

without standard

immunophenotyping. therapy is found

If therapeutic to be
inferior

outto

Morphology. FAB M 1
+ I
-

Cytochemistry 90% little Blasts >3%

with

SBB, MPO
Very M2 >30%

and/or

My 7/9

>25% Leu Ml <25% Leu Ml

in single lineage leukemia an argument could be made to all cases of AL. the immunophenotype

in future for applying cannot

studies (in immunoas

maturation mature

(<10%).
maturation

phenotyping

Blasts

At this time,
Cells, >25%

be regarded

>10%
<20%

More

myeloid My 4

NSE and <25% Auer rods >25%,

M3a

Hypergranular Multiple
contrast

Promyelocytes
(Wright-stain, CAE, or phase microscopy)

the gold standard mia. Unfortunately, most widely accepted and classification

for classifying all cases of acute leukethere is no gold standard. To date, the classification reason system the design
ofour

has been

the FAB was with will

for this

of this study leukemia The further process

paper

AML M4 >30%

2.23

Ia <25%,

My4

<25%

Blasts
differentiation

to compare the immunophenotyping the FAB classification. The purpose data which may help
will have

of acute systems.7 however,

is to provide

Monocytic

improve to

current relevance;

NSE >20%.
My 4 between

<80%
45-55%.

and/or

My 4 >25%,
6/29 >25%

<55%

hopefully meaning
studies

establish

a classification
to determine whether

with

biologic controlled

PMN

or prognostic based
as

MS

>30%
Monocytic

Blasts differentiation and/or My 4 >55%

PMN 6/29 <25% 45-55%,

to be done

therapeutic

decisions
the outcome

upon
is

immunophenotyping
possible in ALL in

in adults childhood.6263 classification phenotyping groups

successful Griffin

improve Such is firmly

NSE >80%
My 4 between

M6

>30% >50% McAb

Blasts Erythroid

(Nonerythroid precursors

population) (Total marrow cells) E

studies The with

cannot possibility monoclonal

with

be

done that the

has

until

the

established.

positive Blasts or FVIIIR >25% blasts positive

antigenic may
been

of AML which are of

A

M7

>30% PLT1,

antibodies
My

identify
recently and

or PLM1,

prognostic correlation

importance

reported.#{176}6 remission has

1 reactivity

possible

in over

80%

ofcases

(Fig

I). Using

the

immunophe-

induction

has been of My7 noted

suggested.#{176} In addition, reactivity previously with with

noted

notype together with the FAB morphology and cytochemistry (Table 7) it is possible to obtain almost perfect concordance
scheme (75 AML

a correlation an observation fication groups

poor prognosis in AML, My4.6 The FAB classisuccessful of probes in identifying to test for

between
is based

observers.25 on the cases). Combining

It should testing FAB when classification

be emphasized small M2, the as be FAB the and MI, using

that M4

this as

of a relatively

sample

has only of clinical

the of

been minimally reIevance.#{176}

recent introduction

Following rearrangement

so-called

M734

is unnecessary

combined various

FA B/immunophenotypic

receptor distinguish
certainty;

genes,9 acute

the immunoglobulin or it seemed that a test would lymphoid from myeloid immunoglobulin have been Thus in cases or
may

T cell antigen be available to leukemia with and T cell noted in cases with a somatic T cell
be

myeloid subtypes can be distinguished. Should all cases of acute leukemia typed? A major difficulty of the encountered in cases with This was found in 30 ofour observers
the

immunophenoclassification is staining. our two in five of

between

negative I 38 cases
there was

cytochemical (22%). Though 30 cases, the were therefore

disagreement

however, recently antigen receptor rearrangement with the AML phenotype.7#{176} rearrangement receptor gene,

were

concordant
observations

in 20 of these and eight may

of the immunoglobulin acute lymphoid leukemia a germ-line nonlymphoid morphological

contribute

antigen
suspected

concordant

the type. (Ml

concordant or L2).

FAB

classification ten cases,

immunophenoundifferentiated be useful in the diagto be given A controlled

this results in an

but is not proven. Cases with DNA analysis would suggest addition
logic

configuration leukemia.4 and

detailed to the

on The
cellu-

Of the remaining

of karyotyping
assessment should

to the
also

immuno-

Immunophenotyping

the cases with negative cytochemistry. Clarifying nosis in this group of patients will allow therapy
according to the type of

lar phenotype

in acute

leukemia.47

leukemia
Acute cases

diagnosed.
whether

ACKNOWLEDGMENT

study
improved mia is

will

be required therapeutic

to ascertain outcome. 1 3% of our

mixed
and

lineage
could rarely

Ieukebe

We wish to thank Dr Bryan Clarke of McMaster University for supplying his monoclonal antibody and the Hematology technologists at the Hamilton General Hospital for performing the tests.

found

in

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