A standardized endogenous megakaryocytic erythroid colony assay for the diagnosis of essential thrombocythemia.

Abstract

The reliability of assays of endogenous megakaryocytic colony (EMC) and endogenous erythroid colony (EEC) formation for the diagnosis of thrombocytoses remains controversial. We tested the suitability of a recently developed collagen-based assay of EMC formation for the diagnosis of essential thrombocythemia (ET).
This was a multicenter (8 laboratories) study including 121 patients: 82 with ET and 39 with reactive thrombocytoses (RT). EMC and EEC were assessed in each laboratory in serum-free, cytokine-free, standardized collagen gel assays; bone marrow (BM) and peripheral blood (PB) were tested in parallel.
In PB cultures, only EEC were specific for ET. In BM cultures, both EMC and EEC were specific for ET and present in assays of 77.8% (EMC) and 33.3% (EEC) of ET patients. Altogether, 80.2% of ET patients had BM EMC and/or EEC, whereas none of the patients with RT did.
When performed with BM progenitors for the diagnosis of thrombocytoses, positivity of the standardized EMC/EEC assay in collagen is specific (100%) and detects 80% of ET.

Full text

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haematologica 2004; 89(10):October 20041207
[haematologica]
2004;89:1207-1212
ABS
TRACT
IRÈNE DOBO
NATHALIE BOIRET
ERIC LIPPERT
FRANÇOIS GIRODON
PASCAL MOSSUZ
MAGALI DONNARD
LYDIA CAMPOS
DANIELLE PINEAU
ELISABETH BASCANS
VINCENT PRALORAN
SYLVIE HERMOUET
From the Laboratoires d' Héma-
tologie of the Centres Hospital-
iers Universitaires d'Angers (ID),
Clermont-Ferrand (NB),
Bordeaux (EL), Dijon (FG),
Grenoble (PM), Limoges (MD),
Saint-Etienne (LC), Nantes (DP ,
SH), France.
Correspondence: Dr. Sylvie
Hermouet, Laboratoire
d'Hematologie, Institut de
Biologie, CHU Nantes, 9 quai
Moncousu, 44093 Nantes
cedex, France. E-mail: sylvie.her-
mouet@chu-nantes.fr
@2004, Ferrata Storti Foundation
A standardized endogenous megakaryocytic
erythroid colony assay for the diagnosis of
essential thrombocythemia
E
Vera (PV) Study Group (PVSG) and the World
Health Organization (WHO),1,2a diagnosis of
ET is made on two positive criteria (platelets
> 600×109/L, bone marrow (BM) histology
in favor of ET) and exclusion of other caus-
es of thrombocytosis: reactive thrombocy-
tosis (RT), other myeloproliferative disorders
(MPD) (PV, chronic myeloid leukemia, idio-
pathic myelofibrosis), or myelodysplastic
syndromes. No additional test can facilitate
the diagnosis of ET. Evidence of X-linked
clonality can be useful but it is not a crite-
rion for ET for several reasons: it is limited to
females; some cases of ET are oligoclonal or
polyclonal; monoclonality can be found in
healthy older females.3-6Similarly, because
of its problematic interpretation, partly due
to the absence of standardization,2,7-9the
ssential thrombocythemia (ET) is char-
acterized by chronically raised platelet
counts. According to the Polycythemia
endogenous colony assay is not a criterion
for diagnosing ET. Yet formation of endoge-
nous megakaryocytic colonies (EMC) and
endogenous erythroid colonies (EEC) is well
described in ET. EEC, specific to MPD, are
clearly in favor of the diagnosis of ET but are
present in only a minority of ET patients.6,10–16
The specificity and diagnostic value of EMC,
depending on culture conditions in plasma
clot or methylcellulose, with BM or periph-
eral blood (PB) cells, remain controver-
sial.8,9,11,17-21
We recently described serum-free,
cytokine-free collagen-based assays with
improved detection of EEC and EMC due to
in situ colony staining.14,15,17,22,23The collagen
EEC assay now standardized for the diag-
nosis of PV is specific (100%) and sensitive
(83%).15,22Preliminary studies indicated that
the collagen assay allows formation by BM
progenitors of EMC which could be specif-
Background and Objectives. The reliability of assays of endogenous megakaryocytic
colony (EMC) and endogenous erythroid colony (EEC) formation for the diagnosis of
thrombocytoses remains controversial. We tested the suitability of a recently developed
collagen-based assay of EMC formation for the diagnosis of essential thrombocythemia
(ET).
Design and Methods. This was a multicenter (8 laboratories) study including 121
patients: 82 with ET and 39 with reactive thrombocytoses (RT). EMC and EEC were assessed
in each laboratory in serum-free, cytokine-free, standardized collagen gel assays; bone
marrow (BM) and peripheral blood (PB) were tested in parallel.
Results. In PB cultures, only EEC were specific for ET. In BM cultures, both EMC and EEC
were specific for ET and present in assays of 77.8% (EMC) and 33.3% (EEC) of ET patients.
Altogether, 80.2% of ET patients had BM EMC and/or EEC, whereas none of the patients
with RT did.
Interpretation and Conclusions. When performed with BM progenitors for the diag-
nosis of thrombocytoses, positivity of the standardized EMC/EEC assay in collagen is spe-
cific (100%) and detects 80% of ET.
Keywords: endogenous megakaryocytic colony formation, endogenous erythroid
colony formation, essential thrombocythemia, collagen, standardization.
Chronic Myeloproliferative Disorders • Research Paper
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haematologica 2004; 89(10):October 2004 1208
I. Dobo et al.
ic to ET.17,22The present multicenter study tested the
value of a standardized collagen EMC/EEC assay for
the diagnosis of ET. Because PB EMC formation also
occurs in ET,21,24,25we compared the use of BM and PB
for the EMC/EEC assay.
Design and Methods
Patients
BM and PB samples were obtained from 121 patients
diagnosed in 16 hospitals as having RT (n=39) and ET
(n=82) using the WHO ET criteria2(Table 1). Samples
collected in clinics far from the 8 participating labo-
ratories were sent by overnight delivery.
Cultures
BM and PB mononuclear cells (BMMC and PBMC)
were prepared as described previously.22Fresh BM and
PB samples were centrifuged at 100 g for 10 min con-
tinuously at room temperature. Platelet-rich super-
natants were removed, nucleated cells were carefully
pipetted from the surface of the red blood cell pellet,
suspended in Hank's buffer and separated on Ficoll
medium (1.077 g) by centrifugation at 400 g for 30
min continuously at room temperature. In order to
eliminate platelets, cells were washed once with
Hank's buffer without calcium or magnesium, once
with PBS + 1% EDTA at 4°C and once in Iscove’s medi-
um. For the EMC/EEC assay, BMMC (105/mL) and PBMC
(2×105/mL) were then plated in 1 mL of collagen-based
semisolid medium without serum or cytokines (medi-
um 1: MegaCult with lipids and human collagen
(StemCell Technologies, Meylan, France; catalog ref-
erences 4850 and 4802) in triplicate (330 µL/well) in
4-well plates (Nunc-Ruskilde, Denmark) and grown for
10 days. As controls of progenitor growth, BFU-E and
CFU-MK assays were performed in the same serum-
free, collagen-based medium supplemented with
appropriate cytokines: medium 2 for BFU-E assays
contained 50 ng/mL stem cell factor, 10 ng/mL inter-
leukin-3 (IL-3), 10 ng/mL IL-6, 10 ng/mL granulocyte-
macrophage colony stimulating factor (GM-CSF), 10
ng/mL G-CSF and 3 U/mL erythropoietin (EPO); medi-
um 3 for CFU-MK assays contained 10 ng/mL IL-6, 10
ng/mL IL-3 and 50 ng/mL thrombopoietin. In BFU-E
assays, 2×104BMMC/mL or 5×104PBMC/mL were plat-
ed, whereas for the CFU-MK assays, 105BMMC/mL or
2×105PBMC/mL were plated. BFU-E were counted on
day 14, CFU-MK on day 10. Culture media and colla-
gen were provided by the manufacturers. PB cultures
were not performed for 7 ET patients; BM was not
available for 21 RT patients. When the number of pro-
genitors available for culture was insufficient, the con-
trol CFU-MK assay was omitted. The gels were har-
vested on glass slides, dried and stained with May-
Grünwald-Giemsa (MGG) as described elsewhere,15,22,23
and colonies were counted on triplicate gels. The num-
bers of colonies were expressed as averages per 105
cells. The identification of MGG-stained CFU-MK (> 4
megakaryocytes) in collagen (Figure 1) is as accurate
as immunocytochemical labeling with anti-CD41/61
antibodies.17,26For erythroid colonies, both CFU-E (> 8
erythroblasts) and BFU-E (> 100 erythroblasts or sev-
eral clusters of more than 20 erythroblasts) were
counted. EMC/EEC assays were positive when CFU-
MK, CFU-E or BFU-E were identified on MGG-stained
gels. The EMC/EEC assay could be considered definitely
negative when these CFUwere not found but the num-
ber of cytokine-stimulated BFU-E in control cultures
was above the threshold of 50/105BMMC or 20/105
PBMC.22A negative BM EMC/EEC assay from 1 ET
Table 1. Diagnostic criteria.
Diagnostic
Criteria
Essential Thrombocythemia (ET)WHO ET criteria
Platelet counts: > 600×109/L.
Characteristic changes in bone marrow histology:
increased numbers ofmegakaryocytes, enlarged with
multilobulated nuclei, clustered in small groups;
increase in reticulin, or myelofibrosis.
Exclusion of other causes of thrombocytosis: RT,
myeloproliferative disorders other than ET (polycythemia vera,
chronic myeloid leukemia, idiopathic myelofibrosis)
and myelodysplastic syndromes.
Reactive Thrombocytosis (RT)Platelet counts: > 600×109/L.
All patients were recovering from recent surgery. All had normal
platelet counts prior to surgery and it was verified that platelet
counts came back to normal after surgery
(usually within weeks, occasionally within months of surgery).
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haematologica 2004; 89(10):October 2004
Standardization of the EMC/EEC assay in collagen
1209
patient with a control culture containing < 50 BFU-
E/105cells was eliminated from the study.
Statistical analysis
Mann-Whitney's and Spearman's tests were used to
compare groups, Fisher's exact test was used to com-
pare ratios. The SPSS software was used.
Results
Control assays with cytokines (media 2 and 3)
There was no significant difference between ET and
RT in cytokine-induced growth of CFU-E, BFU-E and
CFU-MK in PB and BM cultures (Tables 2 and 3). CFU-
MK assays frequently gave very low numbers of
colonies and therefore we could not define a practical
CFU-MK threshold to evaluate progenitor growth. In
contrast, 98.8% (81/82) of ET and all RT BM cultures
had > 50 BFU-E/105BMMC. Hence, the BFU-E assay
remains the best, most practical control of the growth
capacity of progenitors.
Analysis of EMC formation (medium 1)
EMC in cultures of PB progenitors (Table 2) were not
specific since they were found for 87.8% of ET but
also 53.1% of RT patients. Although the numbers of PB
EMC in RT patients was always ≤ 10/105PBMC, this
threshold and the PB EMC assay had no practical diag-
nostic value because only 23% of ET patients had > 10
Figure 1. EMC/EEC formation by
BMMC of ET patients in serum-
free,cytokine-free,collagen-based
media. Typical
megakaryocytic (A,B) and ery-
throid (C,D) colonies observed at
day 10 in MGG-stained collagen
gels of BM cultures. EEC were
usually small, well differentiated;
both EMC and EEC occasionally
included lysed cells. (A,C) × × 10
magnification; (B,D) enlarged view
of colonies A and C.
endogenous
Table 2. EMC and EEC formation by PB progenitors.
Peripheral blood
Endogenous colonies
EMC/105cells
Cytokine-stimulated colonies
CFU-MK/105cellsCFU-E+BFU-E/105cells Diagnostic EEC/105cells
ET
n =
mean±SD
median (range)
nbr+ (%)
74
7435 74
9.1±21.5
3.0 (0-168)
65 (87.8%)
1.8±6.4
0 (0-50)
19 (25.7%)
23.8±40.9
8.0 (0-341)
28 (80.0%)
78±68
56 (0-1905)
73 (98.6%)
RT
n =
mean±SD
median (range)
nbr+ (%)
3232
0
0
0
20 32
1.3*±2.3
0.5 (0-10)
17 (53.1%)
9.9±11.3
6.0 (0-43)
18 (90.0%)
85±71
72 (11-384)
32 (100%)
For EMC/EEC assays, 2×105PBMC were seeded per mL of serum-free, cytokine-free, collagen-based medium 1. Cultures were incubated for 10 days, then gels were dried,
stained with MGG and CFU-MK, CFU-E and BFU-E were scored. Cytokine-stimulated assays were performed in medium 3 (CFU-MK) and in medium 2 (CFU-E, BFU-E);
CFU-MK assays could not be performed for all patients. (nbr +) means “number of cultures with colonies” (ie. positive). (*) p < 0.001, Mann-Whitney's rank sum test,
compared to ET patients.
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haematologica 2004; 89(10):October 20041210
I. Dobo et al.
EMC/105PBMC. Because of the difficulty of obtaining
BM from RT patients, the number of cultures of BM
progenitors performed for RT patients was smaller
than that for ET patients (Table 3); nevertheless, in this
series, as in previous studies using the same assay,15,17
BM progenitors of RT patients never formed EMC. In
contrast, BM EMC were present in 77.8% (63/81) of ET
cultures. Hence, BM cultures confirmed the specifici-
ty (100%) of the collagen EMC assay performed for the
diagnosis of ET.
Analysis of EEC formation (medium 1)
PB EEC were not formed in RT cultures and were
specific to patients with ET (Table 2). However, EEC
were present in only 25.7% of PB ET cultures. Cultures
of BM progenitors (Table 3) confirmed that EEC for-
mation in the collagen assay is specific to myelopro-
liferative syndromes.15,22BM progenitors of RT patients
never formed EEC whereas BM EEC were present in
33.3% (27/81) of ET cultures. Altogether, 80.2%
(65/81) of ET patients had EMC and/or EEC in BM cul-
tures. The numbers of BM EMC and EEC were corre-
lated (n = 81, r = 0.41, p < 0.001).
Association of BM EMC and EEC
Among ET patients with BM EMC, 39.7% (25/63)
also had EEC; 11.1% (2/18) of ET patients without BM
EMC had BM EEC (difference not significant). In col-
lagen, BM EMC associated with EEC are also found in
a minority of patients with PV.17,18,22One common
hypothesis concerning this EMC+EEC+ET is that it
might be a latent form of PV.12In our ET series, there
was no significant difference in hematocrit or serum
erythropoietin level (measured at diagnosis) between
EMC+EEC+(n = 25) and EMC+EEC−(n = 38) patients.
Rather, EMC+EEC+ET patients had significantly (p =
0.017) higher numbers of BM EMC than did EMC+EEC−
ET patients: the median number of BM EMC in
EMC+EEC+ET was 13 EMC/105cells whereas that in
EMC+EEC−ET was 5 EMC/105cells.
Discussion
We have described a simple, standardized, serum- and
cytokine-free collagen EMC/EEC assay suitable for the
diagnosis of thrombocytoses. This assay must be per-
formed with BM progenitors. Using the assay on a larg-
er series of patients, eight laboratories confirmed the
finding of a preliminary, single-center study of 17
patients with thrombocytosis:17among patients with
platelet counts higher than 600×109/L, a positive BM
EMC assay is specific to ET. The collagen EMC/EEC assay
in the ready-to-use commercial culture media described
here was easily performed by all participating laborato-
ries. In addition, since not all clinics are equipped with
cell culture facilities, it is worth nothing that a 24 hr-
delay did not affect the EMC/EEC assay, thus allowing
shipment of BM samples to a specialized laboratory (in
this study, samples were collected in 16 hospitals and
sent to 8 laboratories).
As shown previously,22controlling the quality of pro-
genitors, by using cytokine-stimulated cultures, is an
important part of the standardized EM/EEC assay in
order to eliminate false negatives. The cytokine-stimu-
lated CFU-MK assay was not sufficiently reliable, per-
haps because of the choice of cytokines in medium 3
(lack of SCF). Hence, the BFU-E assay remains the pre-
ferred control for assessing progenitor quality. As in pre-
vious studies of erythrocytoses,22two commercial media
Table 3. EMC and EEC formation by BM progenitors.
Bone marrow
Endogenous colonies
EMC/105cells
Cytokine-stimulated colonies
CFU-MK/105cellsDiagnostic
EEC/105cells
CFU-E+BFU-E/105cells
ET
n =
mean±SD
median (range)
nbr+ (%)
8181 3381
10.4±15.4
5.0 (0-84)
63 (77.8%)
10.3±31.9
0 (0-207)
27 (33.3%)
42.9±36.7
31 (0-251)
32 (97.0%)
247±269
158 (8-1771)
81 (100%)
RT
n =
mean±SD
median (range)
nbr+ (%)
18*
0
0
0
18*
0
0
0
310**
190±181
112 (67-700)
10 (100%)
25.7±13.8
15 (15-47)
3 (100%)
For EMC/EEC assays, 105BMMC were seeded per ml of serum-free, cytokine-free, collagen-based medium 1. Cultures were incubated for 10 days, then gels were dried,
stained with MGG and CFU-MK, CFU-E and BFU-E were scored. Cytokine-stimulated assays were performed in medium 3 (CFU-MK) and in medium 2 (CFU-E, BFU-E);
CFU-MK assays could not be performed for all patients. (nbr +) means “number of cultures with colonies” (ie. positive). (*) Using the same assay, we previously reported
the absence of EMC and EEC in BM cultures of 18 healthy donors.15,17(**) For 8 RT patients, cytokine-stimulated BM cultures showed sufficient CFU-E/BFU-E growth by
direct, inverted microscopy but gels were not harvested and therefore MGG-stained colonies were not counted.
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haematologica 2004; 89(10):October 2004
Standardization of the EMC/EEC assay in collagen
1211
were tested for the diagnosis of thrombocytoses; EMC
and EEC formation observed in PB and BM cultures of
RT and ET patients with the two media were compara-
ble (data not shown).
Previous studies using the same collagen-based medi-
um had established that BM progenitors of healthy
donors and patients with secondary erythrocytosis do
not form EEC or EMC (based on the observation of more
than fifty BM cultures).15,22The sensitivity (80.2%) and
specificity (100%) of the collagen BM EMC/EEC assay
performed for the diagnosis of thrombocytoses were
excellent and comparable to those of BM and PB EEC
assays performed for the diagnosis of erythrocytoses.15,22
In particular, the sensitivity of the assay means that
additional cultures testing the sensitivity of progenitors
to low doses of thrombopoietin are not necessary.17,19,20
Like EEC, found mainly in PV but also in ET and in idio-
pathic myelofibrosis with myeloid metaplasia, BM EMC
in collagen assays are not exclusive to ET. Using the
same commercial culture medium, we previously report-
ed BM EMC growth in about one third of PV patients.15,22
Interestingly, in these studies we reported six patients
with absolute erythrocytosis and no BM EEC but with
BM EMC (< 4 EMC/105BMMC). At diagnosis, three of
these patients had platelet counts over 500 000×109/L
and BM histology, available for five patients, was in
favor of ET.
The study also confirmed that EEC are seen in a minor-
ity of ET but never in RT patients; in ET, BM EEC with-
out EMC were rare (2/81 ET patients). In our series of ET
patients at diagnosis, patients with both BM EMC and
EEC did not have a significantly higher hematocrit com-
pared to those without EEC. They had high numbers of
EMC, which would suggest a more severe (advanced)
form of ET rather than a latent form of PV.12However,
one cannot exclude that overt PV might appear several
years after diagnosis and long term follow-up of
EMC+EEC+vs. EMC+EEC−ET patients is necessary in order
to draw definitive conclusions. Alternatively, compared
to BM EEC only PV and BM EMC only ET, BM EMC+EEC+
patients might define MPD subsets originating from a
pool of early progenitors common to erythroid and
megakaryocytic lineages (CFU-Meg, CFU-GEMM); this
would be consistent with the variable presentation of
EMC+EEC+patients (polyglobulia and/or thrombocyto-
sis). Besides the EMC assay, more detailed and stan-
dardized evaluation of BM histology, as well as several
recent molecular assays (clonality assays, PRV-1 mRNA
expression assays),16,21should be evaluated for their rel-
evance to the diagnosis of ET in large, multicenter series;
furthermore, the results of these assays should be com-
pared to those of the BM EMC/EEC assay. In this regard,
our multicenter group has begun systematic measure-
ments of c-mplexpression in platelets and PRV-1mRNA
expression in neutrophils of patients with thrombocy-
tosis, in parallel with the BM EMC/EEC assay. Also,
although we limited our study to patients whose BM
histology was in favor of ET, one can argue that because
of the relative difficulty of establishing the diagnosis of
ET using the current WHO criteria, the sensitivity of the
collagen EMC/EEC assay could be over- or under-esti-
mated, as some of the patients diagnosed as having ET
in our series might not acually have had ET. Again, large
multicenter studies of thrombocytic patients including
several potential diagnostic assays and long-term clin-
ical follow-up should answer these questions.
In conclusion, the present study shows, in a large
series of patients, that the standardized collagen BM
EMC/EEC assay is suitable for the diagnosis of throm-
bocytoses. The sensitivity (80.2%) of the assay for the
diagnosis of ET is similar to the sensitivity of the EEC
assay for the diagnosis of PV (83%).15,22The simplicity of
this assay should encourage its use as a diagnostic test
in thrombocytoses; further studies on larger series of
thrombocytic patients will determine whether the stan-
dardized BM EMC assay should become a criterion for
ET, as the EEC assay is for PV.
ID: designed the study, collected and analyzed the data, and wrote
the paper; VP: designed the study; read and revised the manuscript;
SH: designed the study and analyzed data; wrote the paper. The fol-
lowing authors carried out or supervised cell cultures. They are list-
ed in decreasing order of the number of patients contributed to the
study: NB, EL, FG, PM, MD, LC. These authors carried out cell cultures
in their hospital: DP, EB. The authors reported no potential conflicts
of interest.
We are indebted to our colleagues from the Departments of Inter-
nal Medicine and the Departments of Clinical Hematology of the
Centres Hospitaliers of Angers, Bordeaux, Châlon-sur-Saône, Cler-
mont-Ferrand, Dijon, Grenoble, Guéret, Laval, Libourne, Limoges, Lori-
ent, Nantes, La Roche-sur-Yon, Saint-Etienne, Saumur and Vannes
for providing samples. This work was supported by a grant from the
Programme Hospitalier de Recherche Clinique 2001 (Région Bour-
gogne). We also thank Dr. Richard Garand (Nantes) for precious help
with image processing.
Manuscript received April 15, 2004. Accepted July 7, 2004.
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