AJCP / REVIEW ARTICLE

Laboratory Investigation of Myeloproliferative

Neoplasms (MPNs)
Recommendations of the Canadian MPN Group
Lambert Busque, MD,1 Anna Porwit, MD,2,3 Radmila Day, MSc,5 Harold J. Olney, MD,6

Brian Leber, MD,7 Vincent Ethier, MD,1 Shireen Sirhan, MD,8 Linda Foltz, MD,9
Jaroslav Prchal, MD,10 Suzanne Kamel-Reid, PhD,2,3 Aly Karsan, MD,11 and Vikas Gupta, MD4

Downloaded from https://academic.oup.com/ajcp/article-abstract/146/4/408/2236122 by guest on 22 June 2020

From the 1Department of Laboratory Hematology, Hôpital Maisonneuve-Rosemont, Université de Montréal, Montréal, Canada; 2Department of Pathology,
University Health Network, 3Department of Laboratory Medicine and Pathobiology, and 4Department of Medical Oncology and Hematology, Princess
Margaret Cancer Centre, University of Toronto, Toronto, Canada; 5Fusion MD Medical Science Network, Montréal, Canada; 6Department of Hematology
and Transfusional Medicine, Centre Hospitalier de l’Université de Montréal, Montréal, Canada; 7Department of Medicine, Hematology and
Thromboembolism, McMaster University, Hamilton, Canada; 8Division of Hematology, Jewish General Hospital, Montréal, Canada; 9Division of
Hematology, St Paul’s Hospital, University of British Columbia, Vancouver, Canada; 10Department of Oncology, McGill University, Montréal, Canada;
and 11Pathology & Laboratory Medicine and Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, Canada.

Key Words: Myeloproliferative neoplasms; Myelofibrosis; Polycythemia vera; Essential thrombocythemia; JAK2 V617F; CALR; MPL;
BCR-ABL negative

Am J Clin Pathol October 2016;146:408-422

DOI: 10.1093/AJCP/AQW131

ABSTRACT BCR-ABL1–negative myeloproliferative neoplasms

Objectives: To standardize diagnostic investigations for
myeloproliferative neoplasms (MPNs) to increase homogen-
eity in patient care and to streamline diagnostic approaches
in the most efficient and cost-effective manner.
Methods: The development of Canadian expert consensus
recommendations for the diagnosis of MPNs began with a
review of the following: clinical evidence, daily practice,
existing treatment guidelines, and availability of diagnostic
tools. Each group member was assigned a specific topic,
which they discussed with the entire group during several
consensus meetings.
Results: This document provides the Canadian MPN
group’s recommendations, proposed diagnostic algorithms,
and background evidence upon which decisions were made.
Conclusions: Standardization of diagnostic investigations
will increase homogeneity in patient care and provide a
foundation for future clinical research in this rapidly evolv-
ing therapeutic area. Streamlining diagnostic approaches in
the most efficient and cost-effective manner will also result
in significant cost saving for the health care system.
(MPNs) represent several clonal hematologic conditions
with overlapping features and the absence of the
Philadelphia chromosome, which is the hallmark of chronic
myelogenous leukemia (CML). This article focuses on three
main classic entities: polycythemia vera (PV), essential
thrombocythemia (ET), and primary myelofibrosis (PMF).
Recent discoveries in the molecular pathogenesis of
each MPN phenotype accompanied with the new molecular
diagnostic technologies greatly expanded the knowledge
about these conditions and created a need for more stream-
lined diagnostic approaches. Standardization of diagnostic
approaches for MPNs across the country will avoid unneces-
sary investigation and subsequently reduce the cost to the
Canadian health care system. As the latest data indicate that
the presence of various mutations carries significant prog-
nostic value,1-5 classification of patients according to their
mutational, clinical, morphologic, and cytogenetic features
could also assist in selecting appropriate therapeutic
approaches that could lead to better treatment outcomes.
Although focused on the Canadian reality, the recommenda-
tions of our group can apply to many countries that place an
important value on cost-effectiveness.
Currently, diagnostic criteria for MPNs are based on
the 2008 World Health Organization (WHO) classification
that includes clinical features, laboratory investigations,

408 Am J Clin Pathol 2016;146:408-422 © American Society for Clinical Pathology, 2016. All rights reserved.
DOI: 10.1093/ajcp/aqw131 For permissions, please e-mail: journals.permissions@oup.com

bone marrow morphology, and the presence of the JAK2
V617F mutation Table 1 .6,7 Recent discovery of another
MPN driver mutation in the calreticulin (CALR) gene in
addition to MPL mutations has led to the proposal of new
diagnostic criteria now included in the recently published
2016 revision of the WHO diagnostic criteria (Table 1).8,9
Materials and Methods
The development of expert consensus recommendations
for the diagnosis of MPNs began with a review of the follow-
ing: clinical evidence, daily practice, existing treatment
guidelines, and availability of diagnostic tools. Each group
member was assigned a specific topic, which he or she dis-
cussed with the entire group during several consensus meet-
ings occurring between October 2014 and January 2016. All
human experiments were approved by the institutional
review board where the experiments were conducted or were
in accord with the Helsinki Declaration of 1975.
Basic Physical and Laboratory Investigation
The assessment of patients with suspected MPNs begins
with the careful clinical evaluation of signs and symptoms
associated with these pathologies as well as those that are
part of the differential diagnosis. However, owing to the
main focus of this article being on laboratory investigations,
only the importance of documenting age, history of throm-
bosis, presence of constitutional symptoms, and splenomeg-
aly will be emphasized, since these features are integrated
into diagnostic criteria and/or prognostic risk scores.
Basic laboratory investigations for MPNs should in-
clude CBC, the peripheral blood smear, an erythropoietin
(EPO) level, and biochemistry tests (lactate dehydrogenase
[LDH], uric acid, vitamin B12, and iron status).
CBC
A CBC is the first step in establishing the diagnosis of
MPNs, and it could point to a specific MPN subtype. Most
MPNs have elevated values in one or more cell lineages
(erythrocytes, platelets, or neutrophils). For example, while
a consistently elevated platelet count of more than 450 
103/mL (>450  109/L) can indicate ET, erythrocytosis is a
feature of PV.7 Myelofibrosis (MF) can present with anemia
(listed by the WHO as one of the minor criteria for PMF;
Table 1)6,7 or thrombocytopenia.
There is, however, an ongoing debate regarding a reli-
able indicator of absolute erythrocytosis in patients with
suspected PV.10,11 The procedure for RBC mass determin-
ation by isotope dilution has been abandoned in Canadian
clinical practice due to the unavailability of isotopes and the
© American Society for Clinical Pathology
409
AJCP / REVIEW ARTICLE
difficulty in the standardization of this technique. Although
both hemoglobin and hematocrit levels have some limita-
tions, they are accepted as reasonable surrogates and indica-
tors of increased RBC mass. According to WHO criteria,
persistent hematocrit/hemoglobin above the diagnostic
thresholds or other evidence of increased RBC volume, es-
pecially if a patient has a history of thrombosis or other
disease-related symptoms (ie, pruritus), is indicative of PV.7
Recently, several investigators have documented that
some patients with JAK2 V617F–positive PV have
hemoglobin levels lower than the current WHO criteria
Downloaded from https://academic.oup.com/ajcp/article-abstract/146/4/408/2236122 by guest on 22 June 2020
(18.5 g/dL [185 g/L] for men and 16.5 g/dL [165 g/L] for
women).12 These patients with masked PV (mPV) have a
similar clinical evolution course as patients with PV, includ-
ing an increased risk of thrombosis.13 As patients with mPV
are missed by current WHO criteria, a retrospective analysis
of a large cohort of patients with MPN has suggested that
lowering the hemoglobin threshold to 16.5 g/dL (165 g/L)
for men or 16.0 g/dL (160 g/L) for women would include
most of these mPV cases.13 These lower thresholds have
been included in the 2016 revision to the WHO diagnostic
criteria.9,14 The Canadian MPN group acknowledges the ne-
cessity of appropriately diagnosing mPV but is concerned
with the large number of individuals who would be sub-
jected to further testing to rule out mPV.
For the purpose of this publication, two large Montreal-
based hospitals (Centre Hospitalier de l’Université de
Montréal and Maisonneuve-Rosemont Hospital) performed
an analysis over a 1-week period that showed that approxi-
mately 4.1% of all CBC results from unselected male par-
ticipants (outpatients) had hemoglobin levels of more
than 16.5 g/dL (>165 g/L) Figure 1A vs only 0.35% who
meet current criteria (hemoglobin >18.5 g/dL [>185 g/L]).
This indicates that 12 times more males would be suspected
as potential patients with PV and as such would be subjected
to further investigation. The proposed change to WHO crite-
ria would have less impact on females since only 0.35% had
hemoglobin levels greater than the proposed 16.0 g/dL
(160 g/L) Figure 1B . This is only three times more frequent
than the current cutoff of 16.5 g/dL (165 g/L), which ac-
counted for 0.13% of unselected females in this analysis.
The Canadian MPN group is concerned about unneces-
sary laboratory evaluation, optimal utilization of clinical re-
sources, and the psychological burden placed on individuals
who are highly unlikely to have PV. Therefore, it is important
that the cost-effectiveness of the proposed criteria be carefully
evaluated before being adopted in routine clinical practice. It is
important to emphasize that the new criteria should not be
used as a screening tool for PV, and as such, hemoglobin levels
above the suggested threshold should not be taken in isolation
but rather in the context of other potential signs and symptoms
indicative of PV. It is also important to stress that the intent of
Am J Clin Pathol 2016;146:408-422 409
DOI: 10.1093/ajcp/aqw131
Busque et al / LABORATORY INVESTIGATION OF MPNS

Table 1
2008 WHO Criteria and 2016 Revision of the WHO Criteria for PMF, PV, and ET7,9
2008 WHO Criteria
PMF
Diagnosis requires meeting all three major criteria and two minor criteria.
Major criteria
1. Presence of megakaryocyte proliferation and atypia,a usually accompanied by either reticulin and/or collagen fibrosis, or, in the absence
of significant reticulin fibrosis, the megakaryocyte changes must be accompanied by an increased bone marrow cellularity characterized
by granulocytic proliferation and often decreased erythropoiesis (ie, prefibrotic cellular-phase disease)
2. Not meeting WHO criteria for PV, CML, MDS, or other myeloid neoplasmb
3. Demonstration of JAK2 617VF or other clonal marker (eg, MPL515WL/K) or, in the absence of a clonal marker, no evidence of bone mar-
row fibrosis due to underlying inflammatory or other neoplastic diseasesc
Minor criteria

Downloaded from https://academic.oup.com/ajcp/article-abstract/146/4/408/2236122 by guest on 22 June 2020

1. Leukoerythroblastosis
2. Increase in serum lactate dehydrogenase leveld
3. Anemiad
4. Palpable splenomegalyd
PV
Diagnosis requires the presence of both major criteria and one minor criterion or the presence of the first major criterion together with two
minor criteria.
Major criteria
1. Hemoglobin >18.5 g/dL (>185 g/L) in men, >16.5 g/dL (>165 g/L) in women, or hematocrit >0.52 in men and >0.48 in women, or other
evidence of increased RBC volumee
2. Presence of JAK2 V617F or other functionally similar mutation such as JAK2 exon 12 mutation
Minor criteria
1. Bone marrow biopsy specimen showing hypercellularity for age with trilineage growth (panmyelosis) with prominent erythroid, granulo-
cytic, and megakaryocytic proliferation
2. Serum erythropoietin level below the reference range for normal
3. Endogenous erythroid colony formation in vitro
ET
All four criteria must be met.
1. Sustained platelet count 450  103/mL (450  109/L)
2. Bone marrow biopsy specimen showing proliferation mainly of the megakaryocytic lineage with increased number of enlarged, mature
megakaryocytes. No significant increase or left-shift of neutrophil granulopoiesis or erythropoiesis
3. Not meeting WHO criteria for PV, PMF, BCR-ABL–positive CML, or MDS or other myeloid neoplasm
4. Demonstration of JAK2 V617F or other clonal marker or, in the absence of JAK2 V617F, no evidence for reactive thrombosis

2016 Revision of the WHO Criteria

Prefibrotic/early PMF (pre-PMF)
Diagnosis requires meeting all three major criteria and at least one minor criterion.
Major criteria
1. Megakaryocytic proliferation and atypia, without reticulin fibrosis more than grade 1,f accompanied by increased age-adjusted BM cellu-
larity, granulocytic proliferation, and often decreased erythropoiesis
2. Not meeting the WHO criteria for CML, PV, ET, MDS, or other myeloid neoplasms
3. Presence of JAK2, CALR, or MPL mutation or, in the absence of these mutations, presence of another clonal markerg or absence of
minor reactive BM reticulin fibrosish
Minor criteria
1. Anemia not attributed to a comorbid condition
2. Leukocytosis 11  109/L
3. Palpable splenomegaly
4. LDH increased to above upper normal limit of institutional reference range
Overt PMF
Diagnosis requires meeting all three major criteria and at least one minor criterion.
Major criteria
1. Presence of megakaryocyte proliferation and atypia, accompanied by either reticulin and/or collagen fibrosis grade 2 or 3f
2. Not meeting WHO criteria for ET, PV, CML, MDS, or other myeloid neoplasms
3. Presence of JAK2, CALR, or MPL mutation or, in the absence of these mutations, presence of another clonal markerg or absence of
reactive myelofibrosish
Minor criteria
1. Anemia not attributed to a comorbid condition
2. Leukocytosis 11  109/L
3. Palpable splenomegaly
4. LDH increased to above upper normal limit of institutional reference range
5. Leukoerythroblastosis

(continued)

410 Am J Clin Pathol 2016;146:408-422 © American Society for Clinical Pathology

410 DOI: 10.1093/ajcp/aqw131
 AJCP / REVIEW ARTICLE

Table 1 (cont)
2016 Revision of the WHO Criteria
PV
Diagnosis requires meeting either all three major criteria or the first two major criteria and the minor criterion.i
Major criteria
1. Hemoglobin >16.5 g/dL (>165 g/L) in men, >16.0 g/dL (>160 g/L) in women, or hematocrit >0.49 in men and >0.48 in women, or
increased RBC massj
2. Bone marrow biopsy specimen showing hypercellularity for age with trilineage growth (panmyelosis), including prominent erythroid,
granulocytic, and megakaryocytic proliferation with pleomorphic, mature megakaryocytes (differences in size)
3. Presence of JAK2 V617F or JAK2 exon 12 mutation
Minor criteria
1. Subnormal serum erythropoietin level

Downloaded from https://academic.oup.com/ajcp/article-abstract/146/4/408/2236122 by guest on 22 June 2020

ET
Diagnosis requires meeting all four major criteria or the first three major criteria and the minor criterion.
Major criteria
1. Platelet count 450  109/L
2. Bone marrow biopsy specimen showing proliferation mainly of the megakaryocytic lineage with increased number of enlarged, mature
megakaryocytes with hyperlobulated nuclei. No significant increase or left-shift of neutrophil granulopoiesis or erythropoiesis and very
rarely minor (grade 1) increase in reticulin fibers
3. Not meeting WHO criteria for CML, PV, PMF, MDS, or other myeloid neoplasms
4. Presence of JAK2, CALR or MPL mutation
Minor criteria
1. Presence of a clonal marker or absence of evidence for reactive thrombocytosis

BM, bone marrow; CML, chronic myelogenous leukemia; ET, essential thrombocythemia; LDH, lactate dehydrogenase; MDS, myelodysplastic syndrome;
PMF, primary myelofibrosis; PV, polycythemia vera; WHO, World Health Organization.
a
Small to large megakaryocytes with an aberrant nuclear/cytoplasmic ratio and hyperchromatic, bulbous, or irregularly folded nuclei and dense clustering.
b
Requires the failure of iron replacement therapy to increase hemoglobin level to the PV range in the presence of decreased serum ferritin. Exclusion of PV is based on hemoglo-
bin and hematocrit levels. RBC mass measurement is not required. Requires the absence of BCR-ABL. Requires the absence of dyserythropoiesis and dysgranulopoiesis.
c
Secondary to infection, autoimmune disorder or other chronic inflammatory condition, hairy cell leukemia or other lymphoid neoplasm, metastatic malignancy, or toxic
(chronic) myelopathies. It should be noted that patients with conditions associated with reactive myelofibrosis are not immune to PMF, and the diagnosis should be considered in
such cases if other criteria are met.
d
Degree of abnormality could be borderline or marked.
e
Hemoglobin or hematocrit greater than the 99th percentile of method-specific reference range for age, sex, altitude of residence, or hemoglobin greater than 17.0 g/dL (170 g/L)
in men, 15.0 g/dL (150 g/L) in women if associated with a documented and sustained increase of at least 2.0 g/dL (20 g/L) from an individual’s baseline value, or elevated RBC
mass greater than 25% above mean normal predicted value.
f
See Table 4.
g
In the absence of any of the three major clonal mutations, the search for the most frequent accompanying mutations (eg, ASXL1, EZH2, TET2, IDH1/IDH2, SRSF2, SF3B1) is of
help in determining the clonal nature of the disease.
h
Fibrosis secondary to infection, autoimmune disorder or other chronic inflammatory conditions, hairy cell leukemia or other lymphoid neoplasm, metastatic malignancy, or toxic
(chronic) myelopathies.
i
Criterion number 2 (BM biopsy) may not be required in cases with sustained absolute erythrocytosis: hemoglobin levels more than 18.5 g/dL in men (hematocrit 55.5%) or more
than 16.5 g/dL in women (hematocrit 49.5%) if major criterion 3 and the minor criterion are present. However, initial myelofibrosis (present in up to 20% of patients) can only be
detected by performing a BM biopsy; this finding may predict a more rapid progression to overt myelofibrosis (post-PV myelofibrosis).
j
More than 25% above mean normal predicted value.

lowering the thresholds is to more accurately differentiate be-
tween JAK2-positive ET and mPV rather than to serve as a
base for population screening.
Peripheral Blood Smear
Several characteristics of the peripheral blood smear in
patients with MPNs can be used for diagnostic purposes.7
The peripheral smear of patients with PMF includes RBCs of
variable shape (including teardrop cells) and size as well as
variable degrees of polychromasia. Leukoerythroblastosis is
present in most cases and is recognized by the presence of
immature cells of both the myeloid and the erythroid lin-
eages. The major abnormality seen in blood smears of pa-
tients with ET is platelet anisocytosis, ranging from very
small to giant in size. The PV smear usually shows a mild to
overt excess of normochromic, normocytic RBCs. However,
if iron deficiency is present, the RBCs might be hypochromic
and microcytic. Circulating blasts are generally not present in
PV/ET. Due to predominant thrombocytosis, some cases of
early phase PV might mimic ET. Such cases eventually
evolve into an overt polycythemic stage.
Biochemistry
Patients with MPNs may have nonspecific abnormal-
ities in a variety of biochemistry tests, including increased
serum LDH (listed by the WHO as one of the minor criteria
for PMF),6,7 uric acid, and vitamin B12. It is noteworthy that
increased LDH is not specific for PMF and can also be seen
in PV and ET. An increase in LDH may be due to ineffect-
ive hematopoiesis or a hemolytic process occurring in the

© American Society for Clinical Pathology Am J Clin Pathol 2016;146:408-422 411

411 DOI: 10.1093/ajcp/aqw131
Busque et al / LABORATORY INVESTIGATION OF MPNS

A 100 B 100

84.22

80 80 77.38

60 60

Downloaded from https://academic.oup.com/ajcp/article-abstract/146/4/408/2236122 by guest on 22 June 2020

Patients (%)

Patients (%)
40 Meeting proposed 40 Meeting proposed
revised WHO revised WHO
threshold for PV threshold for PV

21.83
Meeting Meeting
20 current 20 current
WHO WHO
11.03 threshold threshold
for PV for PV

3.71
0.69 0.35 0.44 0.22 0.13
0 0
≤80 81-120 121-165 166-185 >185 ≤80 81-120 121-165 166-185 >185
Hb (g/L) Hb (g/L)

Figure 1 Distribution of hemoglobin levels in general population. Two large Montreal-based hospitals (Centre Hospitalier de
l’Université de Montréal and Maisonneuve-Rosemont Hospital) performed an analysis over a 1-week period that showed that
approximately 4.1% of all CBC results from unselected male participants (outpatients) had hemoglobin levels higher than
16.5 g/dL (>165 g/L) (A) vs only 0.35% that meet current criteria (hemoglobin higher than >18.5 g/dL [>185 g/L]). Only 0.35%
of females had hemoglobin levels greater than the proposed 16.0 g/dL (160 g/L) (B). Hb, hemoglobin; PV, polycythemia vera.

spleen. Hyperuricemia is due to enhanced turnover of hem-
atopoietic tissue.
Other Tests
A clinical laboratory finding that can assist in confirm-
ing a diagnosis of PV is a below-normal EPO level (listed as
a minor WHO diagnostic criterion for PV).6,7,9 Although
not necessary for diagnostic purposes, imaging of the abdo-
men with ultrasound, computed tomography scan, or mag-
netic resonance imaging can be used to document and
confirm organomegaly.
Recommendations for Laboratory Investigations
Basic laboratory investigations for MPNs should in-
clude CBC, EPO levels, peripheral blood smear, and the fol-
lowing biochemistry tests: LDH, uric acid, and vitamin B12.
Other tests such as serum ferritin and C-reactive protein
(CRP) may be useful in establishing a diagnosis.
• According to the WHO criteria, raised hemoglobin and/
or hematocrit levels, confirmed on separate occasions,
are indicators of erythrocytosis and, along with subopti-
mal EPO levels, are key criteria for PV diagnosis.
• We suggest using a hemoglobin level of 18.5 g/dL
(185 g/L) in men and 16.5 g/dL (165 g/L) in women
as the threshold for PV diagnosis. Newly proposed
hemoglobin threshold levels (16.5 g/dL [165 g/L]
in men and 16.0 g/dL [160 g/L] in women) should
not be taken in isolation but rather in the context of
other potential signs and symptoms indicative
of PV.
• A persistent and otherwise unexplained elevation in
platelet count (>450  103/mL [>450  109/L]) in a

412 Am J Clin Pathol 2016;146:408-422 © American Society for Clinical Pathology

412 DOI: 10.1093/ajcp/aqw131

Table 2
Standardized Morphologic Features of Distinctive Value Regarding World Health Organization–Defined ET vs Early Prefibrotic
Stage of PMF Criteria16
Feature ET
Cellularity No or only slight increase in age-matched cellularity
Granulopoiesis and No significant increase
erythropoiesis
Megakaryocytes Prominent large to giant mature with hyperlobulated or
deeply folded nuclei, dispersed or loosely clustered
in the marrow space
Reticulin fibers No or very rare minor focal increase (0-1)
ET, essential thrombocythemia; PMF, primary myelofibrosis.
patient with normal serum ferritin and CRP levels is sug-
gestive of ET.
• Anemia, splenomegaly, elevated LDH, and leukoery-
throblastosis are often present in patients with PMF.
Bone Marrow Aspiration and Core Bone
Biopsy
An MPN diagnosis can often be suspected based on clin-
ical presentation and abnormal CBC results. However, a bone
marrow examination is usually needed to confirm the diagno-
sis. In addition, the recently published revision of the WHO
criteria stresses the important role of bone marrow biopsy in
establishing the diagnosis of all MPNs.8,9,14-16 For example,
bone marrow biopsy may be necessary to clearly distinguish
between ET and early prefibrotic PMF with thrombocythemia,
the latter being associated with specific diagnostic criteria in
the 2016 revision of the WHO criteria (Table 1).9,16 However,
as there is substantial interobserver variability in the interpret-
ation of bone marrow biopsy specimens,17,18 and specific
changes in four major diagnostic parameters that can help in
distinguishing between these two clinical entities have been
outlined by Thiele et al16 Table 2 . It should also be empha-
sized that no isolated bone marrow feature characterizes any
subtype of MPNs but only distinctive histologic patterns.
Bone Marrow Requirement for PV Diagnosis
In patients with clear PV characteristics (increased
RBC mass corresponding to the WHO 2008 criteria, low
EPO levels, and presence of JAK2 V617F mutation), bone
marrow evaluation provides limited additional value for
diagnostic purposes and is often not performed by Canadian
clinicians.6,9,19 However, the presence of fibrosis in bone
marrow does have some prognostic utility,6 and therefore a
bone marrow evaluation is suggested—especially in rela-
tively younger patients. A bone marrow evaluation should
always be done in patients who are JAK2 V617F mutation
© American Society for Clinical Pathology
413
AJCP / REVIEW ARTICLE
PMF
Marked increase in age-matched cellularity
Pronounced proliferation of granulopoiesis and reduction of
erythroid precursors
Dense or loose clustering and frequent endosteal transloca-
tion of medium sized to giant, showing hyperchromatic,
hypolobulated, bulbous, or irregularly folded nuclei and an
aberrant nuclear/cytoplasmic ratio
No significant increase in reticulin fibers (0-1)
Downloaded from https://academic.oup.com/ajcp/article-abstract/146/4/408/2236122 by guest on 22 June 2020
negative or patients who have hemoglobin values below the
2008 WHO defined threshold.
Diagnostic Approach and Features of Bone Marrow
Report
The International Committee for Standardization in
Hematology guidelines for bone marrow sampling should be
followed.20 Bone marrow aspiration is useful for the assess-
ment of cell morphology and iron stains for the evaluation of
ring sideroblasts, as well as for obtaining marrow cells for
other tests such as cytogenetic and genetic mutational ana-
lyses. However, due to extensive marrow fibrosis, it may be
difficult to aspirate in some patients (ie, those with advanced
PMF), yielding a “dry” tap. Thus, a pretreatment bone mar-
row core biopsy is necessary both to establish a correct diag-
nosis and as a baseline for follow-up examinations.
Evaluation of bone marrow smears is important to ex-
clude dysplasia, which should not be present at diagnosis of
MPNs but can be seen after treatment and upon progres-
sion.7 A determination of the percentage of blasts is of par-
ticular importance since it can indicate either the
accelerated phase of the disease (10%-19% blasts) in pa-
tients with a previously established diagnosis of PMF or leu-
kemic transformation (>20% blasts).
Currently, there is a need to standardize bone marrow
reporting to ensure that the basic investigational criteria are
met and included in the report. The major requirements for
a bone marrow biopsy report include age-adjusted cellular-
ity Table 3 ,21 presence of fibrosis (reticulin and collagen
stains), evaluation of granulopoiesis with special reference
to blast clusters, and evaluation of erythropoiesis (frequency
and distribution).22 Evaluation of megakaryocytopoiesis
should include frequency, size, distribution, and presence of
atypia and maturation defects. A consensus proposal for a
standardized reporting strategy was recently published.22
The WHO classification criteria for MPNs include specific
bone marrow features as diagnostic parameters for the char-
acterization and distinction between MPN subgroups.6,7,9
Am J Clin Pathol 2016;146:408-422 413
DOI: 10.1093/ajcp/aqw131
Busque et al / LABORATORY INVESTIGATION OF MPNS
Appendix 1 provides a summary of the key distinguishing
bone marrow features for MPNs. 7 Normal Ranges of Bone Marrow Cellularity for Selected Age
Standardized Evaluation of Fibrosis
Bone marrow fibrosis is present in approximately 15%
to 20% of patients with PV23 and can also be seen in patients
with ET.24,25 In PMF, bone marrow fibrosis may vary con-
siderably at different stages of the disease and is not a
required criterion for the diagnosis but instead parallels dis-
ease progression. Presence of bone marrow fibrosis is rou-
tinely assessed by using histochemical staining methods that
produce qualitative rather than quantitative results.
Currently, quantitative measurements of fibrosis are being
adapted in clinical trials.
The fibrosis is typically visualized with a reticulin sil-
ver stain (Gorden-Sweet, Gomori). Trichrome stain should
be added to visualize collagen. The quality of the reticulin
stain should be assessed by detection of normal staining in
vessel walls as internal control. Progressive fibrosis is gen-
erally associated with atypical megakaryocytic hyperplasia
and a thickening and distortion of the bony trabeculae
(osteosclerosis).7,21 Fibrosis should be graded according to
the WHO classification based on the European consensus
criteria Table 4 .9,21
Recommendations for Histopathologic Investigations
A bone marrow biopsy should be an integral part of the
evaluation of a patient with MPN.
• The pretreatment bone marrow core biopsy is a basic re-
quirement for adequate diagnosis.
• The bone marrow biopsy report should include megakar-
yocyte atypia (number, morphology, and distribution)
and the presence of fibrosis (reticulin and collagen
stains), as well as age-matched bone cellularity, iron dis-
tribution, myeloblast description (blast clusters and per-
centage of CD34 stain), granulopoiesis, and
erythropoiesis (frequency and distribution).
Molecular Diagnostics
The discovery of the Philadelphia chromosome in 1960
precisely defined CML as a clinical entity characterized by
extreme leukocytosis, splenomegaly, and a high risk of pro-
gression toward acute leukemia.26-28 This discovery paved
the way for the development of diagnostic tools and targeted
therapies that have dramatically changed the clinical course
of this disease. Similarly, significant advances made over
the past decade, including the breakthrough discovery of the
JAK2 V617F mutation,29-32 now allow for the identification
of molecular markers in almost all patients with PV as well
as most patients with ET or MF. These discoveries have
414 Am J Clin Pathol 2016;146:408-422
414 DOI: 10.1093/ajcp/aqw131
Table 3
Groups21,a
Age, y % Hematopoietic Area
20-30 60-70
40-60 40-50
70 30-40
a
Obtained from the Haematologica Journal website (http://www.haematologica.org)
as per the rights and permissions outlined.
Table 4
Downloaded from https://academic.oup.com/ajcp/article-abstract/146/4/408/2236122 by guest on 22 June 2020
Grading of Bone Marrow Fibrosis9,21
Grading Descriptiona
MF-0 Scattered linear reticulin with no intersections (cross-
overs) corresponding to normal bone marrow
MF-1 Loose network of reticulin with many intersections, espe-
cially in perivascular areas
MF-2 Diffuse and dense increase in reticulin with extensive
intersections, occasionally with focal bundles of thick
fibers mostly consistent with collagen and/or focal
osteosclerosisb
MF-3 Diffuse and dense increase in reticulin with extensive
intersections and coarse bundles of thick fibers consist-
ent with collagen, usually associated with
osteosclerosisb
MF, myelofibrosis.
a
Fiber density should be assessed only in hematopoietic areas.
b
In grades MF-2 or MF-3, an additional trichrome stain is recommended.
made molecular testing the cornerstone of MPN
investigation.
Figure 2 provides a practical diagnostic algorithm that
suggests a sequence in which mutational testing for MPNs
can be ordered.
Detection of JAK2 V617F
Testing for JAK2 V617F mutation should be performed
early as part of a diagnostic workup of suspected MPNs. As
tests for JAK2 V617F are simple and cost-effective, they
can be performed in all patients in whom basic clinical in-
vestigation suggests a potential MPN (ie, increased hemo-
globin level or thrombocytosis with platelet count 450 
103/mL [450  109/L]).
Laboratory detection of the JAK2 V617F mutation is
highly sensitive and specific for an MPN, and a positive re-
sult in the appropriate clinical setting supports the diagnosis
of an MPN.33-37 However, a negative result does not ex-
clude the possibility of an MPN, and such patients should
undergo further molecular testing.
The assays for the JAK2 V617F mutation are divided
into qualitative and quantitative. All of the assays, however,
must meet standards of specificity and sensitivity. Assays
commonly use in Canada for the detection of the JAK2
© American Society for Clinical Pathology
 AJCP / REVIEW ARTICLE

Basic investigations
• Medical history (history of thrombosis)
• Physical examination including palpation of spleen
• CBC and blood smear examination
• Iron status and LDH and EPO levels

Anemia, splenomegaly, elevated Elevated platelet count (>450 × 109 /L), Elevated hemoglobin/hematocrit,
LDH, and leukoerythroblastosis history of thrombosis history of thrombosis

Suspected PMFa Suspected ET Suspected PV

Bone marrow investigations S-erythropoietin JAK2 V617F

Downloaded from https://academic.oup.com/ajcp/article-abstract/146/4/408/2236122 by guest on 22 June 2020

High Positive
Stop Stop
JAK2 V617F
Use bone marrow Positive
Stop
morphology for
JAK2 exon 12 mutation
diagnosis CALR
clonal markers if Positive
feasible Stop
MPLb Bone marrow
biopsyd
Positive
Stop
BCR-ABL c

Positive
Stop

Negative for all 4 mutations

Consider EEC, X-inactivation in females, clonality by NGS

Consider homeostatic evaluation at the time of diagnosis and

as a part of ongoing routine evaluation

Figure 2 Diagnostic workup of non–BCR-ABL myeloproliferative neoplasms (MPNs). A practical diagnostic algorithm that sug-
gests a sequence in which mutational testing for MPNs can be ordered. CALR, calreticulin; EEC, endogenous erythroid colony;
EPO, erythropoietin; ET, essential thrombocythemia; Hb, hemoglobin; LDH, lactate dehydrogenase; MF, myelofibrosis; NGS,
next-generation sequencing; PMF, primary myelofibrosis; PV, polycythemia vera; WHO, World Health Organization. aAbnormal
karyotype in myelofibrosis can be used to confirm clonal myeloproliferation and, in some instances, facilitates the distinction
between MF and bone marrow fibrosis associated with another myeloid malignancy such as chromosome 5q deletion syn-
drome. bTesting for the MPL mutation should be reserved for patients who are negative for JAK2 V617F and CALR mutations
and in whom bone marrow biopsy and other investigations support the diagnosis of ET or PMF. cConsider performing BCR-
ABL testing as an initial step in all patients as recommended by other groups. dA diagnosis of PV can be made without bone
marrow biopsy if a patient meets the criteria for an increase in RBC volume (hemoglobin/hematocrit, as defined in the 2008
WHO guidelines) and is JAK2 V617F/exon 12 positive. Using less stringent Hb-level criteria mandates a bone marrow biopsy.
Furthermore, biopsy is highly recommended since degree of fibrosis can carry valuable prognostic information.

V617F mutation at diagnosis include single allele-specific
polymerase chain reaction (PCR) and quantitative TaqMan
assays.37
As levels of JAK2 V617F vary between patients, the
question has been raised as to the minimum level of JAK2
V617F that should be used to diagnose an MPN. Setting a
cutoff too high may lead to some MPN cases not being cor-
rectly diagnosed, and conversely, setting the cutoff too low
runs the risk of greater numbers of false positives. This is of
particular importance as low levels of JAK2 V617F can be
found in approximately 1% of otherwise hematologically
normal individuals.33 Thus, when choosing an assay for the
JAK2 V617F mutation, it has to be sensitive enough to be
able to identify a JAK2 V617F mutant allele burden as low
as 1% to 3%. This threshold has been demonstrated to be
pathogenically relevant and carry clinical significance.34-37

© American Society for Clinical Pathology Am J Clin Pathol 2016;146:408-422 415

415 DOI: 10.1093/ajcp/aqw131
Busque et al / LABORATORY INVESTIGATION OF MPNS
Patients evaluated for MPN who present with a nondiagnos-
tic, low level of JAK2 V617F mutational burden (<3%)
should be reevaluated (within 6 months).
Quantitative assays such as quantitative TaqMan offer the
possibility of determining the precise JAK2 V617F allele bur-
den,37 which, according to an increasing body of evidence, has
significant prognostic value.38 Several studies reported that a
high allele burden is associated with worse outcomes with re-
gard to symptom severity, disease transformation, and the need
for more aggressive therapies.38-40 Quantitative assays may
also have diagnostic value since it is rare to have ET with an
allele burden of more than 40%.41 Another advantage of quan-
titative assays is that they can be used to monitor the response
to a particular therapy (ie, interferon or ruxolitinib in PV).42,43
With new targeted therapies that have an impact on disease
burden, the need for quantitative assays will likely increase in
the near future.44,45
Although DNA extracted from either peripheral blood
or bone marrow is acceptable for JAK2 V617F mutation
testing, peripheral blood (2-10 mL) is the preferred option,
and EDTA is the usual anticoagulant of choice. The sample
should be received within 24 to 48 hours, although samples
of up to 1 week old are usually acceptable for nonquantita-
tive DNA analysis. Isolation of granulocytes is not required
if the assay is sufficiently sensitive (sensitivity of 1%-3% or
better). When reporting results, it is important to indicate
the assay sensitivity.
Supplementary Tests for JAK2 V617F–Negative
Patients
Mutations in JAK2 Exon 12
In 2007, Scott et al46 described mutations within exon 12
of JAK2 in some patients with JAK2 V617F–negative PV as
well as in patients previously categorized as having idiopathic
erythrocytosis, raising the possibility that all patients with PV
carry a mutation within JAK2.47-49 Since then, at least 17 dif-
ferent mutations have been described within exon 12.49 Thus
far, mutations within exon 12 of JAK2 have been reported only
in patients with PV.46-49 Given that the presence of a JAK2
exon 12 mutation in a patient with erythrocytosis is diagnostic
for PV, it has been suggested that all patients with unexplained
erythrocytosis who are JAK2 V617F negative should be
screened for mutations within JAK2 exon 12.50,51
Gene sequencing using the Sanger method is currently
used for identification of exon 12 mutations but lacks sensitiv-
ity compared with the techniques used for JAK2 V617F or
CALR mutation testing.46 Other, more sensitive, methods
have been developed, including high-resolution melting
analysis52-55 and denaturing high-performance liquid chroma-
tography.56 A technique using identification of different frag-
ment lengths generated by insertion/deletion in the mutant
416 Am J Clin Pathol 2016;146:408-422
416 DOI: 10.1093/ajcp/aqw131
clone has been developed and could increase the cost-
effectiveness of this analysis (L. Busque, MD, laboratory
review meeting, December 2015). Bone marrow specimens
are preferred for exon 12 mutation identification since they in-
crease sensitivity.
Calreticulin
In 2013, two independent laboratories identified the
gene encoding CALR as a new cancer gene that is mutated
in most patients with PMF or ET who are JAK2 V617F or
MPL negative.1,2 Among patients with ET or PMF with
Downloaded from https://academic.oup.com/ajcp/article-abstract/146/4/408/2236122 by guest on 22 June 2020
nonmutated JAK2 V617F or MPL, CALR mutations were
detected in 67% of those with ET and 88% of those with
PMF. Thus, CALR mutations have significant diagnostic
value, and its inclusion in diagnostic algorithms (Figure 2)
will alleviate the need for additional testing in a substantial
significant percentage of patients with ET or PMF, which in
turn may will allow significant cost savings. Assays for
CALR mutations are DNA based and use fragment length
analysis. They can be performed on peripheral blood speci-
mens. However, as germline mutations in CALR have been
described,57 one has to be careful about interpreting results
in the appropriate clinical context.
Mutations in the MPL Gene
At least five different pathogenetic mutations within
exon 10, affecting codon S505 or W515, of the thrombopoie-
tin receptor, MPL, have been described in around 5% to 10%
of patients with ET and PMF.58,59 When performed by
Sanger sequencing, testing for MPL mutation can be costly
and labor intensive, especially if used as a screening proced-
ure. It should be performed only after bone marrow biopsy
and other investigations in selected MPN-suspected cases
negative for JAK2 V617F and CALR mutations (Figure 2).
MPL mutation testing can be performed using a peripheral
blood specimen. The use of an amplification-refractory muta-
tion system PCR technique (allele-specific PCR that includes
S505N, W515L, W515A, W515R, and W515K variants) is a
cost-effective alternative to Sanger sequencing (L. Busque,
MD, laboratory review meeting, December 2015).
BCR-ABL1 Assessment
Historically, exclusion of CML was the first and only
step in the molecular investigation of MPNs. The Canadian
MPN group is questioning the sequential place of this test in
the laboratory investigation. We recommend that BCR-ABL
testing be performed in patients who are JAK2, CALR, and
MPL negative (triple negative) or in those with features sug-
gestive of CML, as concomitant BCR-ABL and JAK V617F
has been reported in extremely rare instances.60 This ap-
proach is in line with our global cost-effectiveness guide-
lines since screening all patients using fluorescence in situ
© American Society for Clinical Pathology

hybridization (FISH) or karyotype is particularly costly. If
universal screening for BCR-ABL is preferred, we suggest
the use of the less costly reverse transcription–PCR (RT-
PCR) technique.
Recommendations for Molecular Investigations
Patients with suspected MPN should be tested for three
mutations in specific order, starting with the JAK2 V612F
mutation. Consider screening all patients for BCR-ABL1 with
RT-PCR, as recommended by other groups.6,61-63
• Patients who are JAK2 V612F negative and meet other
criteria for ET or PMF should be screened for CALR
mutations.
• Patients who are JAK2 V612F negative and meet other
diagnostic criteria for PV should be screened for JAK2
exon 12 mutations.
• MPL mutation testing should be reserved for patients
who are negative for JAK2 V617F and CALR mutations
and in whom bone marrow biopsy and other investiga-
tions support a diagnosis of ET or PMF.
• BCR-ABL1 screening by RT-PCR should be done in all
patients who have the triple mutation (JAK2 V617F,
CALR, and MPL) or in those with unusual clinical fea-
tures suggestive of CML.
Investigation of Triple (JAK2 V617F, CALR,
MPL) Negative Cases
Approximately 10% to 15% of patients with ET or MF
have a suspected MPN diagnosis but without a proven clonal
marker.2,64 In these cases, other tests may assist in confirming
the clonal nature of their hematopoietic stem cell disorders
and/or specific aspects of their MPN-related phenotype.
Recently, novel gain-of-function mutations in JAK2 and MPL
were identified in 5% to 10% of triple-negative cases using
whole-exome sequencing, highlighting the heterogeneous na-
ture of this disease entity and illustrating the potential utility of
new molecular biology techniques.65,66
Endogenous Erythroid Colony Formation
The discovery that ET and PV erythroid progenitors
could proliferate in vitro, in the absence of erythropoietin,
confirmed the autonomous nature of these disorders.67,68
Endogenous erythroid colony (EEC) formation is listed
among the 2008 WHO criteria as one of the minor criteria for
PV.7 The positive predictive value for the diagnosis of PV by
spontaneous growth of EECs of peripheral blood and bone
marrow cells was approximately 80% to 85% when either
peripheral blood or bone marrow assays were performed and
up to 94% when both assays were performed.69 In addition,
EEC number is a sensitive and specific marker reflecting the
© American Society for Clinical Pathology
417
AJCP / REVIEW ARTICLE
abnormal hematopoietic clone burden induced by these dis-
orders and, as such, provides important diagnostic value.69 In
patients with PV, EEC number correlates with hemoglobin
levels, time required for treatment response, the incidence of
vascular thrombosis, and time to relapse. However, this test
is not routinely available in most laboratories, and it is diffi-
cult to standardize. This analysis had been eliminated in the
2016 revision of the WHO diagnostic criteria.9,14
Karyotype Analysis
Although in MPNs, karyotype carries prognostic rather
Downloaded from https://academic.oup.com/ajcp/article-abstract/146/4/408/2236122 by guest on 22 June 2020
than diagnostic values, cytogenetic analysis can be used to
confirm clonal hematopoiesis, especially in patients who are
triple negative.70 Furthermore, it may facilitate the distinction
between MPN and other myeloid malignancies such as
chromosome 5q deletion syndrome, a chromosome abnor-
mality commonly associated with myelodysplastic syndrome
(MDS) presenting with normal to mild thrombocytosis.71
Next-Generation Sequencing
Recently, several investigators have documented recurrent
mutations in a limited number of genes that occur in several
myeloid cancers, including MDS,72 acute myeloid leukemia,73
and MPNs.5 Next-generation sequencing (NGS) has the poten-
tial to very efficiently identify mutations by simultaneously
analyzing several dozen candidate genes (myeloid gene panel).
Mutational analysis at diagnosis and during follow-up could
detect clonal evolution and identify patients at high risk of dis-
ease progression.74 In addition, this technique has recently
been used to identify nondriver mutations in 40% of patients
with PV or ET, the presence of which might help predict sur-
vival.75 The documentation of an acquired mutation will con-
firm the clonal nature of the hematopoiesis but will not
indicate the specific clinical entity. In addition, the recent find-
ings that some clonal mutations might be a result of the aging
process should be taken into consideration.76-79 It is presently
unclear how to use the information gathered from NGS plat-
forms in routine clinical practice in a meaningful way. NGS
techniques are currently being investigated at some Canadian
centers as part of ongoing observational studies.
Prognostic Markers
Cytogenetic
In general, two approaches can be used for cytogenetic
investigations: FISH and karyotype analysis. The cytogen-
etic investigation for MPNs should be based on karyotype
analysis with a minimum of 20 metaphases at a banding
resolution of ideally 450 bands or higher. Performing
Am J Clin Pathol 2016;146:408-422 417
DOI: 10.1093/ajcp/aqw131
Busque et al / LABORATORY INVESTIGATION OF MPNS
cytogenetic analyses on the aspirate specimen is preferred,
but when an aspirate is not available, the peripheral blood or
a core biopsy specimen is suitable.
The value of cytogenetic investigation in PV and ET is
limited to mutation-negative cases or patients with early
signs of transformation. In patients with MF, cytogenetic
abnormalities carry significant prognostic value and are
included in the Dynamic International Prognostic Scoring
System.70 Approximately one-third of patients with PMF
have cytogenetic abnormalities, none of which are disease
specific. The most frequent findings are del(20q), del(13q),
gains of chromosomes 8 and 9, and abnormalities of
chromosome 1, including duplication of 1q. Other less fre-
quent lesions include –7/del(7q), del(5q), del(12p), þ21,
and der(6)t(1;6)(q21;p21.3).70,80-83 A recently presented
genetics-based prognostic scoring system distinguishes four
distinct cytogenetic risk categories Table 5 .83,84 Unfavorable
karyotypes are associated with thrombocytopenia, leuko-
penia, circulating blasts of 1% or more, and lower hemoglo-
bin levels.80 Available data also suggest that patients with
unfavorable karyotypes are at high risk of leukemic
transformation.80
Mutations in Specific Genes With Prognostic Value
In the context of MPN, recent studies identified that
mutations in several genes (ASXL1, EZH2, SRSF2, and
IDH1/2) had a significant impact on disease progression and
survival.1,5,81 Harboring more than one mutation in any of
these genes is considered a “high molecular risk” and has
been associated with reduced survival and an increased risk
of blast transformation in PMF.4,5 Thus, it has been pro-
posed to incorporate these mutations in prognostic scoring
systems.4,5,85 The 2016 revision of the WHO criteria lists
some of these mutations as potentially helpful to confirm
the clonal nature of PMF.9 Currently, most centers do not
have access to clinically validated NGS testing.
Recommendation for Prognostic Evaluation
• Cytogenetic testing should be performed in all cases of
MF and considered in cases of PV/ET.
• When available, screening for mutations of prognostic
significance should be conducted.
Hemostasis Evaluation
Routine coagulation evaluation, including the pro-
thrombin time (PT), activated partial thromboplastin time
(APTT), thrombin time, and fibrinogen level, should be per-
formed in every MPN case.
418 Am J Clin Pathol 2016;146:408-422
418 DOI: 10.1093/ajcp/aqw131
Table 5
Cytogenetic Risk Categories According to the Genetics-Based
Prognostic Scoring System83,84
Risk Category Abnormality
Very high Monosomal karyotype, inv(3), i(17q), –7/7q–, 11q,
or 12p abnormalities
High Complex nonmonosomal, two abnormalities not
included in very high-risk category, 5q–, þ8,
other autosomal trisomies except þ9, and other
sole abnormalities not included in other risk
categories
Intermediate Sole abnormalities of 20q–, 1qþ, or any other sole
translocation and –Y or other sex chromosome
Downloaded from https://academic.oup.com/ajcp/article-abstract/146/4/408/2236122 by guest on 22 June 2020
abnormality
Low Normal or sole abnormalities of 13q– or þ 9
The thrombocytosis associated with MPNs can increase
the risk of bleeding due to acquired von Willebrand disease
(vWD), where the clearance of the von Willebrand factor
(vWF) is increased proportionately to platelet count.86-88 To
detect this, it is important to investigate the functional activity
as well as the antigen level of vWF, since the clearance of the
most effective high molecular weight multimers is preferen-
tially affected and the total decrease in antigen level may not
be remarkable.86-88 This phenomenon is most commonly
observed with very high platelet counts (>1,000  103/mL
[>1,000  109/L]).89 However, it has also been reported in
patients with PV who have platelet counts that are only mar-
ginally elevated, perhaps reflecting a contribution of hyper-
viscosity to the increased possibility of cleavage of vWF by
ADAMTS13 at high sheer rates.90 This acquired vWF is re-
sponsive to cytoreductive therapy directed at reducing the
platelet count.86-90 Laboratory testing for acquired vWD typ-
ically includes the screening tests of coagulation (PT and
APTT) and specific tests for vWD (vWF antigen and ristoce-
tin cofactor activity, with vWF multimer analysis as needed)
and factor VIII activity.91
If fibrinogen level is below the normal value, it may be
necessary to rule out the possibility of increased fibrinolysis.
This may be particularly necessary in the context of a preop-
erative workup. Testing for fibrinolysis includes a euglobu-
lin lysis time assay, with a shortened lysis time indicating a
hyperfibrinolytic state and an increased bleeding risk.
Recommendations for Hemostasis Evaluation
• Basic coagulation screening (PT, APTT, fibrinogen
level) should be done in all patients.
• Screening for vWD should be done in all patients with a plate-
let level greater than 1,000  103/mL (1,000  109/L) or in
patients with clinical signs of increased mucocutaneous
bleeding.
© American Society for Clinical Pathology

Conclusion
The recent and rapid accumulation of data relative to the
molecular pathogenesis of MPNs has led the Canadian MPN
group to develop a set of recommendations specific to the in-
vestigation of these diseases. Moreover, the recently acquired
molecular insights on MPNs have led to the development of
novel tests that should be added to the routine diagnostic arma-
mentarium. The use of these tests should be based on scientific
evidence and according to the cost-effective strategy proposed
in these guidelines. Furthermore, the novel WHO 2016 diag-
nostic criteria for PV should be judiciously applied to the clin-
ical context; taking these criteria at face value will lead to the
unnecessary investigation of a large segment of the healthy
population. The precision of MPN diagnosis will become pro-
gressively more important as novel therapeutics arise with the
potential to change the clinical outcome of these disorders.
Corresponding authors: Lambert Busque, MD, Dept of Laboratory
Hematology, Hôpital Maisonneuve-Rosemont, Universté De
Montréal, 5415 Blvd de l’Assomption, Montréal, QC H1T 2M4,
Canada; lbusque.hmr@ssss.gouv.qc.ca; Vikas Gupta, MD,
Princess Margaret Cancer Centre, 610 University Avenue,
Toronto, ON M5G 2M9, Canada; vikas.gupta@uhn.ca.
This work was supported by an unrestricted grant from
funding: Novartis Canada Pharmaceuticals.
Conflicts of interest: Vikas Gupta received research funding from
Incyte and Novartis, received an honorarium from Novartis/Incyte,
and served on the advisory board panel for Novartis. Brian Leber
received an honorarium from Novartis Canada and served on the
medical advisory board for Novartis Canada. Anna Porwit received
an honorarium from Novartis and served on the advisory board
panel for Novartis. Suzanne Kamel-Reid received an honorarium
from Novartis and served on the advisory board panel for Novartis.
Shireen Sirhan received an honorarium from Novartis and served
on the advisory board panel for Novartis. Lambert Busque received
an honorarium from Novartis and served on the advisory board
panel for Novartis. Harold J. Olney received an honorarium from
Novartis and served on the advisory board panel of Novartis. Lynda
Foltz received an honorarium and research funding from Novartis
and served on the advisory boards for Novartis. The other authors
declare no relevant conflict of interests.
The Canadian MPN group is a not-for-profit, charitable na-
tional organization whose mission is to improve care and research
for patients with MPN through interprofessional collaboration.
For more information about the MPN group and its activities,
please visit http://mpncanada.com.
References
1. Nangalia J, Massie CE, Baxter EJ, et al. Somatic CALR mu-
tations in myeloproliferative neoplasms with nonmutated
JAK2. N Engl J Med. 2013;369:2391-2405.
2. Klampfl T, Gisslinger H, Harutyunyan AS, et al. Somatic
mutations of calreticulin in myeloproliferative neoplasms. N
Engl J Med. 2013;369:2379-2390.
© American Society for Clinical Pathology
419
AJCP / REVIEW ARTICLE
3. Tefferi A, Lasho TL, Finke CM, et al. CALR vs JAK2 vs
MPL-mutated or triple-negative myelofibrosis: clinical, cyto-
genetic and molecular comparisons. Leukemia.
2014;28:1472-1477.
4. Vannucchi AM, Lasho TL, Guglielmelli P, et al. Mutations
and prognosis in primary myelofibrosis. Leukemia.
2013;27:1861-1869.
5. Guglielmelli P, Lasho TL, Rotunno G, et al. The number of
prognostically detrimental mutations and prognosis in pri-
mary myelofibrosis: an international study of 797 patients.
Leukemia. 2014;28:1804-1810.
6. Tefferi A, Thiele J, Orazi A, et al. Proposals and rationale
for revision of the World Health Organization diagnostic
criteria for polycythemia vera, essential thrombocythemia,
Downloaded from https://academic.oup.com/ajcp/article-abstract/146/4/408/2236122 by guest on 22 June 2020
and primary myelofibrosis: recommendations from an
ad hoc international expert panel. Blood.
2007;110:1092-1097.
7. Swerdlow S, Campo E, Harris N, et al, eds. WHO
Classification of Tumours of Haematopoietic and Lymphoid
Tissues. 4th ed. Lyon, France: IARC; 2008:40-53.
8. Tefferi A, Thiele J, Vannucchi AM, et al. An overview on
CALR and CSF3R mutations and a proposal for revision of
WHO diagnostic criteria for myeloproliferative neoplasms.
Leukemia. 2014;28:1407-1413.
9. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to
the World Health Organization (WHO) classification of mye-
loid neoplasms and acute leukemia. Blood. 2016;127:2391-2405.
10. Silver RT, Chow W, Orazi A, et al. Evaluation of WHO cri-
teria for diagnosis of polycythemia vera: a prospective ana-
lysis. Blood. 2013;122:1881-1886.
11. Johansson PL, Safai-Kutti S, Kutti J. An elevated venous
haemoglobin concentration cannot be used as a surrogate
marker for absolute erythrocytosis: a study of patients with
polycythaemia vera and apparent polycythaemia. Br J
Haematol. 2005;129:701-705.
12. Barbui T, Thiele J, Carobbio A, et al. Discriminating be-
tween essential thrombocythemia and masked polycythemia
vera in JAK2 mutated patients. Am J Hematol.
2014;89:588-590.
13. Barbui T, Thiele J, Carobbio A, et al. Masked polycythemia
vera diagnosed according to WHO and BCSH classification.
Am J Hematol. 2014;89:199-202.
14. Barbui T, Thiele J, Vannucchi AM, et al. Rationale for revi-
sion and proposed changes of the WHO diagnostic criteria
for polycythemia vera, essential thrombocythemia and pri-
mary myelofibrosis. Blood Cancer J. 2015;5:e337.
15. Barbui T, Thiele J, Vannucchi AM, et al. Rethinking the
diagnostic criteria of polycythemia vera. Leukemia.
2014;28:1191-1195.
16. Thiele J, Kvasnicka HM, Mullauer L, et al. Essential
thrombocythemia versus early primary myelofibrosis: a multi-
center study to validate the WHO classification. Blood.
2011;117:5710-5718.
17. Wilkins BS, Erber WN, Bareford D, et al. Bone marrow path-
ology in essential thrombocythemia: interobserver reliability
and utility for identifying disease subtypes. Blood.
2008;111:60-70.
18. Kvasnicka HM. WHO classification of myeloproliferative
neoplasms (MPN): a critical update. Curr Hematol Malig Rep.
2013;8:333-341.
19. Passamonti F. How I treat polycythemia vera. Blood.
2012;120:275-284.
Am J Clin Pathol 2016;146:408-422 419
DOI: 10.1093/ajcp/aqw131
Busque et al / LABORATORY INVESTIGATION OF MPNS
20. Lee SH, Erber WN, Porwit A, et al. ICSH guidelines for the
standardization of bone marrow specimens and reports. Int J
Lab Hematol. 2008;30:349-364.
21. Thiele J, Kvasnicka HM, Facchetti F, et al. European consen-
sus on grading bone marrow fibrosis and assessment of cellu-
larity. Haematologica. 2005;90:1128-1132.
22. Raya JM, Montes-Moreno S, Acevedo A, et al. Pathology re-
porting of bone marrow biopsy in myelofibrosis: application of
the Delphi consensus process to the development of a standar-
dised diagnostic report. J Clin Pathol. 2014;67:620-625.
23. Barbui T, Thiele J, Passamonti F, et al. Initial bone marrow
reticulin fibrosis in polycythemia vera exerts an impact on
clinical outcome. Blood. 2012;119:2239-2241.
24. Campbell PJ, Bareford D, Erber WN, et al. Reticulin accu-
mulation in essential thrombocythemia: prognostic signifi-
cance and relationship to therapy. J Clin Oncol.
2009;27:2991-2999.
25. Thiele J, Kvasnicka HM, Vardiman JW, et al. Bone marrow
fibrosis and diagnosis of essential thrombocythemia. J Clin
Oncol. 2009;27:e220-e221.
26. Nowell PC, Hungerford DA. A minute chromosome in
human chronic granulocytic leukemia. Science.
1960;132:1497.
27. Rowley JD. A new consistent chromosomal abnormality in
chronic myelogenous leukaemia identified by quinacrine fluor-
escence and Giemsa staining. Nature. 1973;243:290-293.
28. Kurzrock R, Kantarjian HM, Druker BJ, et al. Philadelphia
chromosome–positive leukemias: from basic mechanisms to
molecular therapeutics. Ann Intern Med. 2003;138:819-830.
29. Levine RL, Wadleigh M, Cools J, et al. Activating mutation
in the tyrosine kinase JAK2 in polycythemia vera, essential
thrombocythemia, and myeloid metaplasia with myelofibro-
sis. Cancer Cell. 2005;7:387-397.
30. Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation
of the tyrosine kinase JAK2 in human myeloproliferative dis-
orders. Lancet. 2005;365:1054-1061.
31. James C, Ugo V, Le Couedic JP, et al. A unique clonal JAK2
mutation leading to constitutive signalling causes polycy-
thaemia vera. Nature. 2005;434:1144-1148.
32. Kralovics R, Passamonti F, Buser AS, et al. A gain-of-
function mutation of JAK2 in myeloproliferative disorders.
N Engl J Med. 2005;352:1779-1790.
33. Xu X, Zhang Q, Luo J, et al. JAK2(V617F): prevalence in a
large Chinese hospital population. Blood. 2007;109:339-342.
34. Wang YL, Vandris K, Jones A, et al. JAK2 mutations are present
in all cases of polycythemia vera. Leukemia. 2008;22:1289.
35. Mason J, Akiki S, Griffiths MJ. Pitfalls in molecular diagnosis
in haemato-oncology. J Clin Pathol. 2011;64:275-278.
36. Bench AJ, White HE, Foroni L, et al. Molecular diagnosis of
the myeloproliferative neoplasms: UK guidelines for the de-
tection of JAK2 V617F and other relevant mutations. Br J
Haematol. 2013;160:25-34.
37. Hammond E, Shaw K, Carnley B, et al. Quantitative deter-
mination of JAK2 V617F by TaqMan: an absolute measure
of averaged copies per cell that may be associated with the
different types of myeloproliferative disorders. J Mol Diagn.
2007;9:242-248.
38. Vannucchi AM, Antonioli E, Guglielmelli P, et al. Clinical
correlates of JAK2V617F presence or allele burden in myelo-
proliferative neoplasms: a critical reappraisal. Leukemia.
2008;22:1299-1307.
420 Am J Clin Pathol 2016;146:408-422
420 DOI: 10.1093/ajcp/aqw131
39. Silver RT, Vandris K, Wang YL, et al. JAK2(V617F) allele
burden in polycythemia vera correlates with grade of myelo-
fibrosis, but is not substantially affected by therapy. Leuk Res.
2011;35:177-182.
40. Passamonti F, Rumi E, Pietra D, et al. A prospective study of
338 patients with polycythemia vera: the impact of JAK2
(V617F) allele burden and leukocytosis on fibrotic or leu-
kemic disease transformation and vascular complications.
Leukemia. 2010;24:1574-1579.
41. Hussein K, Bock O, Theophile K, et al. JAK2(V617F) allele
burden discriminates essential thrombocythemia from a sub-
set of prefibrotic-stage primary myelofibrosis. Exp Hematol.
2009;37:1186-1193.
42. Kiladjian JJ, Cassinat B, Chevret S, et al. Pegylated
Downloaded from https://academic.oup.com/ajcp/article-abstract/146/4/408/2236122 by guest on 22 June 2020
interferon-alfa-2a induces complete hematologic and mo-
lecular responses with low toxicity in polycythemia vera.
Blood. 2008;112:3065-3072.
43. Guglielmelli P, Pacili A, Rotunno G, et al. Mutational pro-
file of patients with polycythemia vera treaated with ruxoliti-
nib in the phase III controlled response study [abstract].
Blood. 2015;126:4087.
44. Vannucchi A, Passamonti F, Al-Ali H, et al. Reductions in
JAK2 V617F allele burden with ruxolitinib treatment in
comfort-II, a phase 3 study comparing the safety and efficacy
of ruxolitinib with best available therapy (BAT) [abstract].
Blood. 2012;120:802.
45. Verstovsek S, Passamonti F, Rambaldi A, et al. A phase 2
study of ruxolitinib, an oral JAK1 and JAK2 Inhibitor, in pa-
tients with advanced polycythemia vera who are refractory or
intolerant to hydroxyurea. Cancer. 2014;120:513-520.
46. Scott LM, Tong W, Levine RL, et al. JAK2 exon 12 muta-
tions in polycythemia vera and idiopathic erythrocytosis. N
Engl J Med. 2007;356:459-468.
47. Scott LM, Beer PA, Bench AJ, et al. Prevalence of JAK2
V617F and exon 12 mutations in polycythaemia vera. Br J
Haematol. 2007;139:511-512.
48. McMullin MF. The classification and diagnosis of erythrocy-
tosis. Int J Lab Hematol. 2008;30:447-459.
49. Wong CL, Ma ES, Wang CL, et al. JAK2 V617F due to a
novel TG ! CT mutation at nucleotides 1848-1849: diag-
nostic implication. Leukemia. 2007;21:1344-1346.
50. Passamonti F, Elena C, Schnittger S, et al. Molecular and
clinical features of the myeloproliferative neoplasm associ-
ated with JAK2 exon 12 mutations. Blood.
2011;117:2813-2816.
51. Cazzola M. Somatic mutations of JAK2 exon 12 as a molecular
basis of erythrocytosis. Haematologica. 2007;92:1585-1589.
52. Jones AV, Cross NC, White HE, et al. Rapid identification
of JAK2 exon 12 mutations using high resolution melting
analysis. Haematologica. 2008;93:1560-1564.
53. Rapado I, Grande S, Albizua E, et al. High resolution melting
analysis for JAK2 exon 14 and exon 12 mutations: a diagnos-
tic tool for myeloproliferative neoplasms. J Mol Diagn.
2009;11:155-161.
54. Ugo V, Tondeur S, Menot ML, et al. Interlaboratory devel-
opment and validation of a HRM method applied to the de-
tection of JAK2 exon 12 mutations in polycythemia vera
patients. PLoS One. 2010;5:e8893.
55. Schnittger S, Bacher U, Haferlach C, et al. Detection of
JAK2 exon 12 mutations in 15 patients with JAK2V617F
negative polycythemia vera. Haematologica.
2009;94:414-418.
© American Society for Clinical Pathology

56. Laughlin TS, Moliterno AR, Stein BL, et al. Detection of
exon 12 mutations in the JAK2 gene: enhanced analytical
sensitivity using clamped PCR and nucleotide sequencing.
J Mol Diagn. 2010;12:278-282.
57. Szuber N, Lamontagne B, Busque L. Novel germline muta-
tions in the calreticulin gene: implications for the diagnosis
of myeloproliferative neoplasms. J Clin Pathol. 2016. doi:
10.1136/jclinpath-2016-203940 [epub ahead of print].
58. Pardanani AD, Levine RL, Lasho T, et al. MPL515 muta-
tions in myeloproliferative and other myeloid disorders: a
study of 1182 patients. Blood. 2006;108:3472-3476.
59. Beer PA, Campbell PJ, Scott LM, et al. MPL mutations in
myeloproliferative disorders: analysis of the PT-1 cohort.
Blood. 2008;112:141-149.
60. Hassan A, Dogara LG, Babadoko AA, et al. Coexistence of
JAK2 and BCR-ABL mutation in patient with myeloprolifer-
ative neoplasm. Niger Med J. 2015;56:74-76.
61. Harrison CN, Bareford D, Butt N, et al. Guideline for inves-
tigation and management of adults and children presenting
with a thrombocytosis. Br J Haematol. 2010;149:352-375.
62. Tefferi A, Vardiman JW. Classification and diagnosis of mye-
loproliferative neoplasms: the 2008 World Health
Organization criteria and point-of-care diagnostic algo-
rithms. Leukemia. 2008;22:14-22.
63. Tefferi A, Skoda R, Vardiman JW. Myeloproliferative neo-
plasms: contemporary diagnosis using histology and genetics.
Nat Rev Clin Oncol. 2009;6:627-637.
64. Campregher P, Helman R, Pereira W, et al. Genomic profile
of patients with triple negative (JAK2, CALR and MPL) es-
sential thrombocythemia and primary myelofibrosis [ab-
stract]. Blood. 2014;124:4589.
65. Cabagnols X, Favale F, Pasquier F, et al. Presence of atypical
thrombopoietin receptor (MPL) mutations in triple-negative
essential thrombocythemia patients. Blood. 2016;127:333-342.
66. Milosevic Feenstra JD, Nivarthi H, Gisslinger H, et al.
Whole-exome sequencing identifies novel MPL and JAK2
mutations in triple-negative myeloproliferative neoplasms.
Blood. 2016;127:325-332.
67. Zanjani ED, Lutton JD, Hoffman R, et al. Erythroid colony
formation by polycythemia vera bone marrow in vitro: de-
pendence on erythropoietin. J Clin Invest. 1977;59:841-848.
68. Ciaudo M, Hadjez JM, Teyssandier I, et al. Prognostic and diag-
nostic value of endogenous erythroid colony formation in essen-
tial thrombocythemia. Hematol Cell Ther. 1998;40:171-174.
69. Bai J, Shao ZH, Liu H, et al. Endogenous erythroid colony
assay in patients with polycythemia vera and its clinical sig-
nificance. Chin Med J (Engl). 2004;117:668-672.
70. Gangat N, Caramazza D, Vaidya R, et al. DIPSS plus: a
refined dynamic international prognostic scoring system for
primary myelofibrosis that incorporates prognostic informa-
tion from karyotype, platelet count, and transfusion status.
J Clin Oncol. 2011;11392-11397.
71. Van den Berghe H, Cassiman JJ, David G, et al. Distinct
haematological disorder with deletion of long arm of no. 5
chromosome. Nature. 1974;251:437-438.
72. Papaemmanuil E, Gerstung M, Malcovati L, et al. Clinical
and biological implications of driver mutations in myelodys-
plastic syndromes. Blood. 2013;122:3616-3627, quiz 3699.
73. Mardis ER, Ding L, Dooling DJ, et al. Recurring mutations
found by sequencing an acute myeloid leukemia genome. N
Engl J Med. 2009;361:1058-1066.
© American Society for Clinical Pathology
421
AJCP / REVIEW ARTICLE
74. Lundberg P, Karow A, Nienhold R, et al. Clonal evolution
and clinical correlates of somatic mutations in myeloprolifer-
ative neoplasms. Blood. 2014;123:2220-2228.
75. Tefferi A, Lasho T, Finke C, et al. Targeted next-generation
sequencing in polycythemia vera and essential thrombocyth-
emia [abstract]. Blood. 2015;126:354.
76. Busque L, Patel JP, Figueroa ME, et al. Recurrent somatic
TET2 mutations in normal elderly individuals with clonal
hematopoiesis. Nat Genet. 2012;44:1179-1181.
77. Xie M, Lu C, Wang J, et al. Age-related mutations associated
with clonal hematopoietic expansion and malignancies. Nat
Med. 2014;20:1472-1478.
78. Jaiswal S, Fontanillas P, Flannick J, et al. Age-related clonal
Downloaded from https://academic.oup.com/ajcp/article-abstract/146/4/408/2236122 by guest on 22 June 2020
hematopoiesis associated with adverse outcomes. N Engl J
Med. 2014;371:2488-2498.
79. Genovese G, Kahler AK, Handsaker RE, et al. Clonal hem-
atopoiesis and blood-cancer risk inferred from blood DNA
sequence. N Engl J Med. 2014;371:2477-2487.
80. Tefferi A, Jimma T, Gangat N, et al. Predictors of greater
than 80% 2-year mortality in primary myelofibrosis: a mayo
clinic study of 884 karyotypically annotated patients. Blood.
2011;118:4595-4598.
81. Caramazza D, Begna KH, Gangat N, et al. Refined
cytogenetic-risk categorization for overall and leukemia-free
survival in primary myelofibrosis: a single center study of 433
patients. Leukemia. 2011;25:82-88.
82. Hussein K, Pardanani AD, Van Dyke DL, et al. International
Prognostic Scoring System–independent cytogenetic risk
categorization in primary myelofibrosis. Blood.
2010;115:496-499.
83. Tefferi A, Guglielmelli P, Finke C, et al. Integration of muta-
tions and karyotype towards a genetics-based prognostic scor-
ing system (GPSS) for primary myelofibrosis [abstract].
Blood. 2014;124:406.
84. Tefferi A, Wassie E, Begna K, et al. Revised cytogenic risk
stratification in primary myelofibrosis: a mayo clinic study of
903 patients [abstract]. Blood. 2014;124:631.
85. Vannucchi A, Guglielmelli P, Rotunno G, et al. Mutation-
enhanced international prognostic scoring system (MIPSS)
for primary myelofibrosis: an AGIMM & IWG-MRT project
[abstract]. Blood. 2014;124:405.
86. van Genderen PJ, Budde U, Michiels JJ, et al. The reduction
of large von Willebrand factor multimers in plasma in essen-
tial thrombocythaemia is related to the platelet count. Br J
Haematol. 1996;93:962-965.
87. van Genderen PJ, Prins FJ, Lucas IS, et al. Decreased half-life
time of plasma von Willebrand factor collagen binding activity in
essential thrombocythaemia: normalization after cytoreduction of
the increased platelet count. Br J Haematol. 1997;99:832-836.
88. Budde U, Scharf RE, Franke P, et al. Elevated platelet count
as a cause of abnormal von Willebrand factor multimer distri-
bution in plasma. Blood. 1993;82:1749-1757.
89. Tsantes AE, Nikolopoulos GK, Tsirigotis P, et al. Direct evi-
dence for normalization of platelet function resulting from
platelet count reduction in essential thrombocythemia. Blood
Coagul Fibrinolysis. 2011;22:457-462.
90. Tefferi A, Smock KJ, Divgi AB. Polycythemia vera–associ-
ated acquired von Willebrand syndrome despite near-normal
platelet count. Am J Hematol. 2010;85:545.
91. Kumar S, Pruthi RK, Nichols WL. Acquired von Willebrand
disease. Mayo Clin Proc. 2002;77:181-187.
Am J Clin Pathol 2016;146:408-422 421
DOI: 10.1093/ajcp/aqw131
Busque et al / LABORATORY INVESTIGATION OF MPNS

Appendix 1
Summary of the Key Characteristic Bone Marrow Features for MPNs7
Clinical Entity
Polycythemia vera
Essential thrombocythemia
Bone Marrow Features
The major features are attributable to proliferation in the erythroid, granulocytic, and megakaryocytic lineages.
Bone marrow cellularity can range from 30% to 100% with a median cellularity of about 80%. In general, the
bone marrow biopsy specimen is hypercellular for patient’s age.
Reticulin stains usually show a normal reticulin fiber network in about 80% of patients, and in a minor fraction
of patients, fibrosis grade 1 may be seen. Twenty percent of patients may develop increased reticulin and
borderline to mild collagen fibrosis after long-term disease.
Stainable iron is lacking in more than 95% of cases.
The features of exon 12 mutated PV include an increased erythrocyte proliferation with accompanying
increased granulocytic and megakaryocytic proliferation.
In most cases, the bone marrow biopsy specimen is normocellular or moderately hypercellular for the

Downloaded from https://academic.oup.com/ajcp/article-abstract/146/4/408/2236122 by guest on 22 June 2020

Prefibrotic and early stage PMF This early stage is characterized by bone marrow hypercellularity with an increase in the number of neutro-
Primary myelofibrosis
MPN, myeloproliferative neoplasm; PMF, primary myelofibrosis; PV, polycythemia vera.
patient’s age.
The most pronounced abnormality is a significant proliferation of megakaryocytes with predominance of large
forms with abundant, mature cytoplasm. The nucleus is deeply lobulated and hyperlobulated (stag-horn
like). These large megakaryocytes are usually dispersed throughout the bone marrow but might also occur
in loose clusters.
Granulopoiesis and erythropoiesis should be present in accordance to age.
phils and abnormal megakaryocytes with reduction of erythropoiesis.
The megakaryocytes in PMF are characterized by marked atypia, vary in size and shape, and often form dense
clusters that are often adjacent to the bone marrow vascular sinuses and the bone trabeculae.
This stage is characterized by the obvious presence of fibrosis.
With progression of fibrosis, bone marrow is becoming hypocellular with patches of active hematopoiesis.
Atypical megakaryocytes are often the most prominent findings, occurring in large clusters often within vas-
cular sinuses.
Although foci immature cells might be noticeable, myoblasts account for <10% of the bone marrow cells.
Immunohistochemical finding of increased number of CD34þ cells indicates an accelerated phase of the dis-
ease and the presence of  20% of CD34þ cells is considered a transformation to acute leukemia.
In some patients, increased numbers of blasts at transformation may be seen in blood, but bone marrow will
show only hypocellular fibrosis.

422 Am J Clin Pathol 2016;146:408-422 © American Society for Clinical Pathology

422 DOI: 10.1093/ajcp/aqw131