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REVIEW ARTICLE INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY
REVIEW ARTICLE
INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY

The classification and diagnosis of erythrocytosis

M. F. M C MULLIN

Department of Haematology, The Queen’s University, Belfast, UK

Correspondence:

Prof. M. F. McMullin, Department of Haematology, ‘C’ Floor, Belfast City Hospital, Queen’s University Belfast, Lisburn Road, Belfast BT9 7AB, UK. Tel.: +44 289 026 3732; Fax: +44 289 026 3870; E-mail: m.mcmullin@qub.ac.uk

doi:10.1111/j.1751-553X.2008.01102.x

Received 27 August 2008;

accepted for publication 27 August

2008

Keywords Polycythaemia vera, erythrocytosis, erythropoietin

SUMMARY

An absolute erythrocytosis is present when the red cell mass is raised and the haematocrit is elevated above prescribed limits. Causes of an absolute erythrocytosis can be primary where there is an intrinsic problem in the bone marrow and secondary where there an event outside the bone marrow driving erythropoiesis. This can further be divided into congenital and acquired causes. There remain an unexplained group idiopathic erythrocytosis. Investigation com- mencing with thorough history taking and examination and then investigation depending on initial features is required. Clear simple criteria for polycythaemia vera are now defined. Those who do not fulfil these criteria require further investigation depending on the clinical scenario and initial results. The erythropoietin level provides some guidance as to the direction in which to proceed and the order and extent of investigation necessary in an individual patient. It should thus be possible to make an accurate diagnosis in the majority of patients.

an Hct above the normal range. The normal range is defined and quoted as 0.45 ± 0.05 in males and 0.41 ± 0.05 in females and thus there will be varia- tion beyond this range which may or may not be abnormal. An Hct >0.51 in a male and 0.48 in a female is judged to be above the normal range and thus elevated. To establish if there is a definite increase in red cell mass as reflected by an elevated haemoglobin/Hct a red cell mass can be measured. This was previously expressed in terms of total body weight (ml/kg) but this leads to low measured values and high predicted normal values in the obese as fat is relatively avascular. Therefore measurements are related to formula for prediction

447

INTRODUCTION

Definition of an erythrocytosis

Red cells make up the majority of cells in the blood and as such influence the viscosity. Raised viscosity may lead to pathology. An increase in the quantity of red cells or red cell mass is therefore an erythro- cytosis. An elevated haemoglobin or haematocrit (Hct) raises the possibility of an erythrocytosis. The Hct reflects whole blood viscosity most accurately (Pearson et al. , 1981) but it can be underestimated by some analysers in the presence of iron deficiency (Guthrie & Pearson, 1982). An individual may have

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of normal RCM using height and weight and based on measurements on normal populations (Pearson et al., 1995). Using these formulae a RCM greater that 125% of predicted for an individuals height and weight is judged to be abnormal and thus the individual has an absolute erythrocytosis. Those who have a raised Hct but fall below this level have an apparent erythrocytosis. The higher the Hct the more likely the individual on measuring RCM will be shown to have a true ery- throcytosis and it has been shown that a male with a Hct of 0.60 and 0.56 in a female will always have a true erythrocytosis and it can then be judged that measurement of RCM is unnecessary in these individ- uals (Johansson, Soodabeh & Kutti, 2005). In this instance an absolute erythrocytosis can be assumed. Thus an absolute erythrocytosis is defined as a RCM above 125% of predicted. This can be assumed if the Hct is above 0.60 in a male and 0.56 in a female. It is suspected with an Hct above 0.51 in a male or 0.48 in a female but further tests are required for confirmation of erythrocytosis and related pathol- ogy.

CLASSIFICATION OF ERYTHROCYTOSIS

Having established that an erythrocytosis is present, it is then necessary to establish the aetiology. Initial classification is on the basis of whether it is a primary process where the bone marrow is the unaided source of production of the excess red cell production or sec- ondary where red cell production is driven by some other process and finally an idiopathic group where the aetiology cannot be established (Table 1).

Primary erythrocytosis

Table 1. Classification of the absolute erythrocytoses

Primary erythrocytosis Polycythaemia vera Secondary erythrocytosis Congenital Erythropoietin receptor mediated High oxygen-affinity haemoglobin Bisphosphoglycerate mutase deficiency VHL gene mutation (Chuvash erythrocytosis) PHD 2 mutations HIF -2a mutations Acquired EPO mediated Hypoxia driven Central hypoxic process Chronic lung disease Right-to-left cardiopulmonary vascular shunts Carbon monoxide poisoning Smoker’s erythrocytosis Hypoventilation syndromes including sleep apnoea (high-altitude habitat) Local renal hypoxia Renal artery stenosis End-stage renal disease Hydronephrosis Renal cysts (polycystic kidney disease) Postrenal transplant erythrocytosis Pathologic EPO production Tumours Cerebellar haemangioblastoma Meningioma Parathyroid carcinoma/adenomas Hepatocellular carcinoma Renal cell cancer Pheochromocytoma Uterine leiomyomas Exogenous EPO Drug associated Erythropoietin administration Androgen administration Idiopathic erythrocytosis.

The term primary erythrocytosis is applied when there is an intrinsic defect of the erythropoietic component of the bone marrow. The main cause of primary ery- throcytosis is polycythaemia vera (PV) where the bone marrow is producing too many red cells and also frequently too many white cells and platelets. The presence of clonal abnormalities as shown by G6PD heterozygotes (Adamson et al. , 1976) and cytogenetic abnormalities demonstrated the presence of an abnor- mal clone in the bone marrow which leads to the dis- ease. The JAK 2 V617F mutation which has been

EPO, erythropoietin.

demonstrated in the majority of patients with PV (Baxter et al.,2005; James et al., 2005; Kralovics et al., 2005; Levine et al., 2005) shows that a clone is pres- ent. The erythropoietin (EPO) level in such patients is usually below the normal range supporting the con- tention that a primary bone marrow problem leads to the erythrocytosis. More recently Scott et al. (2007) described mutations in exon 12 of JAK2 in patients with PV who did not have the V617F mutation. These

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patients have a predominantly erythroid phenotype. An increasing number of these mutations are being described (Cazzola, 2007) and they clone may be small, however, their presence demonstrates clonal disease and they fall within the remit of PV.

Secondary erythrocytosis

Secondary erythrocytoses are those which result from

a mechanism other than intrinsic to the bone marrow.

Classification can be further subdivided into congeni- tal or acquired processes. Each of these categories can then be further subdivided on the basis of the patho-

logical process producing the erythrocytosis.

CONGENITAL

Congenital causes of erythrocytosis are those in which

a genetic abnormality has been identified. These cases

usually present at a young age as obviously the muta- tion has been present from birth. There will often be

a family history as the defect is inherited although the possibility of new mutations can arise. The patients who appear to have a congenital cause fall into two sets, those with EPO levels below the normal range who can be assumed to have defects of the EPO signalling pathway and those with inap- propriately normal for the haemoglobin level or ele-

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vated EPO levels who may have defects of the oxygen sensing pathway.

EPO receptor mutations

EPO attaches to its receptor and initiates a series of events which leads to gene transcription and ulti- mately production of more red cells. This process is switched off whenever sufficient red cells have been produced by binding of SHP-1. Truncation of the EPO receptor results in failure of attachment of SHP-1, ongoing production of red cells and thus erythrocyto- sis. Such a mutation was first described by de la Chap- elle who observed a mutation of G to A at base 6002 in the EPO receptor of a Finnish kindred (de La Chap- elle, Traskelin & Juvonen, 1993). This results in a stop codon and a 70 amino acid truncation of the EPO receptor. The same mutation was described arising de novo in a teenage English male (Percy et al., 1998). In total now at least 11 mutations have been described in the EPO receptor associated with erythro- cytosis. All the described mutations are listed in Table 2 with references.

High oxygen-affinity haemoglobins

During oxygenation haem iron moves into the plane of the porphyrin ring and this results in change in

Table 2. Erythropoietin receptor mutations ( Source : Percy, 2007b)

Mutation

Nucleotide position

Mutation type

Truncation

Reference

GT

5881

Termination

110a

Arcasoy et al. (2002) Petersen et al. (2004) Kralovics et al. (1997a) Kralovics, Sokol and Prchal (1998), Cario et al. (2006) Kralovics et al. (1997b) Watowich et al. (1999) Sokol et al. (1995) Kralovics et al. (1997a,b), Arcasoy et al. (1997) Furukawa et al. (1997) de La Chapelle, Traskelin and Juvonen (1993), Percy et al. (1998) Cario et al. (2006)

Deletion

5938–5941

Frameshift

58aa

GT

5959

Termination

84aa

CG

5964

Termination

83aa

Insertion T

5967

Frameshift

65aa

Tandem duplication

5968–5975

Frameshift

79aa

Insertion G

5974

Frameshift

64aa

Deletion

5985–5991

Frameshift

59aa

CT

5986

Termination

74aa

G

A

6002

Termination

70aa

G

A

6003

Termination

70aa

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the shape of the globin chains. A change in one globin chain will facilitate the uptake of oxygen by the other chains. The capacity of a haemoglobin to deliver oxygen to the tissues is expressed by the shape of the haemoglobin-oxygen dissociation curve and shift of the curve to the left indicates an abnor- mal haemoglobin which has increased affinity for oxygen (so called high oxygen affinity haemoglo- bin). The first high affinity haemoglobin associated with erythrocytosis was reported in 1966, Haemoglo- bin Chesapeake, by Charache, Weatherall and Clegg (1966). From that time over 90 haemoglobin vari- ants have been described where there is an altered oxygen affinity. (For full current list see human glo- bin gene server under human haemoglobin mutants http://globin.cse.psu.edu). Defects of a-globin and b-globin genes are described. High oxygen affinity haemoglobins may be stable or unstable, and as they do not release oxygen readily to the tissues the resulting hypoxia drives EPO production and thus erythrocytosis. Erythrocytosis is accompanied by chronic haemolysis where the haemoglobin variant is unstable. Haemoglobin variants can be detected by electro- phoresis but 20–25% are electrophorectically silent. The presence of a left shifted oxygen dissociation curve as demonstrated by measuring the P50 is a means of screening for a high affinity haemoglobin. The globin genes can also be fully sequenced although this may be technically difficult in the case of the a- globin genes (Means, 2004).

Bisphosphoglycerate mutase deficiency

The proximal limb of the 2, 3 bisphosphoglycerate (2, 3-BPG) shunt involves conversion of 1, 3-BPG to 2, 3-BPG. This reaction is catalysed by the enzyme bisphosphoglycerate mutase. 2, 3-BPG binds to the haemoglobin tetramer and converts the haemoglobin molecule to a low oxygen affinity state. The binding of 2, 3-BPG therefore shifts the oxygen affinity curve to the right. Lack of 2, 3-BPG would shift the oxygen affinity curve to the left as the haemoglobin tetramer is kept in a high oxygen affinity state. Deficiency of bisphosphoglycerate mutase would lead to low 2, 3- BPG levels and result in a left shifted oxygen affinity curve, a high affinity haemoglobin and a compensa- tory erythrocytosis. Complete deficiency of the

enzyme was first described by Rosa et al. (1978) and rare cases have been described since with both auto- somal dominant (Galacteros et al., 1984) and autoso- mal recessive inheritance (Hoyer et al., 2004). Bisphosphoglycerate mutase deficiency is thus a rare cause of erythrocytosis with a left shifted oxygen dis- sociation curve.

Mutations in the genes in the oxygen sensing pathway

Chuvash erythrocytosis (VHL gene mutation)

A large group of individuals were identified in the Chuvash region of Russia all of whom had erythrocy- tosis. Investigation identified that affected individuals were all homozygous for a single mutation C598T, amino acid 200 arginine changed to tryptophan in the von Hippel Lindau (VHL ) gene (Ang et al., 2002). The VHL gene has an important role in hypoxia sensing where cells sense a decrease in oxygen and allow the organism to adapt. The protein hypoxia inducible fac- tor (HIF-a) is central to this process. It exists in three isoforms HIF-1a , HIF-2a and HIF-3a. Oxygen activates prolyl hydroxylase which then hydroxylates HIF- a. This leads to the binding of VHL protein and ubiquiti- nation of HIF- a and destruction of the proteins. In contrast, in hypoxia, HIF- a associates with its beta subunit, the complex binds to hypoxia responsive ele- ments within the genome leading to the activation of downstream genes and increased production of pro- teins such as EPO. A mutation in the VHL gene would disrupt the normal mechanism, drive HIF- a down the hypoxia pathway ultimately resulting in increased EPO production and erythrocytosis. Eight families have been identified from outside the Chuvash area where individuals with erythrocytosis were homozy- gous for the ‘Chuvash’ mutation (Percy et al., 2003). All these families were of Pakistani or Bangladeshi origin. Another large cluster with the same mutation has been identified in the island of Ischia, Italy (Perrotta et al. , 2006). It appears that all these sub- jects may have had a single founder (Liu et al., 2004). A number of individuals with erythrocytosis have also been described who are compound het- erozygotes for the Chuvash mutation and other VHL mutations. (Percy, 2007b) Interestingly there are

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also a number of individual who are heterozygous for one VHL mutation, the other allele is active and erythrocytosis is present. Some other mechanism must be present as well in these cases to result in erythrocytosis.

PHD2 mutations

In normoxia, the prolyl hydroxlases (PHDs) hydrox- late the a-subunits of HIF which is the first step in the degradation of HIF. A mutation in one of the PHDs could interfere with this process and drive HIF down the hypoxia pathway and lead to increased EPO and erythrocytosis. A family who had a very mild erythro- cytosis has been described with to a mutation in the PHD2 gene resulting in a proline to arginine change at codon 317 (Percy et al., 2006). A further family with a PHD2 mutation resulting in an arginine to histidine change at position 317 has been shown to have abnormal activity in vitro (Percy et al. , 2007a).

HIF-2a mutations

The transcription factor HIF2a is hydroxylated by the PHDs and then degraded by VHL. In hypoxic condi- tions hydroxylation is inhibited. A family with ery- throcytosis and elevated EPO levels in three generations was found to have a mutation of the oxygen degradation domain of HIF -2a, resulting in a change of glycine to tryptophan at amino acid 537. In vitro studies showed that this mutation would result in major change in function of the protein (Percy et al., 2008). Further mutations in HIF -2a have been described. Thus a number of different genes in the oxygen sensing pathway have been shown to be mutated and cause erythrocytosis. A diagram of these proteins and their relationships in normoxia and hypoxia is shown in Figure 1.

ACQUIRED ERYTHROCYTOSIS

There are a number of different ways where an ery- throcytosis can result from an acquired EPO mediated process. This can be divided up into the categories of central or local renal hypoxic processes, excess EPO production either due to a pathological process or exogenous administration.

M. F. MCMULLIN ERYTHROCYTOSIS 451 Normoxia Hypoxia PHD PHD O H O H N N
M. F. MCMULLIN
ERYTHROCYTOSIS 451
Normoxia
Hypoxia
PHD
PHD
O H
O H
N
N
HIF-2α
HIF-2α
O
O 2
2
VHL
O
O
H
O H
N
N
HIF-2α
HIF-2α
HIF-2α
HRE
EPO

Ubiquitination

Figure 1. Diagram of the relationship between the proteins PHD2, VHL and HIF-2 a in normoxia where the proteins are ubiquitinated and destroyed and hypoxia where erythropoeitin production results.

Central hypoxia driven processes

Any process where there is reduced oxygen supply and thus hypoxia will lead to stimulation of EPO pro- duction and ultimately erythrocytosis. This can be due to high altitude (Leo´ n-Velarde et al., 1991) chronic lung disease (Skouby, 1976; Wedzicha et al., 1985), in smokers (Calverley et al., 1982; Spiers & Levine, 1983), due to nocturnal-hypoxia (Moore-Gillon et al., 1986) and carbon monoxide poisoning (DiMarco,

1988).

Congenital cyanotic heart disease can cause a fail- ure to maintain tissue oxygen delivery and a compen- satory erythrocytosis. The heart defects fall into two main categories. The first group have absent or poorly developed central pulmonary arteries and pulmonary blood flow via collateral arteries from the aorta or branches and a large right to left shunt (pulmonary atresia with a ventricular septal defect and major aor- to-pulmonary collateral arteries). The second group with pulmonary vascular disease where the intra-pul- monary arterioles and capillaries have undergone

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obliterative changes secondary to high pressure and high flow shunts in a variety of simple and complex congenital heart defects and occasionally as a result of palliative surgical shunting procedures (Eisenmenger syndrome). The resultant hypoxia drives EPO produc- tion and a compensatory erythrocytosis results (Ha¨ ga¨ et al., 1987).

Local renal hypoxia driven processes

Erythropoietin is produced by the kidney. A local problem in the kidney leading to hypoxia would lead to increased EPO production and erythrocytosis. Pro- cesses which can have this result include renal artery stenosis (Hudgson, Pearce & Yeates, 1967) and end stage renal disease (Shalhoub et al., 1982; Shih & Leu, 1993). In renal disorders erythrocytosis has also been described with polycystic kidney disease where the cysts may be responsible for EPO production (Chandra et al.,1985). Erythrocytosis is described in 10–15% of renal transplant recipients between 8 and 24 months post-transplant, a condition referred to as postrenal transplant erythrocytosis (PTE; Vlahakos et al., 2003). The pathogenesis of PTE is thought to be multifacto- rial involving as well as increased EPO production, abnormal erythroid precursor sensitivity to EPO and abnormal erythroid sensitivity to angiotensin II or altered angiotensin II production and an elevated con- centration of insulin-like growth factor I and its bind- ing proteins (Gaston, Julien & Curtis, 1994; Julian et al., 1998; Brox et al., 1998).

Pathological EPO production

Erythropoietin production has been documented by a number of malignant tumours and nonmalignant tumours. Raised EPO levels in patients with erythro- cytosis and a tumour raise the suspicion. High expres- sion of EPO mRNA in the tumour tissue and correction of the erythrocytosis with a fall in EPO lev- els with removal of the tumour confirm the link between the tumour and erythrocytosis. Examples of conditions in which it has been described are cerebel- lar haemangioblastoma (Trimble et al., 1991), menin- gioma (Bruneval et al., 1993), parathyroid adenomas (Godeau et al. , 1981), hepatocellular carcinoma (Muta et al., 1994; Matsuyama et al., 2000), renal cell carci-

noma where there may be associated VHL mutations (Hama et al. , 2005; Rad et al., 2008) pheochromocy- toma (Dre´ nou et al., 1995) and uterine leiomyomas (Yoshida et al., 1999; Suzuki et al., 2001).

Exogenous EPO administration

There are situations where an erythrocytosis may improve performance for instance in endurance sports. Therefore athletes may self administer EPO in order to enhance performance and this will result in erythrocytosis. Various strategies are available to detect this practice (Robinson et al., 2006) although it can be challenging. Erythrocytosis has also been asso- ciated with androgen administration (Dickerman et al.,

1999).

IDIOPATHIC ERYTHROCYTOSIS

There remain a group of patients in whom no cause for their erythrocytosis has been identified. This group are termed idiopathic erythrocytosis on this basis. These patients can be divided on the basis of their EPO levels. Approximately one-third of such a group of subjects will have EPO levels below the normal range and are likely to have defects in the EPO signalling pathway. Two thirds have inappropri- ately normal for elevated haemoglobin or raised EPO. This group are likely to have defects in the oxygen sensing pathway (Figure 1). If a cause was to be identified in one of these patients then their classification would move to the secondary group.

DIAGNOSIS

History and examination

The initial step in the investigation and diagnosis of any patient referred with erythrocytosis must be tak- ing a thorough history and carrying out a rigorous and focused examination. Bearing in mind the causes of erythrocytosis, history should include questioning and exploration of all likely causes. It may reveal rele- vant features, for instance, a history of snoring may suggest nocturnal hypoxaemia. Examination should cover all systems and again look to explore for sec- ondary causes of erythrocytosis.

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Diagnostic criteria for polycythaemia vera

Diagnostic criteria for PV were originally devised by

the polycythemia vera study group, in the United States in the 1960’s (Berk et al., 1995). These were devised as criteria for patients to enter trials of treat- ment for PV. They required increased red cell mass and additional criteria. As such they were designed to

be restrictive in that it was necessary that all patients

who entered trials clearly had PV. They may thus exclude patients who actually have PV. They also include tests which have become obsolete such as an

increased leucocyte alkaline phosphatase or are not in widespread use such as unbound B 12 binding capacity. The World Health Organisation (WHO) introduced cri- teria for the diagnosis in 2001 (Pierre et al., 2001). These accepted an increased red cell mass or an Hb above 18.5 g/dl in men or 16.5 g/dl in women as well

as additional criteria. These criteria are over permis- sive as they allow a diagnosis with an Hb, which would be considered as within the normal range.

A White cell count (WCC) of 12 · 10 9 /l is allowed

without specifying the type of white cell. This would allow a lymphocytosis as a criterion which was obvi- ously not the objective. Some of the criteria were tests, which are only available in a few laboratories and not validated such as endogenous erythroid col- ony formation. In the UK Pearson defined criteria which where designed to include only patients who were likely to have the diagnosis and be able to make

a diagnosis using tests which were widely available. These criteria were adapted in the British Committee for Standards in Haematology (BCSH) guidelines for diagnosis and management (McMullin et al., 2005). The discovery of JAK 2 mutations in the majority of

patients with PV shows simply the presence of clonal disease. This has allowed revision and simplification of the diagnostic criteria in those with a JAK 2 mutation. This has been adopted by the BCSH and the criteria are outlined in Table 3. Basically the presence of a raised Hb and a JAK 2 mutation is sufficient to make the diagnosis. In those without a JAK 2 mutation more complex criteria are still required. However, the find- ing of exon 12 JAK 2 mutations in increasing numbers

of

patients with features of PV may eventually result

in

finding of a mutation in all patients and thus ren-

der criteria for JAK 2 negative PV obsolete. The WHO has also revised their criteria in view of the presence

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Table 3. Diagnostic criteria for polycythaemia vera

JAK2-positive polycythaemia vera A1. High haematocrit (>0.52 in men,>0.48 in women) OR raised red cell mass (>25% above predicted) A2. Mutation in JAK2 Diagnosis requires both criteria to be present JAK2 -negative polycythaemia vera A1. Raised red cell mass (>25% above predicted) OR haematocrit 0.60 in men, 0.56 in women A2. Absence of a mutation in JAK2 A3. No cause of secondary erythrocytosis A4. Palpable splenomegaly A5. Presence of an acquired genetic abnormality (excluding BCR-ABL ) in the haematopoietic cells B1. Thrombocytosis (platelet count >450 · 10 9 /l) B2. Neutrophil leucocytosis (neutrophil count >10 · 10 9 /l in nonsmokers, >12.5 · 10 9 /l in smokers) B3. Radiological evidence of splenomegaly B4. Endogenous erythroid colonies or low serum erythropoietin Diagnosis requires A1 + A2 + A3 + either another A or two B criteria

of JAK 2 mutations in the majority of patients. They also require a raised Hb or red cell mass and a JAK 2 mutation but they also require a minor criteria, bone marrow biopsy changes, and serum EPO below the normal range or in vitro endogenous erythroid colony formation (Tefferi et al., 2007). It is debatable whether this is necessary or if physicians and patients will be persuaded to do the extra tests.

INVESTIGATIONS

The diagnostic criteria for PV outlined in Table 3 require only a few tests and there are a number of tests which may be carried out in patients with a likely diagnosis of PV. The rationale and utility of some of these tests will be considered below.

Haemoglobin/haematocrit

A raised Hct is the first criteria for a diagnosis of PV. In PV the issue is the viscosity: the greater the viscosity, the greater the sluggishness of the blood flow and thus the greater the problem. The Hb is a measure of the total amount of haemoglobin pres- ent. The Hct or packed cell volume is a measure of

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the total number of cells present of which the vast majority are red cells and is the most accurate way

of measuring the viscosity. The Hb is used as a sub-

stitute measure of viscosity but there is not always

a direct correlation between the two measurements.

Using the Hb as a measure of viscosity may under- estimate it particularly in iron deficient situations. The WHO criteria which use the Hb only may

therefore underestimate the viscosity and the extent

of the problem and its management.

Red cell mass

The measurement of red cell mass is not required in those who have a raised Hct and a JAK2 mutation as they have sufficient to make a diagnosis. However, in those who are JAK2 negative there is a need to mea-

sure the red cell mass to document if there is a true increase before embarking on further investigation. This is well demonstrated by the study of Johansson, Soodabeh and Kutti (2005) who considered Hb and Hct as surrogate markers for an erythrocytosis in a group of individuals with known red cell mass. In a group who had RCM measured and known Hb and Hct and therefore could clearly be described as abso- lute erythrocytosis or apparent erythrocytosis, they found that only a 35% of male PV patients were shown to have an absolute erythrocytosis as defined by a Hb above 18.5 g/dl, while 14% of individuals with an apparent erythrocytosis would have been mis-diagnosed as having an absolute erythrocytosis. Similarly in females, an Hb of >16.5 g/dl would pre- dict an absolute erythrocytosis in only 65% of PV patients whereas 35% of apparent erythrocytosis would be classified as absolute erythrocytosis on the basis of Hb alone. A RCM, an expensive test at the outset, may avoid lifelong follow-up and treatment in someone who ends up in the wrong category because this test was not done in the first instance. It should be performed

at the outset to confirm an elevated red cell mass in a

patient who is JAK 2 negative.

JAK2 mutations

A JAK 2 V617F screen is a relatively simple test to

carry out. Methods use sensitive polymerase chain reaction (PCR) based technology. Several methods are

available which are based on allele-specific PCR amplification of the mutant band or by removal of a restriction enzyme recognition site by the mutation

(Campbell et al., 2006). The most straightforward way

is to isolate DNA from whole blood leucocytes. This

can be done with an EDTA sample taken for full blood count measurement. This has sensitivity for detection of the mutation of about 20 to 30%. Isolation of mononuclear cells using Ficoll–Paque gradient separa- tion and subsequent separation into granulocyte and lymphocyte lineages allows examination of the mye- loid lineage for the mutation. Using this type of sepa- ration and amplification refractory mutation screening (ARMS) PCR, it may be possible to detect a mutant clone down to a level or sensitivity of <1% (Chen et al., 2007). These methods require separation of granulocytes from a fresh blood sample which may be much more difficult to organise in a routine clinical situation (McLornan, Percy & McMullin, 2006). These methods allow determination as to whether an indi- vidual has a totally mutated JAK2 or a mixture of wild type and mutated, described as homozygous or heterozygous. The quantity of the mutant present may also be of importance. Vannucchi et al. (2007) have shown that homozygous patients with PV have higher counts at presentation and more symptoms and more splenomegaly. This may be important for prognosis and in the future it is likely that measure- ment of the quantity of mutant present will also be used to monitor the response to therapy. Methods like pyrosequencing are in development and will be used for these purposes. Detection of a number of exon 12 JAK 2 mutations has been shown by isolation of granulocytes and lym- phocytes and performing allele-specific PCR using

granulocyte DNA (Scott et al., 2007). This also requires

a fresh sample to be transported rapidly to a labora-

tory which can separate granulocytes and is challeng- ing for routine practice in all hospitals. These methods can only detect known mutations where primers are available. Methods are in development to screen the whole of exon 12. In summary therefore in a patient with an elevated Hb or Hct JAK 2 V617F can be screened using an EDTA sample. Negative patients who otherwise appear to have PV may merit more intensive investigation by more sensitive methodology for smaller clones or for exon 12 mutations.

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ERYTHROCYTOSIS 455

EPO levels

EPO controls the production of red cells and is pro- duced by the kidney. It is synthesised in response to hypoxia resulting in the increased production of red cells. In contrast in PV, EPO levels should be sup- pressed as the increased red cell production is inher- ent from the abnormal clone in the bone marrow. It is currently measured by immunoassay or radio immunoassay. These assays are only available in a few laboratories. They are not standardised between laboratories although there is now in the United King- dom a National quality control system monitoring the assays in use. The Primary thrombocythaemia-1 trial followed up 806 patients with essential thrombocythaemia. Sam- ples were stored and when the JAK 2 mutation was discovered it was possible to look at the mutation sta- tus and EPO levels in this group. There were clearly two groups, those who were JAK 2 positive (53%) and those who were JAK 2 negative (47%). EPO levels were lower in the JAK 2 positive patients who had a disease which have more features of PV (Campbell et al., 2005). This implies that while a low EPO level may back up a diagnosis of PV it will not discriminate between a PV case and essential thrombcythaemia. An EPO level does divide those with secondary or idi- opathic erythrocytosis into two groups and helps guide further investigation.

Bone marrow biopsy

ria. It can help to distinguish between myeloprolifer- ative disorders and aid the distinction from secondary and reactive causes.

Endogenous erythroid colonies

Endogenous Erythroid Colonies (EEC) were described in classical cases of PV >30 years ago (Prchal & Axel- rad, 1974). Mononuclear cells from the nonadherent cell fraction in PV patients form erythroid colonies in the absence of added EPO in contrast to samples from normal patients where EEC will only grow when exogenous EPO is present. This is a technically difficult test which is only available in a few specialized laboratories. It is not standardized between laboratories. It does not now add anything to the investigation of a standard case of PV but is helpful in the further investigation in rare unexplained erythrocytosis cases.

INVESTIGATION OF AN ERYTHROCYTOSIS

Having eliminated those patients with PV there remains a group with erythrocytosis. History and examination should be thorough in looking for sec- ondary causes. Investigation should then proceed depending on any leads provided by history and examination. An obvious cause should be pursued ini- tially. Table 4 lists initial tests in a patient with erythr- ocytosis. The first few tests will make a diagnosis of PV and if this is not the diagnosis an EPO level will

The bone marrow in PV is described as hypercellular with erythroid, megakaryocytic and granulocytic proliferation. Erythropoiesis and myelopoiesis are morphologically normal. Megakaryocytes are con- spicuous, pleomorphic with deeply lobulated nuclei (Pierre et al., 2001). It is proposed that a patient with a raised Hb and or Hct and the presence of a JAK2 mutation is sufficient to make a diagnosis. Examination of the bone marrow is included as a minor criterion in the revised WHO criteria. It seems unlikely that it will be possible to persuade clinicians and patients to carry out a bone marrow examination in all patients who have already been shown to have a raised Hct and a JAK 2 mutation. However, it is a useful further investigation in those with erythrocytosis who do not make the PV crite-

2008 The Author Journal compilation 2008 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2008, 30, 447–459

Table 4. Initial tests in an erythrocytosis with further investigations with a low EPO level

Full blood count JAK 2 mutation screen RCM EPO level Exon 12 JAK 2 mutation screen Bone marrow aspirate and trephine Cytogenetics Endogenous erythroid colonies EPO receptor mutation screen*

*EDTA sample by first class post to Dr M. Percy, Haema- tology Department, Belfast City Hospital, Lisburn Road, Belfast BT9 7AB, UK.

456 M. F. MCMULLIN

ERYTHROCYTOSIS

Idiopathic erythrocytosis

Measure EPO levels

ERYTHROCYTOSIS Idiopathic erythrocytosis Measure EPO levels Low/undetectable E P O s i g n a l

Low/undetectable

EPO signalling pathway

Elevated or inappropriately normal

Oxygen sensing pathway

Figure 2. Approach to investigation of idiopathic ery- throcytosis.

Table 5. Investigation in an erythrocytosis with a nor- mal or high erythropoietin level

Haemoglobin electrophoresis

P50

Biphosphoglycerate mutase level Oxygen sensing pathway gene mutations* VHL

PHD2

HIF2a

Pulse oximetry Carboxyhaemoglobin levels CXR Radiology of head, chest and abdomen Sleep studies

*EDTA sample by first class post to Dr M. Percy, Haema- tology Department, Belfast City Hospital, Lisburn Road, Belfast BT9 7AB, UK.

guide as to further directions (Figure 2). Some of the tests which may be indicated in pursuing an erythro- cytosis are listed in Tables 4 and 5.

Investigation with a subnormal EPO level

When an erythrocytosis is proven to be present and the EPO level is below normal then further investiga- tion would proceed as suggested in following Table 4. More extensive investigations for JAK 2 mutations as in looking for an exon 12 mutation are indicated. It may be necessary to look for a mutation in the EPO receptor. This may be particularly suggested in those presenting at a young age and or a family history of erythrocytosis.

Investigation of an inappropriately normal or elevated EPO

The full list of investigations which may be used to investigate the patient with erythrocytosis and increased EPO are listed in Table 5. Which investiga- tions are done and progress through the list should be dictated by history, examination and clinical suspi- cion. Congenital defects may be particularly suspected in those presenting younger and with other affects family members.

CONCLUSION

The investigation of the patient with erythrocytosis starts with history and examination. After confirming the raised Hb and Hct in those without an obvious secondary cause, it is reasonable to proceed to JAK 2 mutation testing to demonstrate JAK 2 positive PV. If this is not the diagnosis the next step should be EPO level measurement. This will provide guidance for the direction of further specialist investigation either look- ing at the EPO signalling pathway in those with sub- normal EPO levels or secondary causes including the oxygen sensing pathway in those with normal or increased EPO levels. There will remain a small group with idiopathic erythrocytosis in whom a cause can- not yet be identified.

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