1 diagnosis and classification of erythrocytoses and thrombocytoses

1

Diagnosis and classification of erythrocytoses and thrombocytoses

T. C. PEARSON MD, FRCPath Professor of Haematology Department of Haematological Medicine, The Guy's, King's & St. Thomas' Hospitals" Medical School, St Thomas' Hospital, Lambeth Palace Road, London SE1 7EI-I, UK

An erythrocytosis describes an increased peripheral blood packed cell volume (PCV) and is deemed to be absolute or apparent depending on whether or not the measured red cell mass (RCM) is above the reference range. This reference range must be related to the individual's height and weight to avoid erroneous interpretations using ml/kg total body weight expressions in obesity. Absolute erythrocytoses are divided into primary, where the erythropoietic compartment is intrinsically abnormal, secondary, where the erythropoietic compartment is normal but is responding to external pathological events leading to an increased erythropoietin drive, and idiopathic, where neither a primary nor a secondary erythrocytosis can be established. Both primary and secondary erythrocytoses have congenital and acquired forms. The only form of primary acquired erythrocytosis that has been defined is the clonal myeloproliferative disorder, polycythaemia vera (PV). Modified diagnostic markers for PV are proposed.

Thrombocytoses can be classified into primary, where megakaryopoiesis is intrinsically abnormal, secondary, where megakaryopoiesis is normal but increased platelet production is a reaction to some other unrelated pathology, and finally idiopathic. This latter new group would be used for patients not satisfying the criteria for primary or secondary thrombocytoses, if these were more precise and rigidly used than currently is the case. While theoretically congenital and acquired forms of primary and secondary thrombocytoses might exist, only one cause of secondary congenital thrombocytosis has been established, and primary congenital thrombocytosis has not yet been precisely defined. Primary (essential) thrombocythaemia (PT) is one of the forms of primary acquired thrombocytoses. The diagnostic criteria of PT traditionally involve the exclusion of secondary thrombocytoses and other myeloproliferative disorders but marrow histology could hold a key positive diagnostic role if objective histological features of PT were agreed.

Key words: erythrocytosis; thrombocytosis; classification; diagnostic criteria; primary thrombocythaemia; polycythaemia vera.

Dameshek (1951) introduced the term 'myeloproliferative disorders' in recognition of a number of closely interrelated haematological conditions which had previously been described. He suggested that they might be due

BaiUi@re's Clinical Haematology-- 695 Vol. 11, No. 4, December 1998 Copyright © 1998, by Bailli~re Tindall ISBN 0-7020-2462-7 All rights of reproduction in any form reserved 0950-3536/98/040695 + 26 $12.00/00

696 T . C . PEARSON

to some unknown stimulus leading to variable proliferative activity of bone marrow cells. Later, the existence of multipotential haemopoietic precursor cells was established in mice (Becker et al, 1963). This observation, combined with the finding that the erythroid and granulocytic cell series shared a common ancestry in chronic myeloid leukaemia, provided a basis for the 'myeloproliferative disease' concept (Gilbert, 1970). Studies in female patients with polycythaemia vera and primary thrombocythaemia, who were heterozygous for the glucose-6-phosphate dehydrogenase (G6PD) enzyme, confirmed the clonal, or unicellular, origin of these conditions (Adamson et al, 1976; Fialkow et al, 1981). However, the marrow fibrosis that occurred later in some patients was established to be a reactive process and not part of the clonal proliferation (Jacobson et al, 1978).

Over the last 20 years, with improvement in histological techniques, karyotypic analysis and development of molecular biological approaches, conditions such as the myelodysplastic syndromes and chronic granulocytic leukaemia have been separated from polycythaemia vera, primary thrombocythaemia and myelofibrosis, which have now been grouped together under the heading 'myeloproliferative disorders' (MPD). The close relationship between the MPD is demonstrated by the fact that their haematological phenotypes may share similar features in some patients and by their capacity to undergo transformations, notably to acute leukaemia and myelofibrosis.

The present chapter provides a classification of the various causes of erythrocytosis and/or thrombocytosis with proposals for the diagnostic approach to patients with one or other of these haematological features. However, due to an overlap of phenotypic expressions, it is not possible in the occasional patient to put them in a specific diagnostic category.

THE CLASSIFICATION OF THE ERYTHROCYTOSES

While the term 'polycythaemia' has become associated with an increased volume of red cells in the peripheral blood, reflected by the packed cell volume (PCV), the term 'erythrocytosis' has descriptive merit because, in one word, it defines the pathological change precisely and is in line with terms such as leukocytosis and thrombocytosis. The use of adjectives to identify that 'polycythaemia' relates to red cells such as 'polycythaemia rubra vera' leads to potential confusion. Thus, in this chapter 'erythrocytosis' wilt be preferred for the non-specific observation of an increased PCV in the peripheral blood. This will additionally prevent the confusion that occasionally arises when it is assumed that 'polycythaemia' indicates the ctonal myeloproliferative disorder.

Definition of raised packed cell volume (PCV)

Individuals with a sustained elevation of their PCV have some form of erythrocytosis. A variety of physiological factors have been shown to

ERYTHROCYTOSES AND THROMBOCYTOSES 697

modify the PCV value. In practice, the use of only minimal or no venous occlusion when collecting the sample is the most important. In the presence of normal red cell indices, electronic cell counters give almost identical results as the microhaematocrit. However, at reduced mean corpuscular haemoglobin values, some cell counters underestimate the true PCV value (Guthrie and Pearson, 1982). In these circumstances, a correction should be applied or the microhaematocrit used. The upper limits for the reference ranges for PCV values are 0.51 for males and 0.48 for females (Williams et al, 1990; Hall and Malia, 1991).

Definition of raised red cell mass (RCM)

Despite clear demonstrations that results and normal values for RCM expressed in terms of total body weight lack precision (Najean and Cacchione, 1977), many laboratories persist in using ml/kg total body weight expressions. The inaccuracy applies particularly in obese individuals because fat tissue is relatively avascular. Thus, if allowance is not made for this, high predicted values and low measured results are found in these individuals. The radionuclide panel of the International Council for Standardization in Haematology (ICSH) have recently given recom- mendations for the interpretation of RCM (Pearson et al, 1995). These use the surface area of the individual derived from both the height and weight. Examination of the scatter of RCM results in normal individuals shows that 98% of results in males and 99% in females fall within +25% of the predicted normal value for that individual. Thus it has been proposed that a measured RCM can be accepted as above the reference range if it exceeds the predicted normal value for that individual by more than 25% (Pearson et al, 1995). Thus, an individual with a persistently elevated PCV can be said to have an absolute erythrocytosis or apparent erythrocytosis depending on whether their measured RCM is above or below their upper reference value using the proposed ICSH reference values. However, the designation of an individual to one or other of these two groups must be done in full knowledge of the possible errors of measurement (_+5%) and the accuracy of the interpretation. This applies particularly when an RCM result is close to the upper limit of the reference range.

NATURE OF AND MECHANISMS INVOLVED IN APPARENT ERYTHROCYTOSIS

Other descriptive titles for patients with an apparent erythrocytosis, include relative, stress, spurious and pseudo-polycythaemia and Geisbock's syndrome. Approximately 25% of these patients have a reduced PV (Pearson, 1991). The remainder have RCM and PV results falling within their normal range, generally with some elevation of the former and reduction of the latter. The possible causes of apparent erythrocytosis are listed in Table 1. From this list, it is obvious that 'apparent' erythrocytosis does not represent a single disease entity. The greater the observed PCV

698 T. C. PEARSON

Table 1. The possible causes and associations of apparent erythrocytosis.

1. Physiological variant 2. 'Early' absolute erythrocytosis 3. Observed associations: obesity, fluid loss and diuretics, smoking, hypertension,

cardiovascular disease, alcohol, arterial oxygen desaturation, renal disease, psycho- logical stress and excess catecholamine secretion (including phaeochromocytoma)

above the normal range the less likely that the individual represents a 'physiological variant'. The normal RCM range is wide. Therefore, some pathology might be present causing an increased PCV but with a measured RCM falling within the individual's normal reference range. Thus, any of the causes of an absolute erythrocytosis might be present. A number of associations that have been described with apparent erythrocytosis are shown in Table 1. These factors either reduce the plasma volume and/or cause some increase in the RCM and more than one factor may be present in the individual patient (Pearson, 1991).

THE SUBDIVISION OF THE ABSOLUTE ERYTHROCYTOSES

Traditionally, these have been separated into primary and secondary when the underlying pathological process can be identified, leaving a sub- group, 'idiopathic erythrocytosis' (IE), where the mechanism is unclear (Table 2). In primary erythrocytoses there is an intrinsic defect of the erythropoietic compartment. In secondary erythrocytoses, erythropoiesis is responding to increased erythropoietin elaboration due to a physiological or pathological process. Both primary and secondary erythrocytoses can be subdivided into congenital and acquired, as in other medical classifications (Table 2).

Table 2. The classification of the erythrocytoses.

Raised PCV (females >0.48; male >0.51)

RCM (interpreted against ICSH reference values)

Increased RCM Normal RCM Absolute erythrocytosis Apparent erythrocytosis

Primary erythrocytosis tCongenital e.g. Truncation of the Epo receptor* Acquired e.g. Polycythaemia vera*

Secondary erythrocytosis tCongenital e.g. High oxygen affinity Hb, autonomous high Epo production Acquired e~g. Hypoxaemia, renal disease

Idiopathic erythrocytosis

t Sometimes familial; *the only condition to be defined in this category at present.


Primary congenital and acquired erythrocytoses Originally, only one form of primary erythrocytosis was known, namely, the acquired myeloproliferative disorder, polycythaemia vera (PV), but recently congenital erythrocytoses due to mutations within the erythropoietin receptor gene have been recognized. The merit of the term 'polycythaemia vera' is that the translation could be taken to indicate a true increase in many cell lines in the blood, which is a feature of the haematological phenotype of PV. While PV must generally be regarded as an acquired disorder, some familial cases of PV or PV in one individual with another MPD in another family member have been described (Gilbert, 1995; see also pp 849-857 of this volume).

There are several congenital mutations within the erythropoietin receptor gene that have now been described (Prchal and Sokol, 1996; Kralovics et al, 1997). All the mutations lead to truncation of the cytoplasmic portion of the erythropoietin receptor. It is this portion which is responsible for switching off the signal following erythropoietin binding. As a result, the erythroid precursors are hypersensitive to erythropoietin leading to erythroid hyperplasia with an absolute erythrocytosis. The majority of patients with this form of primary congenital erythrocytosis represent a dominantly inherited familial condition as originally recognized in a large Finnish kindred (de la Chapelle et al, 1993). However, individual cases where a spontaneous somatic mutation has occurred have been described (Prchal and Sokol, 1996; Percy et al, 1998). While the abnormality is expressed by increased erythropoiesis, it must be recognized that this is a genomic change and present in all the individual's cells and that the erythroid proliferation is polyclonal.

Over time, further examples of well characterized primary congenital and acquired erythrocytoses will be discovered taking patients out of the idiopathic erythrocytosis (IE) group. Currently, work is centred on familial erythrocytoses where no abnormality of the erythropoietin receptor gene is found, and various candidate genes involved in the post- erythropoietin receptor signalling pathway are being examined (Prchal and Sokol, 1996).

Secondary congenital and acquired erythrocytoses Secondary erythrocytoses are the commonest form of an absolute erythrocytosis. There are a few causes of secondary congenital erythrocytosis, which have been well characterized. These include the high oxygen affinity mutant haemoglobins where the left shift in the oxygen dissociation curve reduces the amount of oxygen released at normal tissue oxygen tensions. Thus, despite the normal arterial oxygen saturation, the renal sensor elaborates increased amounts of erythropoietin with a resultant erythrocytosis.

Many families with autonomous high erythropoietin, with either reces- sive or dominant inheritance, have been described (Distelhorst et al, 198 I; Kulkarni et al, 1985). Various patterns of erythropoietin production have

700 T.C. PEARSON

emerged in these families. Some have permanently elevated levels while others show increased erythropoietin values only following normalization of their haemoglobin (Hb) values. The precise genetic defects in these families remains to be established, although an abnormal oxygen sensing pathway involving hypoxia inducible factor-1 (HIF-1) is being explored (Prchal and Sokol, 1996).

Secondary acquired erythrocytoses fall into two groups. In the first, there is a global reduction in oxygen delivery to both kidneys, as in arterial hypoxaemia, or more localized renal ischaemia, producing an enhanced erythropoietic response, such as in renal artery stenosis and hydronephrosis. In the second group, there is ectopic erythropoietin production in a neoplasm of some kind. Examples include hypernephroma, hepatoma, fibroids and cerebeUar haemangioblastoma (Pearson and Treacher, 1990).

Nature of idiopathic erythrocytosis

The term idiopathic erythrocytosis (IE) applies to a heterogeneous group of patients with an absolute erythrocytosis who cannot be categorized into the primary or secondary erythrocytosis groups. Alternative titles include benign erythrocytosis (Modan and Modan, 1968) and pure erythrocytosis (Najean et al, 1981). The possible underlying mechanisms in these patients are listed in Table 3. By definition, 1% of normal males and 0.5% of normal females will have an RCM above the reference range (Pearson et al, 1995). In approximately 5-10% of IE patients definitive diagnostic features of PV emerge in the few years after presentation (Pearson and Wetherley-Mein, 1979; B erglund and Zettervall, 1992). In a small proportion of patients with IE, a cause of secondary erythrocytosis is present but is not identified. Intermittent hypoxaemia, notably occurring at night, is the usual mechanism that remains unrecognized (Moore-Gillon et al, 1986).

Table 3. Nature of idiopathic erythrocytosis.

1. Physiological variant 2. 'Early' polycythaemia vera 3. Underlying cause of secondary erythrocytosis unrecognized or currently undescribed 4. Clonal proliferation showing only erythropoietic expansion

THE INVESTIGATION OF AN ABSOLUTE ERYTHROCYTOSIS

The starting point is a knowledge of the causes of a secondary erythrocytosis and the diagnostic criteria of PV. Occasionally, particularly in the elderly, two different pathologies causing an erythrocytosis are found.

The commoner causes of a secondary erythrocytosis are listed in Table 4. In general, to prove conclusively that an erythrocytosis is indeed secondary is often difficult. Occasionally there is an opportunity to show amelioration of the erythrocytosis following removal or correction of a presumed precipitating factor.


Table 4. The commoner causes of secondary erythrocytosis*.

Congenital Mutant high oxygen affinity haemoglobin Congenital low 2:3 DPG Autonomous high erythropoietin production

Acquired Arterial hypoxaemia

High altitude Cyanotic congenital heart disease Chronic lung disease

Other causes of impaired tissue oxygen delivery Smoking

Renal lesions Renal tumours Cysts Diffuse parenchymal disease Hydronephrosis Renal artery stenosis Renal transplantation

Hepatic lesions Hepatoma Cirrhosis Hepatitis

Endocrine lesions Adrenal tumours

Miscellaneous tumours Cerebellar haemangioblastoma Uterine fibroids Bronchial carcinoma

Drugs Androgens

* Abbreviated from Pearson and Treacher (1990).

As yet no single diagnostic marker for PV has been established. The Polycythemia Vera Study Group (PVSG) criteria for substantiating the diagnosis were published in 1975 (Berlin, 1975). These have been widely used (Berlin, 1995) and have generally proved to be satisfactory. However, they have some limitations. These authors interpreted measured RCM using ml/kg expressions but such expressions lack accuracy, as previously explained. They did not exclude all forms of secondary erythrocytosis. They used raised neutrophil alkaline phosphatase score as a minor criterion but this measurement shows poor inter- and intra-laboratory reproducibility (Pearson and Messinezy, 1996). Raised serum B12 and unbound Bt2 binding capacity (UBlzBC) were also minor criteria, but Bl2 assays commonly used are inaccurate at high values, and very few laboratories measure UB12 BC. As a result of these observations, and to bring in some newer investigations, modified criteria have been published (Table 5; Pearson and Messinezy, 1996).

702 T. C. PEARSON

Table 5. Proposed modified criteria for the diagnosis of polycythaemia vera*.

A1 Raised red cell mass (>25% above mean normal predicted value)

A2 Absence of cause of secondary erythrocytosis

A3 Palpable splenomegaly

A4 Clonality marker--abnormal marrow karyotype

A1 + A2 + A3 or A4 establishes PV A1 + A 2 + t w o of B establishes PV

B 1 Thrombocytosis (platelet count >400 × 109/1)

B2 Neutrophil leucocytosis (neutrophil count > 10 x 10P/l)

B3 Splenomegaly demonstrated on isotope/ ultrasound scanning

B4 Characteristic BFU-E growth or reduced serum erythropoietin

* Reproduced from Pearson and Messinezy (1996, Leukemia and Lymphoma 22 (supplement 1): 87-93) with permission.

Full blood count

When considering a diagnosis of erythrocytosis it is better to use the PCV than the haemoglobin (Hb) value. The reason for this is that the PCV determines haemorheology, and some patients have iron-deficient red cell changes leading to a proportionately lower Hb than PCV value. The under- estimation of the correct PCV value by some electronic cell counters in the presence of iron-deficient red cell changes has already been discussed (Guthrie and Pearson, 1982). Judicious iron therapy may be administered before measuring the RCM in individuals with normal PCV values and proven iron deficiency, but it must be appreciated that the PCV may rise quite dramatically and the level of PCV positively correlates with the risk of vascular occlusion in PV (Pearson and Wetherley-Mein, 1978). In patients with splenomegaly an absolute erythrocytosis can be shown even when the PCV falls within the normal range due to splenic red cell pooling and plasma volume expansion. Thus, in these patients, it is worthwhile measuring the RCM when the PCV falls in the upper part of the reference range.

In the absence of infection and other 'reactive' situations, a neutrophil leukocytosis (> 10 x 109/1) is a minor criterion for the diagnosis of PV. Occasionally a mild eosinophilia or basophilia is also present, but the measurements lack precision for inclusion as criteria. Smokers have significantly higher WBC counts than do non-smokers (Whitehead et al, 1995). Thus, in the very heaviest smokers, it might be advisable to raise the level of neutrophil count to > 12 x 109/1 as a diagnostic marker of PV.

In the absence of a 'reactive state', a thrombocytosis (>400 x 109/1) is a useful minor criterion of PV. Some tumours, for example hypemephroma, can cause an erythrocytosis as well as a reactive thrombocytosis and/or leukocytosis. Other platelet abnormalities, such as platelet anisocytosis on the blood film, increased PDW and abnormal aggregation have all been described in PV but are insufficiently characterized and standardized to use as diagnostic markets. Most recently, reduced or absent expression of the thrombopoietin receptor, Mpl, on the platelets of patients with PV has been reported, and this finding appeared to distinguish PV from secondary


erythrocytosis (Moliterno et al, 1998). This finding awaits confirmation by other authors.

Red cell mass

This has already been discussed (vide supra). It is important to remember the accuracy of both the measurement and its interpretation.

Serum ferritin, vitamin B12 and folate

Low serum ferritin levels are more commonly seen in PV than in secondary erythrocytoses. Indeed, the absence of iron stores has been regarded as a hallmark of PV marrow histology (Kurnick et al, 1972). Vitamin B12 levels may be elevated in PV due to the release of transcobalamins from an increased granulocyte mass. Folate deficiency is observed in occasional patients with an erythrocytosis and should be recognized because it might limit the erythropoietic response.

Urea and creatinine

Mild renal impairment has occasionally been found to be complicated by a secondary erythrocytosis (Hoppin et al, 1976) but usually the RCM is only marginally elevated.

Liver function tests

Impaired liver function due to cirrhosis has occasionally been found to be complicated by a secondary erythrocytosis. The mechanism is thought to be due to arterial hypoxaemia resulting from the development of pulmonary arterio-venous anastomoses (Wolfe et al, 1977). A secondary erythrocytosis associated with an excess alcohol intake and impaired liver function does occur and is probably related to the increased erythropoiesis observed in inflammatory liver disease (Kolk-Vegter et al, 1971).

Arterial oxygen saturation (SaOz) and carbon monoxy-haemoglobin level (COHb)

The measurement of SaO 2 is most easily achieved with the use of a pulse oximeter. Most of these instruments, however, include COHb levels in the reading. The critical level of SaO 2 taken to implicate it in the cause of a secondary erythrocytosis is 92%. The SaO2 is not a static figure, and secondary erythrocytosis may result from intermittent reductions in SaO~ if these are sufficiently prolonged. It is important to enquire about symptoms relating to nocturnal oxygen desaturation. These include snoring, nocturnal restlessness, wakening unrefreshed and daytime somnolence. This situation is more usually, but by no means exclusively, seen in obesity (Pearson and Treacher, 1990). Nocturnal reduction in SaO2 below 92% with normal daytime values has been shown to be the cause of the increased RCM in

704 T . C . PEARSON

10-20% of patients who would otherwise have been classified in the idiopathic erythrocytosis group (Moore-Gillon et al, 1986).

COHb interfers with oxygen carriage and delivery. The COHb level in non-smokers is less than 2.0%. Smokers have higher values but these are usually not particularly elevated. In general, smokers have PCV values that are +0.02 higher than those of non-smokers. A secondary erythrocytosis due to cigarette smoking per se is extremely uncommon but has been recorded (Smith and Landaw, 1978). More commonly, smoking is just an additional factor leading to stimulation of erythropoiesis as seen in some patients with hypoxaemic lung disease (Calverley et al, 1982) and, in general, it is important to complete all other investigations in smokers.

Abdominal ultrasound

Abdominal ultrasound is an essential investigation in all patients with an absolute erythrocytosis. Careful examination of the kidneys is most important. CT scanning or renal arteriography may be necessary to define a renal lesion more clearly. The commonest renal lesion identified is one or more simple renal cysts. These are increasingly common with age (Laucks and McLachlan, 1981). Unlike polycystic kidney disease, it is exceedingly uncommon for these solitary cysts to cause an erythrocytosis.

Relevant liver pathology, such as a hepatoma, can also be excluded on ultrasound and the spleen size can be measured. The spleen must enlarge significantly before it becomes clinically palpable. In the absence of liver disease, a palpable spleen is a reliable sign of PV and as such has been taken as a major criterion in diagnosis (Table 5). An enlargement of the spleen, which is found in at least two-thirds of PV patients, can be identified by various scanning techniques, although ultrasound is the simplest (Westin et al, 1972; Carneskog et al, 1996; Messinezy et al, 1997). Using ultrasound, the greatest demonstrable spleen length is the easiest measurement, but there is a small intra-observer and a greater inter- observer error and the normal spleen size varies with the individual's size and age. Ideally, these factors should be taken into account before using an enlarged spleen demonstrated by ultrasound as a diagnostic marker of PV (Messinezy et al, 1997). For these reasons, it is proposed that this finding be taken as a minor criterion (Table 5).

Bone marrow examination and karyotype

The marrow histology of patients with PV has been extensively reviewed (e.g. Bartl et al, 1993; Peterson and Ellis, 1995; Georgii et al, 1996 and pp 72t-749 in this volume). Typically, there is trilineage hyperplasia with increased large megakaryocytes with increased ploidy often occurring in clusters and some increase in reticulin. However, there is a considerable variability in the marrow histology of PV patients. While generally there are typical appearances of the marrow histology of PV patients, there are no agreed methods of quantifying the changes. While sampling discrepancies will always remain, intra- and inter-observer differences need


to be eliminated before marrow histology can be widely used in the diagnosis of PV, as has been proposed (Kurnick et al, 1972; Michiels, 1997). Fortunately, there are sufficient clear objective markers of the disease to make the inclusion of marrow histology unnecessary. However, a more detailed examination of the marrow histological appearances may hold a clue to the risk of myelofibrotic or leukaemic transformation. Transitional states between PV and myelofibrotic transformation have been described (Pettit et al, 1979; Najean et al, 1984) and generally myelofibrotic transformation evolves over many years.

The marrow karyotype may be a useful marker to demonstrate the presence of a clonal marrow disorder and holds a key diagnostic position of PV (Table 5). Between 10 and 15% of patients have an acquired abnormal karyotype at presentation. The most common changes are trisomies 1, 8, 9 and abnormalities and deletions of 13q and 20q, but a number of other changes have been found (Swolin et al, 1988; Diez Martin et al, 1991; Pierre and Whang-Peng, 1995).

X-chromosome-linked DNA probes to demonstrate clonality

This technique obviously applies only to women and requires the demonstration in granulocytes that one or other of the X-chromosomes is active using the patient's T cells as a control. Using the HUMARA gene probe, the parental X-chromosomes can be identified in approximately 90% of females. The demonstration of the monoclonality of granulocytes in females suspected of having PV was beginning to assume some diagnostic importance. However, a major drawback has recently been shown in that apparent clonal granulocytes can be demonstrated in 20-30% of normal elderly women (Champion et al, 1997; Gale et al, 1997). A small proportion of patients, identified as PV on other criteria, have polyclonal granulocytes reflecting that some of their granulocytes originate from their normal stem cells or possibly even the presence of bi-clonal disease. X-linked probes may still have relevance in demonstrating clonality in younger patients but this needs exploring using a satisfactory control population.

Serum erythropoietin (Epo)

At diagnosis, the serum Epo levels in PV are either reduced or in the low normal range (Cotes et al, 1986; Najean et al, 1990). With treatment to lower the PCV, the Epo levels remain low in two-thirds of patients (Najean et al, 1990; Messinezy et al, 1995). With the reliability of the available assays, serum Epo levels can now be used as a minor criterion in the diagnosis of PV (Table 5). The hyper-sensitivity of BFU-E to Epo in PV and reduced serum Epo levels are probably linked and thus are not independent criteria.

Culture studies of BFU-E (burst forming units-erythroid)

Culture studies of purified progenitors from patients with PV have shown that their BFU-E are hypersensitive to a number of different growth factors,

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including stem cell factor, IL-3, Epo and insulin-like growth factor (Eaves and Eaves, 1978; Casadevall et al, 1982; Dai et al, 1994). The culture of the mononuclear non-adherent fraction of peripheral blood cells of patients with PV in serum-containing medium without the addition of erythropoietin leads to the growth of BFU-E--so-called 'endogenous erythroid colonies'. This observation, which is usually not found in normal individuals or patients with secondary erythrocytoses, has been shown to be a reliable marker of PV (Lacombe et al, 1980; Partanen et al, 1989). However, the BFU-E grown in these circumstances are rather pale, and inter-observer differences in reading the culture plates can present a problem, particularly since it has been proposed that the finding of one EEC is diagnostic of PV (Reid, 1987). An alternative approach, but more costly and time-consuming, is to demonstrate the hypersensitivity of BFU-E in PV by producing a BFU-E response curve against increasing Epo concen- trations (Eaves and Eaves, 1978), although a similar response of BFU-E can be observed in patients with erythrocytosis due to truncation of the Epo-receptor, who also have low serum Epo levels (de la Chapelle et al, 1993).

Some workers have examined BFU-E growth in serum-free culture conditions. The finding of EEC in this situation loses its specificity for PV (Westwood and Pearson, 1996). A more elaborate technique, involving the use of other growth modifiers in the presence of Epo and in serum-free conditions, has been described and shown to be highly specific for PV (Dudley et al, 1990). However, the reading of the culture plates has to be rigorously standardized.

Overall, culture techniques are expensive and are not standardized or generally available. Thus, as a general diagnostic tool, they have limitations. The measurement of serum Epo levels is significantly cheaper, and because, as proposed, Epo and BFU-E results are linked minor critera in the diagnosis of PV (Table 5), the measurement of serum Epo levels is the preferred option.

Oxygen dissociation curve (ODC)

Examination of the ODC and p50 is an important investigation in patients with an unexplained erythrocytosis. There are a large number of p-chain haemoglobin variants that have increased oxygen affinity but a left-shifted curve has also been observed with an o~-chain variant when inherited in conjunction with c~-thalassaemia (Williamson et al, 1992). These haemoglobin variants occasionally show altered mobility on haemoglobin electrophoresis. Usually the diagnosis of a secondary erythrocytosis due to high oxygen affinity haemoglobins is made in young patients with a relevant family history. However, the adaptations to these abnormal haemoglobins vary from individual to individual (Charache et al, 1978) with some having Hb and PCV values falling in the high normal range. Thus, some individuals may escape detection or are discovered only in later life.


SUMMARY OF INVESTIGATIONS OF AN ABSOLUTE ERYTHROCYTOSIS

Current financial pressures dictate that investigations are carried out in the most cost-effective way possible. Table 6 sets out the investigations in two stages. It is proposed that the tests in stage 1 are carried out in all patients. Following examination of the results of the stage 1 tests, selected investigations from stage 2 are carried out. While not totally exhaustive, these cover the majority of the causes of an absolute erythrocytosis seen in routine clinical practice.

Table 6. Stages in the investigation of an absolute erythrocytosis.

Stage 1 Stage 2

Full blood count Marrow histology Urinalysis Marrow karyotype Ferritin Serum erythropoietin Vitamin B12 Oxygen dissociation curve Folate Sleep study Creatinine Lung function tests Liver function tests Chest X-ray Pulse oximetry Echocardiogram COHb Abdominal ultrasound

THE CLASSIFICATION OF THE THROMBOCYTOSES

A thrombocytosis is defined as a platelet count above 400 × 109/1, which is the upper limit of the reference range. A similar model for the classification of the thrombocytoses, as that proposed for the erythrocytoses, can be used (Table 7). In primary thrombocytoses, the abnormality resides in the megakaryocytic compartment, although not necessarily exclusively. While a small number of families with an inherited thrombocytosis have been described (Eyster et al, 1986; Fernandez-Robles et al, 1990), in none has an intrinsically abnormal megakaryocytic compartment been identified. Thus, currently, primary congenital thrombocytosis is only a theoretical condition

Table 7. Proposed classification of the thrombocytoses.

Primary thrombocytosis Congenital Acquired, e.g. primary thrombocythaemia, polycythaemia vera, chronic granulocytic leukaemia, myelodysplastic syndromes

Secondary thrombocytosis Congenital, e.g. autonomous high thrombopoietin production Acquired, e.g. haemorrhage, rheumatoid arthritis, lymphoma

Idiopathic thrombocytosis

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awaiting further elucidation. Much more clearly defined are the various clonal marrow disorders, where the megakaryocytic population is part of the abnormal clone. Obvious examples are the closely linked myeloproliferative disorders, primary (or essential) thrombocythaemia (PT) and polycythaemia vera (PV), but a thrombocytosis may also be found in chronic granulocytic leukaemia and some myelodysplastic syndromes, notably the 5q- syndrome.

The secondary thrombocytoses are characterized by increased megakaryopoiesis produced by an intrinsically normal megakaryocytic compartment as a result of increased cytokine production arising either autonomously (secondary congenital thrombocytoses) or in response to pathological events (secondary acquired thrombocytoses) occurring typically outside the marrow, for example, haemorrhage, rheumatoid arthritis, but occasionally involving the marrow, but not the megakaryocytic compartment, for example Hodgkin's disease, non-Hodgkin's lymphoma.

Secondary congenital thrombocytoses are currently exceedingly rare, although one family with an autosomal dominant thrombocytosis affecting 11 individuals has been described. Examination of the thrombopoietin (TPO) coding region established the presence in affected individuals of a point mutation that leads to elevated TPO levels (Wiestner et al, 1998). Interestingly, the clinical features (Schlemper et al, 1994) and bone marrow histology (Michiels, personal communication) of these patients are similar to those observed in PT.

By far the commonest cause of an elevated platelet count is a secondary acquired thrombocytosis. An analysis of platelet count results in a large teaching hospital revealed that 6% were greater than 400 × 109/1 and 1% exceeded 600 x 109/1. While a few of these patients might have had an MPD of some kind, the vast majority had a secondary acquired or 'reactive' thrombocytosis.

Following the classification given in Table 9, this leaves 'idiopathic thrombocytosis'. This condition has not been previously described. The existence of idiopathic thrombocytosis largely turns on the diagnostic criteria of PT. These were previously given as a thrombocytosis with the exclusion of secondary and other primary thrombocytoses. If this is accepted then all individuals with an unexplained thrombocytosis have PT and there is no need for an idiopathic thrombocytosis group. However, various authors have proposed positive diagnostic criteria for PT. It follows that if these are not met then it will be necessary to put some individuals in this new group. It will obviously be heterogenous containing, on the one hand, individuals at the extreme end of the physiological range for platelet count to others with an, as yet, unrecognized form of thrombocytosis or with an evolving acquired thrombocytosis of primary or secondary type.

THE INVESTIGATION OF A THROMBOCYTOSIS

Well-established causes of primary and secondary congenital thrombocytoses are either so exceedingly rare or unknown that in practice the


investigation of patients with a thrombocytosis rests on the diagnostic criteria of primary thrombocythaemia (PT). However, when younger patients present, it is worth examining samples from other family members to exclude a familial congenital thrombocytosis. There is no single diagnostic test for PT, and the requirements for making the diagnosis rest on the exclusion of secondary acquired thrombocytoses and other myeloproliferative disorders, including myelodysplasia. Some positive diagnostic criteria have been proposed, but these are not firmly established.

THE DIAGNOSTIC CRITERIA OF PRIMARY THROMBOCYTHAEMIA

The traditional criteria evolved from the studies of the PVSG and recently they have been updated from those originally published (Murphy et al, 1997; Table 8). The individual components will be discussed, including some investigations aimed at providing positive diagnostic tests for PT.

Table 8. Diagnostic criteria of primary thrombocythaemia*.

1. Platelet count >600x 109/1

2. PCV <0.51 for males or <0.48 for females or normal red cell mass in those with a high normal PCV and splenomegaly

3. Stainable iron in marrow or normal serum ferfitin or normal red cell MCV (if measurement suggests iron deficiency then PV cannot be excluded unless a trial of iron therapy fails to increase the RCM into the erythrocytotic range)

4. No Philadelphia chromosome or bcr/abl gene re-arrangement

5. Collagen fibrosis of marrow (a) absent or (b) < 1/3 biopsy area without marked splenomegaly and leuko-erythroblastic reaction

6. No cytogenetic or morphological evidence of a myelodysplastic syndrome

7. No cause for reactive thrombocytosis

* Modified from Murphy et al (1997, Seminars in Hematology 34: 29-39).

Platelet count, platelet characteristics and symptomatology

The current criteria for PT give a persistent platelet count of >600 × 109/1. There has been a suggestion that the platelet count criterion should be reduced to >400× 109/1 (Michiels and Juvonen, 1997) or >450x 109/1 (Lengfelder et al, 1998). The major problems with this approach are that platelet counts above these levels are commonly encountered in hospital practice and that individuals at the extreme end of the physiological range will undergo unnecessary investigation. However, in the evolution of PT there is a period of time when the platelet count still falls in the reference range but is elevated above the patient's own normal value. Very occasionally patients develop vascular complications at this time and only later the diagnosis of PT is made when the platelet count rises (Vadher et al, 1993;

710 T. C. PEARSON

Lengfelder et al, 1998). Therefore, the selection of a platelet count of 600x 109/1 for a diagnostic criterion emerges as an arbitrary decision balancing over-investigation against under-diagnosis and is the most appropriate for the selection of PT patients to be put in therapeutic trials. If a lower platelet Count is taken then marrow histology becomes an important diagnostic marker for PT (Michiels and Juvonen, 1997; Lengfelder et al, 1998; see later discussion).

As a generalization, vascular complications are rare in secondary thrombocytosis but are comparatively common in PT. This difference in complication rate has led some authors to suggest that 'clinical ischaemia' is a useful positive criterion for PT (Dudley et al, 1989).

A large number of qualitative platelet abnormalities have been described in PT (Bribre et al, 1997). For a parameter to be of diagnostic value, it must be widely available, relatively cheap and easily performed. The mean platelet volume (MPV) and size heterogeneity, reflected by the platelet distribution width (PDW), measured by electronic cell counters, meet these requirements. The PDW is higher in PT than in secondary acquired thrombocytoses (ST) but the overlap between groups makes it unreliable as the sole criterion. The MPV is an unhelpful marker (van der Leslie and von dem Borne, 1986; Dudley et al, 1989). Overall ADP levels are lower in PT than in ST, leading to a higher ATP:ADP ratio in PT (Dudley et al, 1989). However, again, there is considerable overlap in the results and the measurements are not routinely available. Other qualitative platelet changes are also of limited value because of the cost of the investigations, lack of standardization or, in many cases, the general unavailability of the tests.

Exclusion of secondary thrombocytoses

The causes of secondary thrombocytoses are given in Table 9. The congenital autonomous high thrombopoietin production has been described in only one family (Wiestner et al, 1998). The majority of the causes of

Table 9. Causes of secondary thrombocytosis.

Congenital Autonomous high thrombopoietin production

Acquired Acute haemorrhage Malignant disease(e:g, carcinoma, Hodgkin~s and non-Hodgkin's lymphoma) Chronic inflammatory disorders (e.g. rheumatoid arthritis, ulcerative colitis, Crohn's disease) Acute inflammation Post-operation Splenectomy and hyposplenism Marrow recovery from drug suppression, or response to haematinic following deficiency Exercise Response to certain drugs (e.g. vincristine) Iron deficiency POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and

skin changes)


secondary acquired thrombocytosis are established by the patient's history and appropriate investigation. Non-specific markers of a 'reactive' thrombocytosis, including ESR, C-reactive protein, serum IL-6 levels and plasma vWF levels, have been widely examined and shown to be raised or higher in ST than in PT (Messinezy et al, 1994). The ESR and C-reactive protein level are the preferred routine tests.

One particular cause of secondary thrombocytosis, namely splenectomy, can cause diagnostic difficulty. Some individuals having undergone splenectomy for a variety of reasons, or with functional hyposplenism, develop a persisting, usually marginal, thrombocytosis. The blood film shows post-splenectomy changes with platelet size heterogeneity and a raised PDW. In one very small series of patients with PT splenic atrophy was frequently observed (Marsh et al, 1966). However, this has not been reflected in larger series of these patients. Thus, PT is an unlikely cause of a persisting thrombocytosis in patients with hyposplenic features. Where diagnostic difficulty remains, positive features of PT should be examined, such as marrow histology, BFU-E and CFU-Mk (vide infra).

Exclusion of polycythaemia vera (PV)

A diagnosis of PV should be considered in patients presenting with a thrombocytosis and raised PCV (>0.51 males; >0.48 females) or, in the presence of splenomegaly, a PCV in the high normal range. A RCM measurement should be undertaken and interpreted appropriately (see earlier: interpretation of measured RCM). However, patients with PV may present with iron deficiency and normal, or occasionally low, Hb and PCV values. Thus, an assessment of iron status should be routine in patients presenting with a thrombocytosis. In those with iron deficiency, judicious iron therapy may be used to see whether the PCV rises to a level that would indicate the need for an RCM measurement. However, it must be remembered that the incidence of thrombotic complications in PV is directly related to the PCV level (Pearson and Wetherley-Mein, 1978). A small proportion of patients initially designated to the PT group develop features of PV some years later (Shih and Lee, 1994).

Exclusion of chronic granulocytic ieukaemia (CGL)

Very occasionally patients with CGL show predominantly a thrombocytosis with little or no increase in white count (Morris et al, 1988; Richards et al, 1993). The presence of basophilia or significant left-shift in the granulocyte series would tend to support a diagnosis of Phi-positive CGL. A marrow cytogenetic analysis should reveal the Ph 1 chromosome. However, there are just a few cases reported where karyotypic analysis did not reveal any abnormality but bcr re-arrangement was detected (Morris et al, 1988; Richards et al, 1993). Thus, in patients with a normal karyotype but with atypical features, or developing a particularly raised granulocyte count during follow-up, examination for bcr re-arrmqgement should be done. This is particularly relevant since patients with an apparent diagnosis of PT but

712 T. C . P E A R S O N

who are phi-positive have a significantly worse prognosis than do other PT patients.

Another clue to the correct diagnosis of phi-positive CGL is the marrow histology. Typically, in these patients, while megakaryopoiesis is increased, as in PT, megakaryocytes are smaller and have a more rounded nuclear shape than normal (Thiele et al, 1990; Georgii et al, 1996).

Exclusion of chronic myelofibrosis

Various terms have been used instead of chronic myelofibrosis and include agnogenic mydoid metaplasia, idiopathic or primary myelofibrosis, chronic megakaryocytic granulocytic myelosis and osteomyelofibrosis. Separation of PT from chronic myelofibrosis can occasionally be difficult because patients with chronic myelofibrosis may present in the cellular phase with increased granulocyte and/or platelet counts and PT may undergo myelofibrotic transformation. The features that supports a diagnosis of chronic myelofibrosis include marked splenomegaly, although not all patients have palpable splenomegaly, and the peripheral blood film appearances, notably tear-drop red cell poikilocytosis and leuko-erythroblastic change. A very occasional myelocyte or metamyelocyte can be found in PT blood films, but the red cell changes are absent. The critical examination is the marrow histology.

It is in the cellular phase of chronic myelofibrosis when there may be diagnostic difficulty with PT. In the diagnostic criteria (Table 8) it is stated that collagen fibrosis should be absent or less than one-third of the biopsy area without marked splenomegaly or leuko-erythroblastic reaction. This statement suggests that collagen fibrosis is in some way different to the deposition of reticulin, which is in fact just the earliest phase of marrow fibrosis. In addition, the statement relating to changes affecting one-third of the biopsy area suggests a degree of precision in reporting marrow histology. The fact that the marrow histology under scrutiny may not be a representative sample (Bartl et al, 1993) and that reporting is subjective not objective should be taken into account when evaluating this criterion.

The finding of a marked increase in reticulin, particularly in a marrow with granulocytic and megakaryocytic hyperplasia, should move the diagnosis from t ~ to chronic myelofibrosis. Some authors have empha- sized the megakaryocytic nuclear and cellular pleomorphy, which is seen in the early stages of chronic myelofibrosis (Georgii et al, 1996). Additionally, these authors recognize an early stage of what they term 'chronic megakaryocytic granulocytic myelosis' where there is little or no reticulin fibre formation. Fortunately, generally, the histological features of PT are quite distinct (see below) from chronic myelofibrosis but there is overlap in the appearances in a few patients.

Exclusion of mydodysplastic syndromes

There are just a few patients with MDS where a thrombocytosis is a feature at presentation. In all patients under evaluation for PT, a blood film should


be carefully examined for evidence of trilineage dysplasia, which is the hallmark of MDS. These dysplastic features are reflected in the marrow. Megakaryocyfic hyperplasia, sometimes with marked reticulin fibrosis, may be found in some patients (Pagliuca et al, 1989; Maschek et al, 1992). However, the megakaryocyte morphology in MDS is quite distinct from that in PT. In MDS, megakaryocytic dysplasia with small hypolobulated forms are characteristically found (Pagliuca et al, 1989; Maschek et al, 1992). Cytogenetic abnormalities are rarely found in PT but are observed in 30-50% of patients with MDS. There are a variety of karyotypic changes that have been described, although patients with 5q- have been described as having a thrombocytosis more consistently (Boultwood et al, 1994). However, a raised platelet count can be observed in MDS patients with other karyotypic changes and also in those with a normal karyotype (Pagliuca et al, 1989).

Splenomegaly Splenomegaly can occur in a wide variety of pathological processes, including the other myeloproliferative disorders. Thus, splenomegaly cannot be taken on its own as a diagnostic marker of PT. The context in which it has been proposed to be of diagnostic value is in the patient with isolated thrombocytosis where other pathologies and myeloproliferative disorders have been excluded (Dudley et al, 1989; Carneskog et al, 1996; Michiels and Juvonen, 1997). Splenic size may be assessed by various scanning techniques but ultrasound is widely available. Splenomegaly is less commonly seen in PT than in PV, but up to 50% of patients with PT have modest splenic enlargement. If ultrasound scanning is going to be used to assess splenic size for diagnostic purposes the problems of establishing a normal reference range and observer error must be appreciated (Messinezy et al, 1997).

Haemopoietic progenitor culture techniques Many authors have shown that the presence of endogenous erythr , ' : colonies (EEC) is a reliable marker of PT and is found even in treated patients (Dudley et al, 1989; Turhan et al, 1992; Juvonen et al, 1993; Florensa et al, 1995). The percentage of PT patients with positive EEC findings is lower than that found in PV, and given the reports so far, gives a diagnostic sensitivity in the order of 65% (Westwood and Pearson, 1996). One group has suggested that BFU-E studies could be used as a positive marker for PT (Dudley et al, 1989). However, culture studies are not widely available, are expensive and technically demanding and are not amenable to external quality assurance. Thus, while there may be a place for the investigation in the occasional patient, there are limitations to their wide- spread use.

Considerable efforts have been expended on culture studies examining CFU-Mk growth, particularly in cultures deprived of relevant growth factors--so-called endogenous megakaryocytic colonies (EMC), analogous

714 T. C. PEARSON

to EEC. In those studies where appropriate controls have been examined, the presence of EMC has been shown to be specific for PT by some authors (Juvonen et al, 1993; Florensa et al, 1995; Bri~re et al, 1997) but not by others (Grossi et al, 1987; Hart et al, 1989; Sawyer et al, 1994). The discrepancies in the results obtained in these studies indicate that subtle nuances in the culture technique and/or colony identification can influence the outcome and interpretation. Thus, if the presence of EMC is to be used as a diagnostic marker of PT careful validation and control of the methodology is essential. Unfortunately, this does not render it suitable for general use.

Examination of clonality

Establishing clonality in a patient suspected of having PT by finding an acquired marrow karyotypic change is rarely possible because chromosome findings are usually normal in PT. Examination of X-chromosome inactivation patterns in the granulocytes of PT females has been achieved using probes for various genes, although using the andogen receptor gene (HUMARA) has proved the most useful. It is essential to use the patient's T lymphocytes as a control (Gale et al, 1994) to avoid the misinterpretation of extreme lyonization (skewed X-inactivation) as indicative of clonality. Approximately two-thirds of females, identified as having PT on other criteria, show monoclonality of their granulocytes (Turhan et al, 1992; E1-Kassar et al, 1995). Determination of the allelic expression of three other X-linked genes (G6PD, IDS, P55) has shown monoclonality in both granulocytes and platelets, although in a small proportion of patients monoclonality could be shown only in their platelets (E1-Kassar et al, 1995; Bri~re et al, 1997). The value of demonstrating monoclonal granulocytes as a diagnostic marker has recently been challenged by the finding that 20-30% of normal elderly females show an apparently clonal granulocyte population (Champion et al, 1997; Gale et al, 1997). However, the useful- ness of X-linked probes in showing clonality in younger women needs to be explored by making comparison with an appropriate age-matched control group.

Marrow histology

There are characteristic changes in the marrow histology in PT. An experienced observer may be able to make a diagnosis of PT by just using the histological appearances (Georgii et al, 1996), although there is a need for a standardized approach to reporting. Cellularity may be increased, normal or rarely reduced (Bartl et al, 1993). Generally, megakaryocytes are moderately to markedly increased in number. A large proportion of the megakaryocytes are enlarged with multilobulated nuclei, and they tend to cluster in small groups close to the sinusoids (Georgii et al, 1 9 9 6 ) - although a variant type, which may represent an earlier stage of the disease, shows a more diffuse distribution of megakaryocytes (Bartlet al, 1993). Erythropoiesis is typically not increased but granulopoiesis is rather


variable, and some authors have proposed a subdivision into those patients with and those without an increase in granulocytic precursors (Burkhardt et al, 1986). In the majority of patients normal or only a modest increase in reticulin is found (Georgii et al, 1996). However, some patients transform to myelofibrosis with a marked increase in reticulin content of their marrows. The difficulty of separating patients presenting de novo with chronic myelofibrosis from patients with PT undergoing transformation to myelofibrosis has been discussed previously and can present diagnostic difficulty.

The ploidy of megakaryocytes has been examined by flow cytometry. The characteristic finding in PT, reflecting the histological findings, is to find a larger proportion of cells with a higher ploidy number than normal (Jacobsson et al, 1996). This separation of PT from ST by examination of megakaryocyte ploidy should be helpful, but occasionally increased ploidy can be observed in ST (Tomer et al, 1989).

Serum thrombopoietin (TPO) and erythropoietin (Epo) levels

It might have been expected that serum TPO levels would be low in ST and PT if a similar feedback mechanism existed for TPO as for the control of Epo production. However, serum TPO levels are normal or elevated in both ST and PT (Cerutti et al, 1997; Horikawa et al, 1997; Pitcher et at, 1997). Thus the control mechanism of TPO production is complex and the measurement of serum TPO levels are not useful in the separation of PT from ST. Reduced levels of the TPO receptor on the platelets of patients with PT have been described (Horikawa et al, 1997) but this was not confirmed by other authors (Moliterno et al, 1998).

Serum Epo levels have been shown to be lower than expected in a significant proportion of patients with PT with results similar to PV patients (Najean et al, 1995). In another study, comparison of serum Epo levels in ST and PT did not reveal a useful separation once the haemoglobin concentration had been taken into account (Messinezy et al, 1994). Thus, the measurement of serum Epo would appear to be of no diagnostic value for PT.

SUMMARY OF THE DIAGNOSTIC TESTS FOR PT

The diagnosis of PT is more difficult than that for PV because the only objective marker of the disease is a raised platelet count and this may be observed in other myeloproliferative disorders and in secondary thrombocytoses. In a clinical trial setting, a platelet count of >600 x 109/1 has been arbitrarily selected as an essential for the diagnosis of PT, although an elevated count >450 x 109/1 with clinical features suggestive of PT might warrant investigation and lead to the diagnosis. Exclusion of a 'reactive state' requires a detailed history and appropriate investigations. The full blood count and film might indicate the possibility of a myeloproliferative disorder, including myelodysplasia. A RCM measurement, after iron

716 T . C . PEARSON

therapy where appropriate, is required to exclude an absolute erythrocytosis. Examination of the bone marrow histology and cytogenetics are key investigations to exclude chronic myelofibrosis, myelodysplasia and chronic granulocytic leukaemia. At the moment there are no agreed objective criteria in the histological appearances of PT for its definitive diagnosis. However, the majority of patients not fitting into any other cause of thrombocytosis have characteristic changes in their marrow histology, which enable a diagnosis of PT to be made.

Acknowledgements

The author would like to thank Monica Nestor for her excellent secretarial assistance. Research in the author's laboratory is supported by The J. L. Beckwith Charitable Trust.

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1 diagnosis and classification of erythrocytoses and thrombocytoses

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