NEW TOOLS FOR CLINICAL EVALUATION OF ERYTHRON FUNCTION IN MAN

British Journal of Huernatology, 1992, 80, 278-284

Annotation

NEW TOOLS FOR CLINICAL EVALUATION OF ERYTlIKON FUNCTION IN MAN

The diagnostic approach to anaemia is based on a battery of tests that define the nature of the abnormality in erythron function (Hillman & Finch, 1985). In particular, the initial separation of anaemia into one of three major categories (hypoproliferative, maturation defect, or haemolytic anaemia) requires measurements of red cell production and destruction (Cazzola & Finch, 1987). Some of these measurements, however, involve laboratory methods that are unreli- able or cumbersome in a clinical setting, so that classification of anaemia is not infrequently inaccurate.

In the last years, new tools have been developed for clinical evaluation of erythron function in man. The basic compo- nents of erythropoiesis are erythroid progenitors, their stimulation by erythropoietin, and adequate supply of iron from which to make haemoglobin (Finch, 1982). Endoge- nous erythropoietin production can be blunted in several anaemic conditions, and determining serum erythropoietin levels has provided clinicians with a convenient means of assessing this mechanism of anaemia. On the other hand, serum erythropoietin has proved to be of value also in the differential diagnosis of erythrocytosis.

Iron delivery to erythroblasts is mediated by the interaction of plasma transferrin with surface transferrin receptors (Huebers & Finch, 19 87). One of the most valuable advances in the clinical assessment of erythropoiesis has been the detection of soluble transferrin receptors in human plasma. This has proved to have important clinical implications, since the soluble receptor level has been shown to be closely related to the number of red cell precursors in the bone marrow and to provide a reliable quantitative assay of the rate of erythropoiesis.

This annotation discusses the clinical significance of serum erythropoietin and circulating transferrin receptor.

Erythropoietin response to anarmia and bhtrited erythropoietin production Assessment of serum erythropoietin has become practicable as a routine diagnostic procedure with the availability of validated immunoassays (Garcia et ul, 1982: Cotes et ul. 1986:Erslevetal. 1987:LappinetaL 1988).These havenow replaced bioassays such as the polycythaemic mouse assay or the mouse spleen cell assay. In fact, although the behaviour of the immunologically active erythropoietin may differ from that of the biologically active glycoprotein under certain circumstances (Rich, 19 91 ), immunoassays are reliable enough for clinical purposes.

Serum erythropoietin levels in normal individuals are in

Correspondence: Professor Mario Cazzola, Clinica Medica 2, Policli- nico S. Matteo. 27100 Pavia. Italy.

the range of 10-30 mU/ml. In the laboratory diagnosis of anaemia, however, serum erythropoietin should not be quantitated in absolute terms but evaluated in relation to the degree of anaemia. In fact, provided that the erythropoietin- generating apparatus in the kidney is efficient, levels increase exponentially as the haematocrit decreases (Beguin et a/ , 1990: Erslev, 1991: Garciaetal. 1982). Consequently, serum erythropoietin levels must be expressed in relation to the haematocrit (Garcia rt al, 1982: Eckardt 0; Bauer, 1989). This can be done by determining the exponential regression of serum erythropoietin versus Hct in reference subjects, and defining the 9570 confidence limits (Fig 1). In our own studies, we computed two regression equations since the relationship between serum erythropoietin and Hct was found to change at the cutpoint Hct of 38%. The regression equation was log(EPO) =4.746-(0.093 x Hct) for Hct values < 3 8%. and log(EPO) = 1.3 8 1 - (0.00 5 x Hct) for Hct values > 38%.

The wide range of appropriate erythropoietin levels for anaemic patients may suggest that factors other than tissue hypoxia are physiologically involved in the regulation of erythropoietin production. Although findings are controver- sial, evidence has been presented for an abnormally high erythropoietin production in patients with erythroid hypo- plasia and in those with iron deficiency (see review by Liberato et al, 1990). The red cell precursor mass might intluence erythropoietin clearance, whereas the iron status might affect the haem protein operating as oxygen sensor (Goldberg et al, 1988). This latter mechanism would explain the rapid fall in serum erythropoietin observed within 24-48 h after starting iron treatment in patients with iron deficiency, before any apparent change in Hb concentration (unpublished observations). It is possible that further studies might lead to defining a reference range which would account for the body iron status and the degree of erythroid proliferation.

In clinical investigations, the adequacy of erythropoietin response to anaemia has been generally evaluated by comparing the regression curve of serum erythropoietin versus haematocrit obtained in a group of patients with that of reference subjects (Baer et a!, 1987: Miller et a/, 19Y0: Beguin et ul. 1991 a, b). When studying a n individual patient, appropriateness oferythropoietin response to anaemia can be evaluated graphically as indicated in Fig 1 , or through the observed/predicted log(erythropoietin) ratio (O/P ratio). The O/P ratio averaged 1.00f0.11 in our reference subjects (95% confidence interval: 0.80-1.19): values lower than 0.80 indicate an inadequate erythropoietin response to anaemia.

Blunted erythropoietin production. Decreased erythropoietin

2 78

Annotation 2 79

15 20 25 30 35 40 45 ! HCt, Yo

Fig 1 . Relationship between serum erythropoietin levels and haematocrit in reference subjects: lines are the 95% confidence limits. Values below this reference range indicate an inappropriate erythropoietin response to anaemia.

stimulation is the cause of anaemia in various disorders, including renal disease, protein deprivation and endocrine deficiencies (Hillman & Finch. 198 5). Erythropoietin production is typically suppressed in chronic renal failure because of a damage to the erythropoietin-generating apparatus (Garcia Pf 01, 1 982), and levels that are inappropriately low for the degree of anaemia have been reported in anaemia of prematurity (Brown et u/, 19x3). pregnancy (Beguin r t N / ,

I Y Y O , 1991b). rheumatoid arthritis (Baer Pt al, 1987: Hochberg et a/.. 1988) , AIDS (Spivak P t a1, 1989; Fischl r t a/. 1990). sickle cell anaemia (Sherwood et u1, 1986). anaemia of cancer (Miller ( I t a/ , 1 YYO), lymphoproliferative disorders (Cazzola et al, 1Y92), and allogeneic bone marrow transplantation (Beguin rf d, 1991a; Bosi et al. 1991). Although different pathogenetic mechanisms are responsible for anaemia in these conditions. blunted erythropoietin production is the common denominator. This constitutes a rationale basis for the treatment of many of these conditions with recombinant human erythropoietin (Winearls ( I t a], 1986: Eschbach e t d, 1987. 1989; Goldberg f t al, 19 YO: Ludwig ef ul, 1990; Oster P t a/, 1990: Fischl rt ul, 1990; Pincus Pt al, 1990; Cazzola r t al, 19 9 2). In our phase 1/11 clinical trial on the use of subcutaneous erythropoietin for treatment of refractory anaemia in haematologic disorders. a low O/P ratio was found to be a good predictor of response (Cazzola et a/. 1 9 9 1 ).

Srrum mytkropoicltin arid the di/fPrrntia/ diagr7osis of Prythroq- tosis The diagnostic approach to erythrocytosis is first based on determination of red cell mass (to distinguish between absolute and relative erythrocytosis) and arterial oxygen saturation (to recognize tissue hypoxia which is a frequent cause), as shown in Fig 2. Moreover, in most patients with

Erythrocytosis (elevated Hb)

cell mass

Relative erythrocytosis

Increased

Yes

erythrocytosis

Decreased

No + Measure Hb PYJ Li

Decreased

No

Inappropriate

production

<->4-] High serum erythropoietin?

No

or genetic disorder

Fig 2. Clinical algorithm for a physiological approach to the diagnosis oferythrocytosis. As shown, measurement of serum erythropoietin is useful in identifying patients with secondary erythrocytosis who have inappropriate erythropoietin sccretion.

secondary erythrocytosis or polycythaemia Vera, diagnosis is indicated by the clinical features of the basic condition or the haematological features of a myeloproliferative disorder. ‘The diagnostic value of determinations of serum erythropoietin has been studied by Cotes et ul (1986). These investigators have shown that measurement of serum erythropoietin in ii

single sample may be misleading. and may not have high discriminatory value in distinguishing between polycythaemia Vera (normal to low values) and secondary erythrocytosis (normal to high values). However, according to the clinical algorithm reported in Fig 2, serum erythropoictin is useful in a late step of the diagnostic approach. Once disorders

2 80 Annotation

of oxygen loading and unloading are excluded, serum erythropoietin allows distinction between erythropoietin- independent erythrocytosis (myeloproliferative disorders, familial disorders with increased sensitivity to erythropoietin) and erythropoietin-dependent erythrocytosis (neoplastic or genetic disorder with inappropriate erythropoietin secretion).

Erythroid marrow activity: from ferrokinetic nieasurenients to circulating transferrin receptor The erythron is a very dynamic organ and undergoes marked changes in disease. In a normal adult of 75 kg. the total number of red cell precursors in the bone marrow is about 1 2 x lo", and every second about 2 .6 x loh newly formed red cells (reticulocytes) enter circulation (Cazzola & Finch, 1987). Virtually no red cell precursor is found in the bone marrow of a patient with severe aplastic anaemia, whereas their number can be as high as 2 0 times normal in a subject with thalassaemia intermedia. Clinicians clearly need a convenient means of assessing such changes, and until recently ferrokinetics has provided the best way of character- izing abnormalities of the erythroid function.

FerrokinPtic measurements. Marrow erythroblasts normally receive about four-fifths of all iron passing through the plasma so that a relationship does exist between erythroid marrow function and iron turnover. The most simple ferrokinetic measurement, the plasma iron turnover (PIT), gives a poor description of the erythroid proliferation in conditions of normal or reduced erythropoietic activity (Finch et a/, 1970). Useful ferrokinetic measurements can be obtained by using complex ferrokinetic models (Cavil1 & Iticketts, 1980; Stefanelli et a/ , 1982): these have been of fundamental importance in obtaining a deeper insight into iron metabolism and erythropoiesis. but require sophisticated processing of experimental data over 10-14 d and are not practical for clinical usage.

We have developed a simple ferrokinetic approach to quantitation of erythropoiesis by applying knowledge con- cerning iron exchange between transferrin and body cells to ferrokinetic measurements (Cazzola eta ] , 1985, 1 9 8 7 ~ ) . The procedure requires only a few hours, during which time the Tl l r radioiron disappearance is determined and blood is drawn for assay of haematocrit, plasma iron and total iron binding capacity (TIBC). Through simple calculations, the erythron transferrin uptake (ETIJ) can be derived. Data have been presented by us and others to indicate the validity of this measurement in quantitating erythroid marrow proliferation in various anaemic states (Cazzola et a/, 1987a, b, c: Eschbach eta/, 198 7; Beguin et al, 1988b: Uchida et al, 1988; Hughes et al, 1990; Howarth et a/, 1991).

The ETIJ determination involves negligible radiation hazard and patient discomfort. However, its clinical use must be confined to situations in which otherwise unavailable information of diagnostic or therapeutic importance can be obtained. The recently developed plasma transferrin receptor assay promises to become the routine measurement of erythropoiesis.

Soluble transferrin receptor. The functional transferrin receptor (TfR) is composed of two monomers linked by two disulphide bridges to form a molecule of 1 9 0 0 0 0 daltons

(Huebers & Finch, 1987; Schneider et al, 1984). Each monomer has a N-terminal cytoplasmic domain, a short trans-membrane segment, and a large extracellular segment. One receptor can bind one or two transferrin molecules (Intragumtornchai et a/, 1988). After binding of iron-bearing transferrin, the receptor-transferrin complex is internalized; iron is released to the cytoplasm. the receptor returns to the cell membrane and apotransferrin to plasma (Seligman et a/, 198 7; Huebers & Finch, 198 7).

Virtually all cells have transferrin receptors on their surface, but in a normal adult about 80% of them are in the erythroid marrow (Intragumtornchai et al. 1988; Huebers & Finch, 1987). Receptor density on cells is regulated by a number of factors, the most important ones being cellular iron status, erythropoietin stimulation, and cell cycle (Rao et a / , 1985; Sawyer & Krantz, 1986; Caaaola et a/ , 1990). Deprivation of iron results in prompt induction of transferrin receptor synthesis, whereas the reverse happens with excess iron (Rao et a / , 1985). Erythropoietin stimulation increases the number of receptors (Sawyer & Krantz, 1986). The proliferative status of the cell is a factor able to affect transferrin receptor expression per se, with receptor number increasing during the S phase of the cell cycle and decreasing following induction of differentiation and slowing of cell proliferation (Cazzoia et nl. 1990).

The complete disappearance of transferrin receptors during maturation of reticuiocytes to mature red cells has been documented (Pan et al, i983) . Receptors appear to be selectively externalized in smal! vesicles which have been retrieved from the circulation of sheep, rat, pig, and rabbit (Pan & Johnstone, 1983; Johnstone rt a/ , 1989; Harding r t a/. 1984). These exosomes contain molecules characteristic of the plasma membrane, but no mitochondria1 or cytoplasmic activities (Johnstone r t a/, 1989). Transferrin receptors also appear to be released from the surface of HL60 and K562 cells in culture, either directly or after release from exosomes (Chitambar & Zivkovic, 1989; Baynes et a/., 1990).

Soluble TfR has been identified in rat (Beguin et al, 1988a) as well as human plastna (Huebers et nl, 1990; Kohgo ot a], 1986, 1987; Flowers r t a / , 1989). in the form of a complex of transferrin and its receptor. Evidence has been presented that plasma T R is a truncated form of tissue receptor, with truncation occurring between aminoacid 100 and 101, at a site just beyond the cell membrane (Shih et ul. 1990). The generation of soluble receptors is likely to be due to a proteolytic mechanism, although the site and nature of the specific protease activity remain to be identified (Baynes et a1. 1990).

The circulating complex is probably made by two receptor monomers binding to one transferrin molecule, with a total molecular weight of about 250 000. The fate of this complex has not been identified so far, and we do not know whether its plasma level depends not only on production but also on the rate of destruction.

To measure plasma TfR levels, ELISAs as well as KlAs have been set up with polyclonal or monoclonal antibodies raised against purified tissue receptors (Beguin r t al, 1988a; Huebers et a/. 1990: Kohgo et a/. 1986: Flowers et a/, 1989). Using a n ELISA based on polyclonal antibodies, receptor level

Annotution 2 8 1

averaged 5000 =k 1 100 pg/l (mean f 1 SD) in a group of 165 normal human subjects, and no difference was observed with sexoragein the 1&78yearagerange(Beguin, 1991).These values are considerably higher than those initially reported by Kohgo et a1 (1 986), hut this may relate to their use of low- affinity monoclonal antibodies (Kohgo et 41, 1986) and problems with standardization (Flowers et al. 1989).

The erythroid marrow is the main source of soluble transferrin receptors. In fact, decreased TfR levels are found in patients with erythroid hyperplasia, such as severe aplastic anaemia, chronic renal failure, or after intensive chemo- therapy, and increased levels in those with erythroid hyperplasia, such as autoimmune haemolytic anaemia, hereditary spherocytosis and thalassaemia (Huebers et al, 1990; Flowers et al. 1989; Kohgo et uI, 1987; Beguin, 1991). The plasma TfR represents a remarkably constant fraction (about 6%) of tissue receptor over a wide range of erythropoietic activity (Huebers et nl. 1990: Intragumtornchai et a!, 1988) . The dependence of receptor levels on the activity of the erythron is further demonstrated by the strong correlation observed between plasma TW (ranging from about 800 to 100000 pg/l) and ferrokinetic measurements of erythropoiesis, in rats (Beguin et d. 198th) as well as in humans (Huebers et ul, 1990). The lowest value of about 1000 pg/l probably represents the contribution of nonerythroid tissues to plasma levels.

The direct relationship to the number of erythroid precursors and the simple methodology should make soluble TW assay the method of choice for evaluation of erythroid marrow activity in a clinical setting. It can be used to study erythropoiesis in situations in which ferrokinetics would not be acceptable, such as pregnancy (Heguin et al, 199 1 b). TfK measurements are also ideal for situations in which serial assessments would be needed, such as monitoring the recovery of erythropoietic activity after bone marrow transplantation (Beguin, 1991) or the erythropoietic response to recombinant human erythropoietin in renal patients (Reguin et ul, 1987). In our phase 1-11 clinical study on subcutaneous erythropoietin for treatment of refractory anaemia in haematologic disorders. circulating transferrin receptor appears to be the earliest and most reliable predictor ofresponse (Cazzola et ul, 1992).

Plasma transferrin receptor level is probably less reliable than the ETU measurement in evaluating reduced erythropoiesis, due to the contribution of nonerythroid tissues (Plowers et ul. 1989). IJnder these circumstances, however, the clinician will normally rely also on a bone marrow biopsy and a reticulocyte count. Moreover, it might be possible to apply some form of correction to TfR levels in order to remove the contribution from nonerythroid tissues and make measurements reliable also in assessing diminished erythropoiesis.

The body iron status is the second major determinant of circulating TfR. As compared to normal values, TfR levels are about 2 5% lower in patients with idiopathic haemochroma- tosis and about 20% higher in non-anaemic iron-deficient subjects (Huebers et a], 1990; Skikne et al, 1990; Beguin, 1991). In a study of normal volunteers undergoing serial phlebotomies, TW levels did not change significantly during

the phase of storage iron depletion, but did increase following appearance of functional iron deficiency, defined as a reduc- tion in body iron beyond the point of depleted iron stores, i.e. iron deficient erythropoiesis (Skikne et al. 1990). It was concluded that the main determinant of such increase was iron deficiency. and that there was little or no influence of increased erythropoiesis in the receptor level. It was also concluded that a battery of tests including serum ferritin, soluble TfR and Hb is very useful in population surveys. In fact, serum ferritin can be used as a measure of iron stores, circulating TfR as a measure of early tissue iron deficiency, and Hb as a measure of advanced iron deficiency.

Other studies, however, have shown that TfK levels in anaemic iron-deficient subjects increase in proportion to the increase documented for erythron receptors, but no more so than in patients with haemolytic anaemia and similar haematocrits (Kohgo et al, 1988; Intragumtornchai et (11, 1988; Reguin et ul. 1990; Huebers et ul, 1990; Beguin, 199 1 ). Thus, although iron deficiency does increase transferrin receptor number on individual erythroblasts. it is uncer- tain whether it affects plasma TfK levels much beyond what is expected from the degree of anaemia and the resulting enhanced erythropoietin stimulation. More detailed studies are warranted to assess the respective role of anaemia, iron depletion and erythropoietin stimulation on plasma TfK levels. For practical purposes, however, interpretation of elevated TfK levels requires evaluation of body iron status to distinguish between iron deficient erythropoiesis and increased erythroid mass. Since normal to low values for soluble TfK are found in patients with the anaemia of chronic disease, a condition known to be associated with defective erythropoietin production. circulating TfK can be useful in the differential diagnosis between this condition and iron deficiency (Skikne (,t (81, 1990).

Increased expression of transferrin receptors has been documented on the surface of nialignant tumour cells as compared to their normal counterparts (l‘rowbridge c ~ t al, 1984). However. data on receptor levels in patients with a variety of myeloid. lymphoid and non-haematologic malignancies suggest that, with the exception of polycythaemia Vera and chronic lymphocytic leukaemia, the possible contribution of neoplastic cells to plasma TfR levels is negligible (Huebers et d. 1990: Klemow et al. 1990; Beguin. 1 Y 9 1 ).

Puthoph~~siol~?qic,Lri classijcation of anuerriiu In the presence of a normal marrow stem cell reserve and of adequate erythropoietin production and iron supply to the erythroid marrow, erythropoiesis i,ncreases in proportion to the degree of anaemia because of enhanced erythropoietin stimulation (Hillman & Pinch. 1985: Cazaola e t nl, 1 9 8 7 ~ ) . Therefore, measurements of serum erythropoietin. circulating TM and reticulocyte count may represent the template on which to base a functional classification of red cell disorders according to whether there is a defect in proliferation (hypoproliferative anaemia). maturation (ineffective erythropoiesis) or red cell survival (peripheral haemolysis) (Pig 3 ) .

Erythroid marrow activity can be assessed by measuring the plasma TfR level, and is expressed in relation to the mciin basal value, dividing the patient’s TfR level by the mean

282 Annotation Circulating transferrin receptor

(times basal

10

1

0.1

Ineffective Peripheral erythropoiesis haemol ysis 00

HYPO- proliferative 0 anaemia

0.1 1 10 Reticulocyte index (Yo)

Pig 3. Schematic representation of a functional classification of anaemia based on the circulating transferrin receptor level (as a measure of the erythroid marrow activity) and reticulocyte index (as a measure of effective erythropoiesis).

normal value of 5000 pg/l. A simultaneous measurement of serum ferritin should be performed to distinguish between iron-deficient erythropoiesis and increased erythroid mass. As shown for serum erythropoietin, it would be useful to determine the exponential regression of plasma TfK versus Hct in reference subjects and to define the 95% confidence limits. This would allow the diagnosis of inadequate erythroid marrow response to anaemia.

Alternatively, an empirical approach can be employed for practical purposes. An erythroid activity equal to or greater than 3 times basal in the presence of longlasting anaemia indicates an appropriate proliferative response to anaemia, or adequate proliferative capacity of the erythroid marrow (Cazzola et al, 1 9 8 7 ~ ; Cazzola & Finch, 1987). Among conditions associated with a normal proliferative response to anaemia, the reticulocyte index (Hillman & Finch, 198 5) can make a distinction between ineffective erythropoiesis and peripheral haemolysis. A reticulocyte index of greater than 3 times basal is taken to indicate an adequate red cell production (i.e. peripheral haernolysis), while an index of less than 2 times basal is assumed to represent impaired red cell production (i.e. ineffective erythropoiesis).

When erythroid activity is lower than 3 times normal in the presence of continual anaemia, a proliferative irnpair- ment of erythroid marrow can be assumed. This condition is defined as hypoproliferative anaemia. Impaired iron supply to the erythroid marrow and blunted erythropoietin production play prominent causative roles (Finch. 1982) that can be recognized by evaluating body iron status and determining serum erythropoietin. Excessive production of suppressive cytokines and stern cell defects represent further causes of erythroid proliferation (Cazzola & Finch, 1987; Cazzola et (11, 198 7a).

Conclusions Determining serum erythropoietin and soluble transferrin

receptor in addition to blood cell counts provides clinicians with convenient means of assessing the pathogenetic mechanisms of anaemia and erythrocytosis. Serum erythropoietin level allows the diagnosis of blunted erythropoietin production which in turn represents a rational basis for erythropoietin treatment. Measurement of serum erythropoietin is also useful in the differential diagnosis of erythrocytosis for identifying conditions with inappropriate erythropoietin production. The soluble transferrin receptor assay is a very simple, noninvasive method of quantitating erythroid marrow activity in a clinical setting. It is particularly useful for serial studies, e.g. for monitoring erythropoiesis during erythropoietin treatment. In addition, it may be also employed in concert with serum ferritin to identify iron- deficient erythropoiesis.

ACKNOWLEDGMENTS

This work was supported by grants from AlKC (Associazione Italiana per la Ricerca sul Cancro) and CNR (Consiglio Nazionale dell Ricerche) to Mario Cazzola, and by grants no. 3.4513.88 and 3.4555.91 from FRSM (Fonds de la Recherche Scientifique Medicale. Belgium) to Yves Beguin.

Department of lnternal Medicine MARIO CAZZOLA and Medical T h u a p y , YVES BBGUIN Universi ty of Pavia, Pavia, Italy, and Department of Medicine, Division of Haematology, Universi ty of Liege, LiPge. Belgium

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