British joirrrrnl o/ Hacmntology. 19x6. 6 2 , 209-2 1 3

Annotation

RED C E L I , F E R R I T I N AS A DIAGNOSTIC' TOOT,

Clinical interest in ferritin was aroused in the early 19 70s by the observation in the Cardiff laboratories that there was a quantitative relationship between the level of serum (or plasma) ferritin and the amount of storage iron (Addison et a!, 1972) . Since then, this measurement has been extensively used to evaluate body iron stores, in particular to detect both iron deficiency and iron overload and to monitor their treatment (Tipschit2 r t a!, 19 74: Jacobs R Worwood, 1975; Worwood, 1982). At the same time, it has become clear that the serum ferritin coiicentration is affected by a number of factors other than the amount of storage iron. In fact, an elevated serum ferritin is most frequently dut: to tissue necrosis, inflammation, neoplastic disease arid increased red cell turnover ratht:r than increased iron stores (Lipschih e l ml. 19 74; Worwood. 1982).

Most recently, attention has focused on red cell fcrritin after a few investigations showed that the ferritin content of red cells is a reflection of body iron status (Peters et al, 1983; Cazzola et ai. 1983a; Van der Weyden et al , 1 98 3a) and appears to be only slightly influenced by those factors which falsely elevate the serum ferritin concentration to a degree disproportionate to that of iron stores (Cazzola et a] . 1984).

Metltociological problems. The red cell ferritin assay is obviously more cumbersome than the seriirn ferritin determination. It is essential to work on pure red cell suspensions to avoid contamination by leucocyte ferritin. since the ferritin content ofa white blood cell can be as much as 1000 times that of a red cell. Pure red cell suspensions can be obtained by means of the classical technique of Beutlcr P t nI ( 1976) or using the simpler method devised by Van der Weydcn et al (1983a). Erythrocytes can be lysed in hypotonic solutions, by sonicatiori or by rapid freeze-thawing in liquid nitrogen. Stroma should be removed from red cell lysates before ferritin assay because they can interfere with the immunoassay.

Unlike serum, which contains almost exclusively basic (L-sabunit rich) ferritin that reacts with antibodies against human liver or spleen ferritin, human red cells contain both basic and acidic (€1-subunit rich) isoferritins, with the latter predominating under riornial conditions (Peters et a!, 198 3 : Caxxola et nl, 198 3a). For research purposes, therefore. red cell ferritin should be determined simultaneoiisly using two irnniurioassays, one for basic and the other for acidic ferritin, whereas a cornrricrcially available assay based on antibodies against liver or spleen ferritin alone provides enough information for clinical purposes.

Biological significunw of red cell isqferritins. Red cell ferritin is a residue of erythroblast ferritin, and in order to better understand its clinical signiticancc it is necessary to be aware of the main pathways of iron metabolism within haem-synthesizing erythroid cells. Iron is

Correspondence: Dr Mario Cazzola, Patologia hledica 1, Policlinico S . hlatteo 1-2 7 100 Pavia. Italy.

209

210 Annotation

delivered to erythroblasts by plasma transferrin which interacts with its receptor on the cell surface. The plasma transferrin iron pool is composed of three molecular species, two different monoferric arid one diferric transferrin, the latter having a far greater capacity to dispense iron to tissues (Hucbcrs et al. 198 3 ) . The preferential uptake of diferric transferrin by tissue receptors determines a progressive increase in erythroblazt and reticulocyte iron uptake when plasma iron and transferrin saturation levels rise. Although most of the iron taken up by immature red cells is used for haemoglobin synthesis, the excess has to be stored in ferritin molecules. Thus, the more iron incorporated by immature red cells, the higher the red cell ferritin content. In conditions such as the thalassaemia syndromes or sideroblastic anaemia, haemoglobin synthesis is variably reduced. As erythroblasts take up normal amounts of iron under such conditions, the portion of the metal not utilized for haemoglobin synthesis must be stored in ferritin molecules, and red cell ferritin is increased. Transferrin saturation and the rate of haemoglobin synthesis, therefore, appear to be the major determinants of the red cell ferritin content. although other factors, e.g. proliferative activity of the erythroid marrow and release of ferritin from immature red cells. are operative and can be important in individual cases.

There is uncertainty about the possible functions of basic and acidic isoferritins within erythroid cells (Cazzola et al , 1984). Iron uptake is maximum in early erythroblasts, and it is believed that acidic ferritin provides a temporary reserve of iron which is later available for haem synthesis (Jacobs et al, 1984). On the other hand, basic ferritin appears to be a more stable intracellular depot for excess iron (Cazzola et al, 1984).

Cliniral application. At present this concerns essentially basic ferritin. Normal values range from 3 to 40 ag/cell (ag = attogram, 1 ag = 10 ~ g). The first possible diagnostic use of the red cell ferritin assay is based on thc concept that red cell fcrritin reflects the balance between iron supply to the erythroid marrow and the need for haemoglobin synthesis. Poor iron supply to the erythron results in low red cell basic ferritin, as is typically found in iron deficiency anaemia and iron deficient erythropoiesis (Camola et ul, 1983b). While there is usually no difficulty in diagnosing overt iron deficiency anaemia, the real challenge is to identify the iron deficient patient before a typical microcytic/hypochromic anaemia appears (Hillman & Finch, 1985). The red cell ferritin content can be used along with red cell protoporphyriii as an indicator of decreased plasma iron supply and iron deficient erythropoiesis. For example, Bodemann et nI (1 9843 found this measurement clinically useful in monitoring blood donors.

lnflammation is characterized by abnormalities in iron metabolism, with low values of plasma iron associated with normal or, more typically, elevated values of serum ferritin (Lipschitz et ul. 19 74; Worwood, 1982). Differential diagnosis between iron deficiency anaemia and anaemia of inflammation can be made easily using plasma iron, TIBC and plasma ferritin (Hillman & Finch, 1985). Red cell ferritin is normal to reduced in inflammation. retlecting the poor iron supply to the erythron (Cazzola et al. 1 983b) but i s not routinely required for differential diagnosis with iron deficiency anaemia. However, longlasting inflammatory anaemia presents a more variable picture and may be difficult to assess accurately (Hillman & Finch, 198 5 ) , and this is one ofthe most frcqucntly encountered conditions in a clinical setting. Arthritic patients, for example, may have a combination of

Annotation 211

true iron deficiency, due to bleeding from anti-inflammatory drug ingestion, and inflamma- tion, with laboratory firidings uncharacteristic of either condition alone. Since plasma ferritin is an inflammatory acute phase reactant, its clinical value as an indicator for iron deficiency is limited in anaemic patients with inflammation and plasma ferritin concentrations above 1 5 pg/L and this makes difficult the decision of whether to give these anaemic patients iron. In these conditions, the red cell ferritin content may provide valuable iinformation. A recent study by Davidson et a1 (1984) on patients with rheumatoid arthritis showed that iron therapy resulted in an appreciable rise in haemoglobin concentration only in patients with microcytic anaemia and low red cell ferritin contents, and that p h m a iron, transferrin saturation and plasma ferritin were not useful indicators for iron deficiency.

Red cell ferritin is increased in patients with iron overload as a consequence of the high intracellular influx of iron into marrow erythroblasts (Cazzola et ul. 1983b). Reasoning that mechanisms of iron uptake from plasma transferrin by parenchymal cells and erythroblasts are similar and that ferritin synthesis is dependent on iron, the red cell ferritin assay has been proposed as a non-invasive means for indirectly assessing tissue iron content in patients with idiopathic haemochromatosis. In a study by Van der Weyden Pt a1 (1 980bJ. the mean red cell ferritin content in patients with untreated idiopathic haemochromatosis was about 70 times that in normal subjects and fell with phlebotomy. lied cell ferritin, however, was still elevated in many patients after phlebotomy despite normal serum ferritin concentration, and it was shown that this reflected persistently high parenchymal iron stores. On the other hand, t.he mean red-cell fcrritin content in patients with alcoholic liver disease and iron overload was increased only sevenfold. To distinguish bctwccn patients with idiopathic hacmochrorriatosis and those with alcoholic liver disease, Van der Weyden et a1 (1983) used the ratio of red cell ferritin (in pg/10i2 cells) to serum ferritin (in pg/l), This ratio was 0 . 5 f 0 . 3 in idiopathic haemochromatosis and 0.03 f 0.04 in alcoholics, and clearly separated the two groups.

Elevated red cell ferritin as an index for potential iron overload should be interpreted with caution in the absence of information regarding a possible thalassaemia syndrome or specific data on folate and vitamin B12 status. In thalassaernia syndromes, in fact. red cell ferritin is significantly higher than in normal subjects and increases progressively with the increasing severity of the disorder (Cazzola et al. 1983b: Piperno et al. 1984). The red cell ferritin assay can be utilized to monitor suppression of erythropoiesis during transfusion therapy in thalassaemia major (Cazzola et rr l , 1983b). Increased values for red cell ferritin have also been found in patients with megaloblastic anaemia, the magnitude of increase being comparable to that in subjects with idiopathic haemochromatosis (Van der Mi'eyden & Fong, 1984; Yamada et ul. 1983). Following vitamin replacement. elevated red cell fcrritiri levels decreased during the period of reticulocytosis and were normal after 4 :months. Disproportio- nately high values for red cell ferritin have also been found in patients with myelodysplastic syndromes. indicating that an abnormality of erythroid iron metabolism is common ir i these conditions (Peters et nl, 1985).

Ked cell ferritin should not be used routinely in the laboratory workup of iron metabolism disorders. but in particular situations, such as those outlined above, it can provide information regarding the iron supply to the erythron and the size of parenchymal iron stores which serum ferritin and other conventional iron mcasurcmcnts can not.

212 Annotution

ACKNOWLEDGMENTS

This work has been supported by grant no. 84.00505.44 from C.N.R., Progetto Finalizzato Oncologia, and a grant from Minister0 della Pubblica Istruzione.

Dipirrtimento di Medicina Interria e Terapia Medica University of Yavin, 27100 Pavia, ItaZy

MARIO CAZZOLA EDOARDO ASCARI

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