BritishJournal ofHaematology, 1981,48,263-272.

Erythropoiesis in Myelofibrosis with Myeloid Metaplasia: Recognition of Different Classes of Patients

by Erythrokinetics

G. BAROSI, M. CAZZOLA, F. FRASSONI, E. ORLANDI AND M. STEFANELLI

Clinica Medica I ‘A. Ferrata’, Istituto di Patologia Medica, Istituto di Informatica e Sistemistica, Univerritd degli Studi di Pavia,

Pavia, Italy

(Received 4 August 1980; accepted f o r publication 1 0 December 1980)

SUMMARY. A quantitative assessment oferythropoiesis was performed by means of a mathematical model of iron kinetics in 26 patients with myelofibrosis with myeloid metaplasia (MMM). A direct relationship between total erythropoiesis and red cell volume was revealed. This ‘inverse’ characteristic of erythropoietic control was the best marker of the proliferative nature of the disease. Three classes of patients were singled out by means of a cluster analysis of the erythrokinetic parameters. In class I (11 patients) the red cell volume was above the predicted normal in all but two patients. Erythropoiesis was sustained a t 5-10 times the normal level with a high degree of ineffective erythropoiesis. A fairly constant absence of erythroid activity over the sacral marrow was demonstrated. In class I1 most of the 12 patients had a decreased red cell volume. Erythropoiesis was sustained at 2-4 times thenormal level with a high degree of peripheral haemolysis. Erythroid activity was recognized over the sacral area in all but two patients. The three patients of class 111 showed a pattern of erythroid failure and had the worst prognosis. It is suggested that these classes represent separate disease forms of MMM.

An abnormal haemopoietic cell proliferation of clonal origin is the primary lesion of myelo- fibrosis with myeloid metaplasia (MMM) (Jacobson et al, 1978). Marrow fibrosis is generally considered to represent a secondary response to the expansion of the abnormal m yeloid population (Jacobson et al, 1978; Groopman, 1980). The end result ofthe cell proliferation and of the disturbed microenvironment is the impairment of normal haemopoiesis. Even if polycythaemia is seen at presentation anaemia is a common finding during the course of the disease.

Several erythroid disturbances have been recognized in MMM by means of radioisotopic studies. Anaemia may be caused by enlargement of the red cell pool in the spleen and by increased plasma volume (Christensen, 1975) but ferrokinetic investigations seem to indicate that in most patients there is increased ineffective erythropoiesis: peripheral haemolysis may also contribute to the anaemia (Storti & Perugini, 1968; Szur & Lewis, 1975; Najean e f al, 1978).

Correspondence: Dr Giovanni Barosi, Clinica Medica I ‘A. Ferrata’, Policlinico S. Matteo, 27100 Pavia, Italy. 0007-1048/81/o6o(M263$02.00 0 1981 Blackwell Scientific Publications

263

264 G. Barosi ct a1

Evidence of erythroid hypoplasia has rarely been reported (Bentley ct a l , 1977). Despite the large number of cases studied there is no conclusive interpretation of the erythrokinetic features a t presentation or during the course of the disease.

In this paper the results from 26 patients with MMM are reported. Effective and ineffective erythropoiesis together with peripheral haemolysis were estimated by means of a mathemati- cal model of ferrokinetics. Erythrokinetic parameters were correlated with clinical and haematological findings, with the purpose of revealing different erythropoietic patterns in the disease. The results from patients suffering from uncomplicated polycythaemia Vera (PV) are also reported for comparison.

METHODS

Patierits Twenty-six patients with MMM underwent erythrokinetic study after giving their fully

informed consent. Clinical and haematological data are reported in Table I. In 22 of these patients an initial diagnosis of M M M was made on the basis of the following clinical and histopathological criteria. They all had splenomegaly, a leuco-erythroblastic blood picture with red cell aniso- and poikilocytosis with typical teardrop cells, increased reticulin in the niarrow and a t least focal fibrosis in the bone marrow trephine biopsy from the posterior iliac crest. Erythroid metaplasia was evidenced by erythrokinetic studies in all but two cases. Decreased red cell volume was not considered a diagnostic criterion, as it has been by other authors (Laszlo, 1975; Silverstein, 1974). None of the patients had the picture of malignant myelosclerosis (Lewis 8; Szur, 1963), a history of chronic granulocytic leukaemia or other identifiable cause of marrow fibrosis. Most anaemic patients (cases 12,14, 15, 18,20,21,25 and 36) received testosterone therapy prior to the study with variable response.

Four cases (cases 1, 3, 9 and 16) \yere originally diagnosed as having PV. The clinical and haematological findings of MMM developed 5, 3, 10 and 3 years later, respectively. These patients had been treated with Busulfan and phlebotomy during the course of their disease.

Two patients (cases 9 and 18) had two separate studies a t 6 and 5 years respectively. The patients were classified according to: (i) the duration of the disease, that is the time

elapsed from the first recognized sign or symptom; (ii) the spleen size, as judged by clinical palpation: class I , no more than a t the level ofthe umbilicus; class I1 a t the transiliac line; class 111, into the iliac fossa; (iii) the histological type of bone marrow at the time of erythrokinetic study. classified according to Ward 8; Block (1971): type I, myeloid hyperplasia with increased reticular fibres; type 11, mveloid hypoplasia and fibrosis; type 111, aplasia with myelofibrosis and osteosclcrosis. A t the time ofwriting, five patients had died in blastic crisis (cases 16,20, 24, 25 and 26). A follow-up more than 3 years after the erythrokinetic study was available in only five patients.

Three patients with uncomplicated PV are reported (Table I). They fulfilled the diagnostic criteria of the Polycythaemia Vera Study Group (Berlin, 1975). In particular, none of theni presented with massive splenomegaly or leucoerythroblastosis or marrow fibrosis. No extra- medullary erythropoiesis was detected by erythrokinetic studies.

Ery thropoiesis in Mye lo jbros i s

TABLE I . Clinical and haematological data

265

Time since diagnosis Spleen Histological Hb MCV Retics W B C Platelets

Case A g e Sex (motrths) size class (gldl) P C V c f l ) (igI1) (10!'11) (1@/1)

Myelofibrosis with myeloid metaplasia 1 ,LT 52 M 72 (14) 2, DL 59 F 24 3,LS 39 M 36 (3) 4, ZG 66 F 108 5,MF 61 M 4 6, PG 69 M 16 7 ,OP 62 M 51 8, FA 65 M 84 9,KI 57 F 360 (240) 9 bis 63 F 432

10,MF 57 M 108 11, FR 38 M 6 12,MS 48 M 24 13,ME 73 M -

14, WU 68 M 10 15,MM 69 M 5 16, BP 65 F 36 (2) 17,NA 58 M 36 18, CC 55 M 2 18 bis 60 M 62 19, RG 61 M 4 20, BC 53 M 96 21,ME 57 M 7 21, MI 53 F 3 23.GE 56 M 40 24, BA 43 M 5 15, MF 43 M 18 26, AE 48 F 12

1,CA 49 M 24 2, RD 43 M 12 3, FF 57 M 120

Polycythaemia Vera

111 - 13.1 0.39 88 60 6.2 38 111 I 18.2 0.57 71 250 16.5 155 11 I 14.3 0.47 80 180 46.2 135

111 - 9.2 0.51 92 60 6.1 87 I11 I 14.7 0.44 81 180 46.9 685

I1 - 9.6 0.33 83 115 10.0 160 111 I 12.0 0.36 89 - 12.6 110

I I 10.3 0.33 69 154 12.3 36 111 I 11.4 0.36 86 400 14.9 104 I11 - 9.3 0.31 85 360 11.5 21 111 I1 9.6 0.30 84 64 3.9 170 111 111 10.4 0.32 91 - 8.2 220 111 I1 13.6 0.41 78 150 18.7 700 111 I 9.0 0.27 92 24 29.9 450 I1 111 12.9 0.41 82 200 3.2 75

111 I1 9.4 0.28 88 - 5.0 160 I1 I 14.1 0.43 75 90 35.5 300

111 I1 12.6 0.38 87 20 2.9 180 I1 I1 9.3 0.30 86 80 4.6 344 I1 - 10.2 0.32 88 94 3.5 440 I1 I1 8.8 0.29 92 24 6.2 490

111 I11 9.7 0.28 89 33 2.0 40 I1 111 9.2 0.28 101 - 14.6 -

I1 I1 9.8 0.30 90 450 7.2 440 111 111 12.2 0.39 95 112 17.1 97 111 111 8.8 0.25 92 13 7.5 40 I1 111 5.2 0.16 79 0 2.8 40

I I1 8.5 0.25 96 30 15.0 150

I - 16.6 0.57 78 80 22.5 696 I - 20.5 0.63 91 21 9.0 542 I - 16.0 0.50 71 - 7.3 302

In patients with MMM secondary to PV, the time given is for PV diagnosis with MMM diagnosis in parentheses.

Erythrokirietic Studies Red cell volume was measured by the 5'Cr method and taking account of the massive

splenomegaly in these patients (ICSH Panel, 1973). Plasma volume was measured by dilution of 59Fe-transferrin. Normal reference values were derived by the method of Nadler et al(1962) and the measured volumes were expressed as a percentage of the reference values.

Ferrokinetics (59Fe-transferrin) and 51Cr-red cell survival studies were carried out simul- taneously. The methods have been described in detail elsewhere (Barosi et al , 1978; Cazzola et al, 1976; Berzuini et al, 1978). In all but three cases (8, 10 and 18), Cavill's method (Cavill, 1971)

266 C. Barosi et a1

for the preparation of labelled transferrin was used. i9Fe-haemoglobin was removed from the plasma (Cavil1 et al , 1976) in all but four studies (cases 8, 10. 17 and 18).

The ferrokinetic data were analysed by means of a seven compartment model of ferrokine- tics (Barosi et al, 1978; Berzuini et a / , 1978). Five erythrokinetic parameters were considered as having pathophysiological relevance: (a) Total erythroid iron turnover (TEIT) as a measure of both intra- and extra-medullary erythropoiesis. This parameter corresponds to marrow iron turnover (MIT) as an index of total erythropoiesis in patients without extramedullary erythropoiesis (Barosi et af, 1978). (b) Non-erythroid iron turnover (NEIT), as an estimate of iron flow to parenchymal tissues. (c) Ineffective iron turnover (IIT) as an estimate ofineffective erythropoiesis. (d) Red cell iron turnover (RCIT), as an estimate of effective erythropoiesis. (e) Mean red cell lifespan (MRCL).

External counting was carried out over the spleen, liver, sacrum and precordium, using four NaI (Tl) crystals shielded with a single hole collimator (5 cm diameter, 15 cm high and 3.75 cm thickness). External monitoring values were expressed as a percentage of the zero time extrapolated value. The amount of radioiron fixed in the erythropoietic tissue at the 24th hour after injection was graded according to the following classification: 0, 1 + , 2+ and 3+ =iso- tope uptake less than once, less than twice, less than three times, and more than three times the zero time value, respectively.

Statistical Analysis To investigate if distinct classes of patients could be recognized on the basis of the

erythrokinetic findings only, cluster analysis and principal component analysis were applied to the five computed erythrokinetic parameters on all the patients. A computer program developed by Jambu & Lebeaux (1978) was used for the hierarchical classification of the cases. This program utilizes the central moment of the second order of a partition as aggregation criterion. Principal component analysis was performed by using the B M D O l M program of the BMD library (Dixon. 1967). Finally, discriminant analysis was applied to the classes obtained through cluster analysis (Habbema et al, 1974; Habbema & Hermans, 1977).

RESULTS

The results of the radioisotopic studies are reported in Table 11. Red cell volume was greater than the predicted normal value in 14 out of the 26 M M M patients although in all but one case the PCV was below normal. Plasma volume was greater than the predicted normal in all but two patients. The excess plasma volume ranged from 113% to 221%.

Total erythroid iron turnover (TEIT), a measure of total erythropoiesis, was increased in all but one patient, and ranged from 1.0 to 10 times the mean normal value. A close direct relationship was found between total erythropoiesis (TEIT) and red cell volume (r =0.75, P<0401), (Fig 1) .

Ineffective erythropoiesis, expressed as a per cent of total erythropoiesis, ranged from 14% to 86%. A significant correlation was found between total erythropoiesis and ineffective erythropoiesis (r=0.61, P<O.OOl). Mean red cell lifespan was reduced in all patients. No significant correlation (P> 0.05) was found between total erythropoiesis and peripheral haemolysis (1 /MRCL) or between ineffective erythropoiesis and peripheral haemolysis.

Erythropoiesis in Myelojibrosis 267

TABLE 11. Iron status and erythrokinetic parameters

Serum Plasma I n vivo erythropoiesis iron volume RBC Volume TEIT NEIT RCIT IIT MRCL

Case (pmol/l) (%) (%) (mI/kg) Sacrum Spleen Liver (pmol/l bloodld)' (Yo) ( d )

Myelofibrosis with mveloid metadasia 1 29.6 2 7.1 3 19.6 4 11.6 5 7.7 6 21.4 7 8.9 8 6.8 9 12.1 9 bis 12.5

10 14.3 11 15.2 12 16.9 13 21.4 14 17.8 15 16.9 16 31.3 17 24.7 18 20.4 18 bis 12.5 19 22.3 20 17.4 21 13.9 22 10.7 23 10.7 24 37.7 25 44.7 26 31.2

i00 128 37.3 144 252 63.9 178 171 52.5 205 142 37.6 155 175 49.3 146 84 25.5 176 111 32.8 156 105 30.4 195 160 42.0 193 127 32.5 139 89 26.9 119 112 33.4 151 140 38.4 221 114 33.7 113 105 304 180 72 19.5 148 176 43.6 158 115 35.5 160 98 29.8 166 95 27.9 187 68 20.8 159 78 25.0 166 77 21.4 116 74 30.5 96 92 3143

153 70 18.9 192 38 10.7 ,119 70 15.9

Polycythaemia Vera 1 3.6 105 143 49.7 2 7.1 120 158 50.2 3 5.4 115 168 54.8

0 0 0 1+ 0 0 0 I + 0 0 0 0 1+ 2+ 0 1+ 3+ I + 2+ 2+ 1+ 1+ 0 I + 2+ 0 0 0

3+ 3+ 3+

3+ 1+ 3+ 1+ 3+ 2+ 1+ 0 3+ 2+ 3+ 0 3+ 0 2+ 1+ 3+ 0 3+ 1+ 2+ 1+ 3+ 3+ 3+ I + 3+ 1+ 2+ 2+

0

0

3+ 0 3+ 2+ 3+ 1+ 3+ 3+ 2+ I + 2+ 0 3+ 0 0 0 0 0

0 0 0 0 0 0

3+ 3+ 0

3+ 3+ 0

971 30 910 22 838 71 709 45 672 44 610 48 531 53 541 51 531 55 480 50 513 46 452 33 472 53 465 119 455 42 398 67 397 94 338 73 364 69 324 46 267 79 252 72 238 91 226 29 200 21 161 108 156 179 92 79

126 6 140 9 155 10

310 68 173 81 221 73 289 59 288 57 85 86

134 77 140 74 249 53 217 54 123 76 156 65 302 36 402 14 330 27 242 39 239 39 257 24 148 59 177 49 166 38 159 37 179 25 115 49 114 43 100 38 84 46 51 36

115 8 108 22 120 20

36 105 56 35 98 60 72 61 50 42 54 61 41 25 27 26 57 43 55 45 31 45 40 54 69 t t t

124 115 104

* Ferrokinetic parameters are expressed per litre of predicted normal blood volume. t Transfused prior to study.

Neither ineffective erythropoiesis nor peripheral haemolysis were correlated with red cell volume. No relationship was found between peripheral haemolysis and the degree of spleno- megaly. The duration of the disease prior to the erythrokinetic study was not correlated with total erythropoiesis.

Clustering analysis resolved the erythrokinetic results of patients with MMM into three distinct classes (Fig 2). The three components used for display accounted for 92% of the total variation. Discriminant analysis showed that the accuracy of this classification was 96%.

The pathognomonic features of each class are shown in Table 111. Eleven patients (class I)

G. Barori et al

60 m Y

9) 5 4 0 - e

0

I 0

0 - 0 . r = 0.75 P < 0.001

0 200 400 600 800 1000

Total erythroid iron turnover (ymol/ I blood/d)

FIG I . Relationship between total erythroid iron turnover (TEIT) and red cell volume in patientr suffenng from myelofibrosis Lvith mycloid metaplasia.

A ‘A 0

0

0

0

0 0

urn

0

A

A A

OMMM class I 0 0 1’ ” II

9 1 1’ Ill A Pv

0 0

0 8

0 0.

F 0

0

FIG 2. Cluster analysis of the erythrokinetic parameters for each patient. The left part shows the results on the first and second principal components of the distribution (plane 21. z2) and the right part shows the first and third principal components (plane 11, 23).

Erytkropoiesis in Myelofbrosis 269

TABLE 111. Erythrokinetic parameters, red cell volume and serum iron in the three classes of patients with myelofibrosis with myeloid metaplasia (MMM), in patients with polycythaemia Vera (PV) and

in 14 normal subjects (mean and range)

M M M

Class I Class I1 Class I11 (cases 1 - 1 1 ) (cases 12-23) (cases 24-26) PV Normal subjem

Total erythroid iron turn- over, TEIT 666 (pmol/l blood/d) (452-971)

Non-erythroid iron turn- over, NElT (pmol/l blood/d) Ineffective iron turnover per cent, IIT (YO) Red cell iron turnover, RCIT (pmol/l blood/d) Mean red cell lifespan, MRCL(d) Red cell volume (ml/kg)

Serum iron (pmol/l)

41

70 (53-86)

197

(22-71)

(85-3 10) 62

(35-105)

39.8 (25.5-63.9)

14.0 (6.8-29.6)

339 (200-472)

67

36 (1 4-59)

221 (1 14-402)

43 (25569)

30.1 (1 9.5-43.6)

18.7 (10.7-31.3)

(21-119)

136 (92-161)

122 (7+179)

40

78 (38-46)

(51-100) *

14.8 (10.7-18.9)

3743 (31.2-44.7)

140 (1 26-1 55)

9

17 ( 6 1 1 )

W 2 )

(108-120) 114

114 ( 104-124)

51.5 (49.7-54.8)

5.3 (3.67.1)

87 (62-105)

30 (1 8-44)

8 (1-17)

80 (57-1 00)

112 (88-1 41)

M:26.8-32.2 F: 25.2-27.1

(12.5-21.4)

* Transfused prior to study.

were characterized by a total erythropoietic activity that was 5 times greater than normal but largely ineffective. In spite of this effective erythropoiesis was increased and in six patients red cell volume would have satisfied the diagnostic criterion of PV in the Polycythaemia Vera Study Group protocol. Mean red cell lifespan was slightly shortened. A peculiar feature was the complete absence of detectable radioiron uptake over the sacral marrow in nine cases. An erythropoietic profile was always found by in vivo counting over the spleen and sometimes over the liver. One of these patients (case 2) had an excess red cell concentration in peripheral blood, while the other had a value which was lower than normal. Serum iron concentration was low. The clinical symptoms appeared to be mainly due to the enlarged spleen. Three of them had a history of PV.

Twelve patients (class 11) were characterized by total erythropoiesis ranging from 2 to 5 times normal, with minor degrees of ineffectiveness. Effective erythropoiesis was increased with respect to normal, but a reduced red cell lifespan counteracted this erythropoietic effort. Although reduced, erythropoiesis was almost always detected over the sacral area. It was also found over the spleen in all patients and over the liver in some. There was a moderate to severe anaemia with red cell volume ranging from slightly increased to decreased. Serum iron was greater than in the patients from class I. Besides the consequences of anaemia and spleno- megaly, more general symptoms (malaise, fever and bone pain) were also present. One case (case 16) had a past history of PV. She had the greatest increase of red cell volume and

270 C . Barosi et a1

maintained a normal degree of erythropoiesis over the sacrum. She died 1 year after the study from acute leukaemia.

Three patients (class 111) were characterized by the erythrokinetic pattern of relative erythroid failure. Total erythropoiesis was less than twice normal with a high non-erythroid iron turnover. In two patients, erythropoiesis was not detected anywhere, and the liver accumulated iron as storage. In the other, erythropoiesis was detected over the spleen. Serum iron was greatly increased with respect to normal. The main symptoms were due to anaemia with a high transfusion requirement.

’There was no significant difference between the three classes of patients with respect to the apparent duration of the disease. The classes could not be completely distinguished by the histological bone marrow picture. However, most of the class I patients had a type I histological picture and two patients belonging to class 111 showed the histological feature of aplasia (histological type 111). No correlation w a s found between the degree of total erythroid activity (TEIT) and WBC and platelet counts.

In the PV patients total erythropoiesis \vas sustained at no more than twice normal with minor degrees of ineffective erythropoiesis and a normal red cell lifespan. Cluster analysis on the ferrokinetic data showed a significant separation o f PV from MMM patients. I n the two patients in whom serial studies were carried out, a modest shrinkage of total erythropoiesis and of red cell volume w a s evident, without significant modification of the erythrokinetic pattern. In both patients the erythrokinetic results obtained a t two different times were grouped in the same class by the cluster analysis.

DISCUSSION

The primary proliferative nature o fMMM can be inferred from the results ofthe present study and the circulating red cell volume was directly correlated with total erythropoiesis through- out i ts whole range (from 1.0 to 10 times the normal). This ‘inverse’ regulatory characteristic of erythropoiesis (Kuni & Graul, 1975) demonstrated that proliferation of erythroid tissue is the main determinant of the circulating haemoglobin level. The erythrokinetic results also indicate that the wastage of a great proportion of erythropoietic effort was the feature that separated MMM from PV. In PV, the increased red cell volume was reached through an orderly proliferative mechanism. As far as MMM is concerned disordered erythropoiesis is only a precipitating factor in the production of anaemia. Thus, neither ineffective erythropoiesis nor peripheral haemolysis were correlated with haemoglobin level or red cell volume.

The quantitative assessment of erythropoiesis allowed us to distinguish three classes of MMM patients. In class I, a large increase in erythroid tissue and massive ineffective erythro- poiesis were the outstanding features. Marked decrease in sacral erythroid activity, suggested centrifugal marrow displacement, an aspect already reported in M M M on the basis of histopathological (Oechslin, 1956) and scintiscan evidence (Shreiner, 1974). Ineffective eryth- ropoiesis may be the result either of erythropoiesis in unusual sites or of the overproduction of erythroblasts itself. Although the red cell volume increase suggested PV in six cases, only three of these patients had had a previous history of PV. Haemodilution and red cell pooling in the massive spleen made polycythaemia unrecognizable by relative measurements such as haemo- globin concentration or PCV.

Erythropoiesis in Myelqfibroris 271

A problem that must be taken into consideration in this class ofpatients is the nosology of the disease. Similar patients have been classified differently in the literature. Their clinical, haematological and erythrokinetic features were characteristic of the ‘Transitional Myelopro- liferative Disorder’ described by Pettit et al(1979). O n the other hand, similar cases have been reported as representing late stages ofPV on the basis ofeither ferrokinetic studies (Pollycove et al , 1966) or histology (Duhamel e f al , 1970). Nevertheless, the absence of a manifest phase of polycythaemia in most of our cases, the disordered erythropoiesis and the haematologic and histologic characteristics of the disease, led us to classify these patients as a particular form of MMM.

Axial erythroid hypoplasia was less evident in class 11. Heavy peripheral haemolysis was peculiar to these patients. A minor maturation disturbance allowed the erythroid cells to undergo complete maturation in the marrow, so that their intrinsic defect was revealed only after delivery of the red cells to the circulation. This class corresponds to the classical description of MMM showing a true anaemia in most patients. The three patients in class 111 showed a marked reduction in erythropoiesis and they had the most dramatic clinical involvement. Such an erythrokinetic pattern was described in approximately 6% of pre- viously reported cases (Bentley e f al , 1977).

The degree of erythropoiesis and consequently the red cell volume decrease progressively from class I to class 111. This could suggest that these three classes represent different stages in the natural history of the disease. However, in the few cases submitted to serial examination, no change in erythrokinetic pattern was observed. Moreover, no correlation was found between the duration of the disease prior to the study and the degree of erythropoiesis. Thus, no previous chronic phase ofillness was evident in class 111 patients. Furthermore, it is unlikely that class I , which did not show sacral erythroid activity, was an earlier stage of class 11, which did show it. The limited number of cases treated prior to the study seemed to exclude the possibility that androgen therapy may have induced sacral erythropoiesis in class 11, or cytostatic treatment may have depressed it in class I. Similarly, no cytotoxic treatment was given to class I11 patients before the study. These findings strengthen the view that MMM occurs in several separate clinical forms. The spontaneous transformation of one form into another still has to be proved.

The brief follow up of our cases did not allow us to formulate a significant statistical analysis of survivals in the different classes of patients. However, a worse prognosis may be inferred for class 111, in which all three patients died with blastic transformation within 1 year after the study. In summary, the functional entities defined by this study give a rational classification of erythroid disorders in MMM which may serve as a basis for the therapeutic and prognostic investigation of the disease.

ACKNOWLEDGMENTS

We thank Professor E. Ascari (Istituto di Patologia Medica, Universiti di Pavia) for his criticism; Professor C. Bernasconi (Divisione di Ematologia, Policlinico S. Matteo, Pavia) who allowed us to study his patients; Professor U. Magrini and Dr A. Castello (Istituto di Anatomia Patologica, Universiti di Pavia), who looked a t the bone marrow biopsies.

This work has been supported by C.N.R. Contract No. 79.01257.86 within the Special Project on Biochemical Engineering.

272 C. Barosi et a1

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