British journal of Hamatology. 199 1, 77, 509-5 14
Are ‘dysplastic’and hypogranular megakaryocytes specific markers for myelodysplastic syndrome? K . F. WONC A N D
I . K . C. CHAN Institute of Pathology, Queen Elizabeth Hospital, Hong Kong
Received 2 October 1990; accepted for publication 20 November 1990
Summary. Dysmegakaryocytopoiesis is an integral component of myelodysplastic syndrome (MDS), and has been shown in some studies to be an independent prognostic factor. Megakaryocytic hypogranulation, a feature we have noticed for some time to be fairly common in MDS and acute myeloid leukaemia (AML). has received little attention in the literature as a dysplastic feature of megakaryocytes. This study was performed to determine how frequently this feature was observed in MDS and the specificity of its
occurrence. On review of archival materials, hypogranular megakaryocytes were observed in 80.3%of MDS, 30.6% of AML and 1.4% of controls. On the other hand, the other well-recognized dysmegakaryocytopoietic features (hypolobulation, multiple separate nuclei, micromegakaryocyte), though frequent in MDS or AML. were also observed in 20% of controls. We therefore propose including megakaryocytic hypogranulation as a cytological feature of myelodysplasia.
Myelodysplastic syndrome (MDS) is characterized by ineffective and dysplastic haemopoiesis which may terminate as acute leukaemia. The diagnosis is based mainly on morphologic study of peripheral blood and bone marrow. The FAB (French-American-British) classification, proposed in 19 76 (Bennett d a/. 1976) and modified in 1982 (Bennett et a/. 1982). has been most widely used as a diagnostic guideline. The qualitative dysplastic features described in the megakaryocytic series include large mononuclear forms, multiple separate nuclei and micromegakaryocytes. However, megakaryocytic hypogranulation. a feature we have noticed frequently in MDS and acute myeloid leukaemia, is not included among them. In this study we address the question of whether megakaryocytic hypogranulation and other wellrecognized dysmegakaryocytopoietic features are specific for MDS or related disease processes.
for MDS or AML. 500 consecutive bone marrow aspirate smears obtained in the year 1987 were examined. The bone marrow aspirate smears were stained with MayGrunwald-Giemsa according to standard techniques (Dacie & Lewis, 1984). The films were air-dried and fixed in methanol for 30 min. and then stained with freshly diluted MayGrunwald stain (BDH Chemicals Ltd. U.K.) for 10 min. The films were transferred without rinsing to diluted Giemsa stain (Koch-Light Laboratories Ltd, U.K.). After 1 5 min the films were left in buffered distilled water for differentiation to take place. They were then rinsed in tap water and allowed to dry. Only good-quality marrow smears with adequate megakaryocytes (at least 1 0 megakaryocytes) were considered satisfactory for proper assessment. The whole smear in each case was scrutinized for the presence of dysplastic changes in megakaryocytes as described by the FAB group. They were recorded as present if one or more cells exhibited the feature. Hypogranular megakaryocytes were defined as megakaryocytes showing pale grey or water-clear cytoplasm with sparse or no granules (Fig 1):immature megakaryocytes with open chromatin and basophilic cytoplasm were excluded. Hypogranular megakaryocytes were quantitated as their percentage out of the total megakaryocytes counted.
MATERIALS AND METHODS During the period January 1987 to September 1990, 78 cases of MDS and 96 cases of acute myeloid leukaemia (AML) diagnosed according to the FAB criteria (Bennett e t a!. 1976. 1982) were retrieved from the haematology laboratory files, Queen Elizabeth Hospital, Hong Kong. The bone marrow aspirate smears were available for study. These cases were assessed for the frequency of the various megakaryocytic abnormalities. To study whether these features were specific
RESULTS Among the 76 cases of MDS with optimal smears for assessment, there were 44 (58%) males and 32 (42%) females. Their ages ranged from 4 to 92 years, with a mean of 6 3 . 5 years. There were 32 cases of refractory anaemia/
Correspondence: Dr K. P. Wong. Institute of Pathology, Queen Eliirabeth Hospital, Wylie Road, Kowloon, Hong Kong.
K. F. Wong and 1. K. C. Chon
Fig 1. A hypogranular megakaryocyte which is practically devoid of cytoplasmic granules. The surrounding cells were well stained, rendering the possibility of artefact unlikely. May-Grunwald-Giemsa. x 750.
Fig 2, Refractory anaemia with excess of blasts. This illustration depicts an agranular megakaryocyte (broad arrow), a megakaryocyte showing uneven granulation (narrow arrow) and one normally granulated megakaryocyte. May-Grunwald-Giemsa, x 600. An increase in blasts and abnormal prornyelocytes is evident in the inset. May-Grunwald-Giemsa, x 5 0 0 .
Hypogranular Megakaryocytes in MDS Table I. Dysmegakaryocytopoieticfeatures and hypogranular megakaryocytes in MIX and AML ~~
Hypogranular megakaryocytes Dysmegakaryocytopoieticfeatures Large mononuclear (hypolobulated) forms Multiple separate nuclei Micromegakaryocytes None of the above four features
58 59 41 3
23 30 9 27 8 41
Total cases adequate for assessment
cytopenia (RA) (including two cases of 5q - syndrome), four cases of refractory anaemia with ringed sideroblasts (RARS). 1 5 cases of refractory anaemia with excess of blasts (RAEB), 23 cases of refractory anaemia with excess of blasts in transformation (RAEB-T). and two cases of chronic myelomonocytic leukaemia (CMML). Twenty-one cases of AML and 58 cases of the controls were not suitable for assessment because of aparticulate smears or sparsity of megakaryocytes. Dysplastic megakaryocytes according to the FAB criteria could be identified in 71 cases (93.474) of MDS (Table I). Megakaryocytic hypogranulation was detected in 6 1 cases (80.3%)of MDS, and involved 2-10074 of the megakaryo-
cytes. In five cases it was a prominent feature, being found in over half of the megakaryocytes (Fig 2). In two cases (RAEB 1 , RAEB-T 1 ), hypogranular megakaryocytes were the only abnormal feature observed in the megakaryocytic series. Some hypogranular megakaryocytes also showed other abnormalities such as nuclear hypolobulation or multiple separate nuclei. Dysmegakaryocytopoietic features were found in 30 cases (40%)of AML, and megakaryocytic hypogranulation in 2 3 cases (30.7%) (Table I; Fig 3). Dysplastic megakaryocytes were also observed in small numbers in 8 7 cases (19.7%)among the controls (442 cases adequate for assessment). The majority of cases were patients proven to have iron deficiency and patients with recent exposure to chemotherapeutic agents (Table 11). The dysplastic features most commonly observed were nuclear hypolobulation and multiple nuclear separation (Fig 4).Rare hypogranular megakaryocytes were found only in six cases (iron deficiency 2. chronic myeloid leukaemia 2, azathioprine effect 1, megaloblastic anaemia 1). Such changes involved less than 5% of megakaryocytes.
DISCUSSION MDS is a clonal disorder characterized by ineffective haemopoiesis and variable degrees of dysplastic changes in the red
Fig 3. Acute myeloid leukaemia with numerous hypogranular and agranular megakaryocytes. The cytoplasm of most megakaryocytes is waterclear. May-Grunwald-Giemsa. x 375.
K. F. W o n g and J. K. C. Chan Table 11. Recognired dysmegakaryocytopoietic changes among the controls (n =442) No. of cases showing dysmegakaryocytopoiesis
Multiple Hypolohulation separate nuclei Micromegakaryocytes Total
33 Iron deficiency Postchemotherapy 8 Megalohlastic anaemia 2 Normal or active marrow 4 Other haemic malignancies' 4 Other disorderst 6
12 9 8 4
17 10 10 5 9
* Including chronic myeloid leukaemia (four cases) and acute lymphoblastic leukaemia at presentation (one case). t Including thalassaemia (four cases), idiopathic thrombocytopenic purpura (two cases), anaemia of chronic disorders (two cases) and systemic lupus erythematosus (one case).
Fig 4. Simple iron deficiency with presence of large mononuclear megakaryocytes. May-Grunwald-Giemsa, x 500.
cell. white cell and megakaryocytic series, and a propensity of transformation into acute leukaemia. The morphologic criteria proposed by the FAB group (Bennett et al, 1982) have been widely used as guidelines for diagnosis. Although dysplastic haemopoiesis usually involves one or more cell lines. dysmegakaryocytopoiesis remains one of the most important morphologic findings both for the purpose of diagnosis (Verwilghen& Boogaerts. 198 7: Galton, 1989) and for the assessment of prognosis (Varela et al, 1985: Jacobs et al. 1986: Rios et al, 1990). Because of the importance of identifying dysmegakaryocytopoiesis. some studies have suggested using ancillary techniques such as immunocytochemical staining to improve its detection (Sato et al, 1986: Erber et al, 1987: Kawaguchi et al, 1990).
As far as dysmegakaryocytopoietic features in MDS are concerned, the emphasis has been put mainly on the nuclear abnormalities such as small size, hypolobulation and multiple separate nuclei. Cytoplasmic abnormalities have not been stressed in the FAB criteria (Bennett et al, 1982) or other studies on MDS (Linman & Bagby, 1978: Streuli et al. 1980: Weber et al. 1980: Juneja et al, 1983: Francis & Hofirand, 1985; Travis & Pierre, 1985: Varela et al, 1985: Bennett, 1986: Mufti & Galton, 1986: Wickramasinghe, 1986: Verwilghen & Boogaerts, 1987: Hamblin & Oscier. 1987: Beris. 1989: Doll & List, 1989). Cytoplasmic vacuolation has been briefly mentioned occasionally (Wolf & Neiman, 1987:Travis & Pierre, 1985). 'Cytoplasmic degranulation' and 'defect of cytoplasmic maturation' have also been mentioned in pass-
Hypogranular Megakaryocytes in MDS ing in two reviews (Verwilghen & Boogaerts, 1987; Burkhardt. 1988): these changes appear to be identical to what we call 'hypogranular megakaryocytes'. We consider the term 'defect in cytoplasmic maturation' too non-specific, and the term 'cytoplasmic degranulation' not entirely accurate because there is no proof of the process of loss of granules. In this study we are able to show that megakaryocytic hypogranulation occurs with a high frequency in MDS and in AML. and is the only megakaryocytic abnormality in some patients with MDS. It is also fairly specific in that it is rarely observed in other diseases or normal marrow. The occurrence of hypogranular megakaryocytes in AML is to be expected because AMI, is often a disease involving haemopoietic stem cells. Some cases may represent AML developing in a background of MDS. while some may represent the entity of de novo Ah4L with trilineage dysplasia (Brito-Babapulle rt a/, 1987). Their occurrence in marrow after chemotherapy can be readily understood because cytotoxic drugs are known to affect nuclear-cytoplasmic maturation. Kegarding the presence of hypogranular megakaryocytes in chronic myeloid leukaemia. other megakaryocytic abnormalities have also been previously observed in some cases of this stem cell disorder (Hamblin & Oscier, 1987). Our results confirm those of previous studies (Sato et a/, 1986: Koike & Takahashi. 1986: Hamblin & Oscier. 1987: Kawaguchi et al. 1 9 9 0 ) that morphologically dysplastic megakaryocytes. especially large mononuclear forms and cells with multiple separated nuclei, can be found in conditions other than MDS or AML and even in normal marrows. Kecently, micromegakaryocytosis is reported to be a common finding in neonates with leucoerythroblastic reaction (Press et d, 1990). In particular, dysplastic megakaryocytes as defined by FAB criteria were demonstrated in 47% of cases of iron deficiency in this study, especially large mononuclear forms, We cannot offer a satisfactory explanation for this phenomenon. Since the May-Grunwald-Giemsa is a fastidious stain which requires meticulous attention to the source of reagents, buffer and timing, one may wonder whether the hypogranulation we have observed in the megakaryocytes merely represents a n artefact resulting from suboptimal staining. However, we consider the hypogranulation genuine because normally granulated megakaryocytes are almost invariably observed in the same smears in which hypogranulated megakaryocytes are detected. To further disprove the possibility of artefact from different batches of stain, we have randomly retrieved 3 0 examples of MDS from our files before 1 9 8 7 (19 8 3-86), and have observed identical megakaryocytic abnormalities. Thus. megakaryocytic hypogranulation. which probably reflects an intrinsic abnormality of the megakaryocyte resulting in failure of synthesis of granules, should be considered another sensitive and specific morphological marker of dysmegakaryocytopoiesis in MDS.
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