Eur J Pediatr (1992) 151 : 668-671

European Journal of

Pediatrics

9 Springer-Verlag1992

May-Hegglin anomaly: a rare cause of thrombocytopenia A. Greinacher 1, J. Bux 1, V. Kiefel 1, J. G. White 2, and C. Mueller-Eckhardt ~ 1Institute for ClinicalImmunologyand TransfusionMedicine,Justus LiebigUniversity, Langhansstrasse7, W-6300 Giessen, Federal Republic of Germany 2Departments of Pathology,LaboratoryMedicine and Paediatrics, Universityof Minnesota, Minneapolis, USA Received August 28, 1991 / Accepted in revised form January 14, 1992

Abstract. A family with four and an unrelated family with three individuals affected by the May-Hegglin anomaly are described. Platelet counts were markedly reduced and were correctly determined only in the counting chamber. Bleeding time and platelet aggregation were always normal, but platelet nucleotide concentrations (ATP and ADP) were elevated. The platelet glycoprotein complexes Ib/IX, IIb/IIIa and Ia/IIa were quantitatively normal. Platelet-associated IgG was slightly elevated, although thrombocytopenia was presumably not caused by an immunological mechanism. Morphological investigations showed giant platelets and spindleshaped inclusion bodies in the granulocytes, while their function (phagocytic capacity, radical production) was normal. To exclude hereditary types of thrombocytopenia, morphological and family investigations are required to avoid misdiagnosis with far-reaching diagnostic and therapeutic consequences. Key words: Thrombocytopenia - Giant platelets - Leucocytes - May-Hegglin anomaly - Hereditary thrombocytopenia

Introduction The May-Hegglin anomaly is a rare cause of hereditary thrombocytopenia. Until now only 36 families with this syndrome have been documented in the literature [8]. The frequency of the syndrome is unclear. Because of the widespread use of automatic particle counters, more clinically asymptomatic patients with hereditary thrombocytopenias are being diagnosed [19]. The pathogenesis of the May-Hegglin anomaly is poorly understood. Possible disturbances of the platelet membrane have been investigated in members of two families [5, 23], with conflicting results. Similarly, there are only a few contradictory reports of granulocyte function studies [3, 18,

22]. We report on studies of function, morphology, immunology and biochemistry of platelets and granulocytes of seven affected members of two families with the MayHegglin anomaly.

Patients Patient 1

In a 3-year-old boy a low platelet count of 20 • 109/1 was determined during an acute infectious episode. Besides easy bruising and some older pretibial haematomas, history and physical examination were normal. Laboratory investigations including electrolytes, creatinine, blood urea, liver status, clotting parameters and differential blood count were normal. Bone marrow showed increased megakaryocytopoesis but no evidence of malignancy. Treatment with intravenous IgG and steroids had no effect on platelet count. The family history did not indicate bleeding abnormalities, but extended family investigation revealed May-Hegglin anomaly in the father and the sister of the father who had cerebral palsy since early infancy. Patient 2

In a 4-year-old girl, thrombocytopenia was detected by chance during a gastro-intestinal infection. Blood smear revealed giant platelets. Family history disclosed easy bruising (recurrent nose bleeding, pre-tibial haematomas) in the 6-year-old brother. On family investigation, the brother, father and grandfather of the patient were found to be affected by the May-Hegglin anomaly, the symptoms being least prominent in the paternal grandfather.

Materials and methods Morphology

Correspondence to: A. Greinacher Abbreviation: PRP = platelet-rich plasma

Blood smears prepared from peripheral blood were stainedwithin 4h by May-Granwald-Giemsastain. Electron microscopicstudies were performed as described [26].

669

Plateletfunction and biochemistry

Results

EDTA anticoagulated blood (nine parts venous blood, one part 130mmol EDTA) was used to determine platelet count and platelet volume by an automatic particle counter (Sysmex, TOA Medical Electronics, Kobe, Japan). The platelet number was concomitantly counted by visual phase microscopy. Bleeding time was measured with a Simplate II device using a venous pressure of 40 mmHg and a vertical incision of the volar surface of the forearm. For platelet function studies blood was collected into prewarmed 0.1vo1135mM trisodium citrate. Platelet rich plasma (PRP) was obtained either by differential centrifugation (120g, 20rain) (PRPI) or by spontaneous sedimentation (PRPII) as described [9]. Platelet aggregation was recorded at 37~ with a four channel aggregometer (Labor, Ahrensburg, YRG) using the following agonists: ADP 10, 5, 2.5, 0.5 gmol/ml (Dade Diagnostics, Aquada, Puerto Rico); epinephrine 10, 5, 2.5gmol/ml (Sigma, Munich, FRG); collagen 4, i gg/ml (Hormon Chemie, Munich, FRG); ionophore A23187 10 ~tmol (Sigma, Munich, FRG); ristocetin 1.5, 0.5mg/ml (Sigma, Munich, FRG); arachidonic acid 1.5 mmol (Sigma, Munich, FRG) (final concentrations). The contents of the dense granules were investigated in PRPI by measuring uptake and release of 14C-hydroxy-tryptamine-creatinine-sulphate (Amersham Buchler, Braunschweig, FRG). Total platelet ATP and ADP were measured with the luciferin-luciferase technique using ethanol extracts [25].

PIatelets

For immunological studies EDTA anticoagulated blood and serum were used for determination of platelet-bound and free antibodies by platelet adhesion immunofluorescence test [24] and a glycoprotein-specific ELISA [15]. Platelet associated immunoglobulin G was quantitatively assessed as described [14]. Platelet glycopr0tein complexes Ib/IX, IIb/IIIa, Ia/IIa were determined semiquantitatively by platelet adhesion immunofluorescence test using monoclonal antibodies specific for these complexes (FMC25, Gi5 and Gi14, respectively). For quantitative glycoprotein analysis platelets were collected as described by Clemetson et al. [4]. By means of a FicotI gradient the giant platelets, which normally would sediment with the red blood cell fi'action, were separated from other ceils. An indirect enzyme immunoassay [14] using the monoclonal antibodies Gi5 (specific for IIb/IIIa), FMC25 (Ib/IX), Gi14 (Ia/IIa), was employed for quantitation of the respective glycoprotein complexes.

By light microscopy, well stained giant platelets were visible in all affected patients. Platelet size varied, m a n y platelets being as large as erythrocytes or small lymphocytes. Platelet counts given by the automatic particle counter were 50% lower than those read in the counting chamber. Values are given in Table 1. Mean platelet volumes were increased up to 16 fl (normal range 6.5-12 fl). These values are probably underestimated as the very large platelets are not measured in the particle counter. Bleeding times were in the upper range of normal or slightly prolonged (Table 1). Platelet aggregation determined with P R P I and P R P I I was normal. A regular shape change was demonstrable after induction of aggregation with A D P and collagen. The platelet volume distribution curve of P R P I I was very similar to whole blood indicating that it contained the majority of giant platelets. The maximal aggregation with P R P I I was about 15% less than with P R P I due to contamination with red and white blood cells. Totai ptatelet A T P and A D P were increased ( A T P 9.3-12.4 gmol/10 n platelets [normal 4 . 5 8.5], A D P 6.5-8.4 gmol/10 n platelets [normal 1.7-3.9]). A T P / A D P ratio was normal. Platelet nucleotides of patient I2 were normal. Serotonin uptake and release were normal in all investigated patients (B2, C2, I2, II2, III1, III2). No platelet auto- or alloantibodies were detected in the serum of either patient, but platelet associated I g G was slightly elevated (2.1-2.6 fg [normal < 1.8fg]) in patient C2 and III2. In comparison with platelets of normal donors, giant platelets did not show abnormalities in density and distribution of gp I b / I X , I I b / I I I a , I a / I I a by fluorescence microscopy. By quantitative analysis, the concentration of the glycoprotein complexes I b / IX, I I b / I I I a , I a / I I a were not reduced (Table 1)i Ultrastructurally the enlarged platelets showed a normal morphology.

Investigations of granulocyte function

Granulocytes

Granulocyte function was assessed by using the granulocyte immunophagocytosis inhibition test [20] and the granulocyte chemiluminescence test using a luminometer (Lumat, Berthold, FRG). Granulocytes were isolated by a modified method of B6yum [1]. For determination of the phagocytic capability, granulocytes of patients (5000/gl) were attached to coated slides (Behring, Marburg, FRG) (30 ~tl of granulocyte suspension/reaction field). After washing, the reaction fields were incubated with sheep red blood cells sensitized with diluted rabbit anti-sheep antibodies. After a second washing and staining (Wright stain), TOOgranulocytes were microscopically analysed for phagocytosis. Inhibition of immunophagocytosis was assumed if less than 5% of granulocytes showed intracellular sheep red blood cells. The production of oxygen radicals (O~, H202) was determined by a luminol enhanced chemiluminescence test. Granulocyte suspension (20001; 2500 cells/lal) and 100gl of a 4 x 105mol luminol solution were mixed and production of oxygen radicals was induced by addition of 100 ~1 sheep red blood cells, 100 lal phorbolmyristate-acetate suspension (final concentration 2.5 gg/ml) or a suspension of opsonized zymosan (final concentration 31.25 gg/ ml), respectively. Chemiluminescence was measured by a luminometer.

A b o u t 40% of the granulocytes in the affected individuals contained spindle-shaped, blue stained inclusion bodies electron microscopically consisting of ribosomes associated with 7 - 1 0 m m filaments in parallel arrangement clearly to be differentiated from toxic D6hle bodies (Fig. 1). In comparison to n o r m a l controls the phagocytic capacity and radical production of granulocytes were normal.

Platelet immunology

Discussion The May-Hegglin anomaly is the classic hereditary giant platelet syndrome [11, 17] characterized by giant platelets, low platelet counts and spindle-shaped inclusion bodies in the leucocytes. Inheritance is autosomal dominant, but expression of the anomly can vary widely within the same family, as demonstrated in patient 2. Hae-

670 Table 1. Results of investigations in seven individuals affected by the May-Hegglin anomaly Control

Age (years)

Family i

Family 2

B1

B2

C2 a

I1

II2

III1

III2b

43

33

3

62

32

6

4

Platelet count (• 109/1)

> 130

60

78

76

135

45

64

60

Bleeding time simplate II (min', sec")

3'30"-8'30"

ND

7'

8'30"

4'30"

8'

11'

1'30"(Duke) (normal)

Mean platelet volume (fl)

6,5-11,5

15,8

16,2

16,3

11

16,1

15,2

14,2

Inclusion bodies in the leukocytes

0

+++

++

+++

(+)

+++

+++

+++

Giant platelets in blood smear (%)

0

45

40

55

2

90

65

60

Immunoflourescence GPIa/IIa GPIIIb/IIIa GPIb/IX

++ ++++ ++++

++ ++++ ++++

++ ++++ ++++

++ ++++ ++++

++ ++++ ++++

++ ++++ ++++

++ ++++ ++++

++ ++++ ++++

Quantitative GP analysis GPIa/IIa GPIIb/IIIa GPIb/IX

6000 30500 33 000

ND ND ND

13000 44000 29 000

ND ND ND

5200 30500 ND

10600 33400 28 000

ND ND ND

ND ND ND

ND, Not determined a Patient 1 b Patient 2

Fig. 1. The spindle-shaped leucocyte inclusion bodies, staining Iight blue with May-Gruenwald-Giemsa stain, consist of ribosomes and small filaments in parallel order which form a cytoplasmic area free of other cell organelles and not surrounded by a membrane. morrhagic diathesis is minor and the diagnosis is mostly established by chance, as in the reported cases. By the widespread use of automatic particle analysers for platelet counting, the n u m b e r of clinically asymptomatic pa-

tients who are diagnosed as having "thrombocytopenia" is increasing. Usually the family history does not reveal other affected family members; they can only be identified by appropriate investigations. The history of recurrent epistaxis and pretibial h a e m a t o m a s in the 6-year-old boy (III1), although noted as haemorrhagic diathesis, is not u n c o m m o n in healthy children of the same age and usually does not initiate further investigations. Peri-operative bleeding complications in the MayHegglin anomaly have been observed mainly after gynaecological operations and during tonsillectomy. In most patients, platelet counts are between 40 and 80 • 109 platelets/1. As automatic particle counters do not measure the very large platelets, their results tend to be approximately 50% lower [8, 12]. The normal or only marginally prolonged bleeding time makes a major function defect of the platelets unlikely. This is consistent with previous reports of normal platelet function [10] and morphology [16], as corroborated in our patients. The only known abnormality of the platelets in these patients is their increased size and consequently elevated levels of granule contents [6, 7]. Since in the May-Hegglin anomaly the megakaryocyte n u m b e r is normal and the platelet survival time is not shortened [7, 10, 13], a platelet production defect seems to be likely. Megakaryocytes show a demarcation m e m b r a n e system which forms m e m b r a n e complexes with the smooth endoplasmic reticulum [2], but the mechanism of production of giant platelets remains unresolved [12, 27]. The density of the well characterized glycoprotein complexes I I b / I I I a , I b / I X and I a / I I a was not reduced in

671 the platelet m e m b r a n e s of our patients. This confirms the results of Coller and Z a r r a b i [5] w h o did not find any abnormalities in the platelet m e m b r a n e s of a patient with M a y - H e g g l i n anomaly. T h e typical inclusion bodies in granulocytes are diagnostic for the M a y - H e g g l i n anomaly. T h e y differ ultrastructurally f r o m inclusion bodies of o t h e r anomalies by their spindle shape and the parallel o r d e r of filaments [8, 21]. T h e reason for their a p p e a r a n c e is as yet u n k n o w n . T h e few granulocyte function studies so far r e p o r t e d for the M a y - H e g g l i n a n o m a l y have yielded conflicting results. In one patient, a r e d u c e d phagocytic capacity of granulocytes in inflamed tissues was n o t e d [22], whereas others described r e d u c e d [3] or n o r m a l chemotaxis. O u r findings argue in f a v o u r of a n o r m a l phagocytic capacity and radical p r o d u c t i o n in these patients. T h e differential diagnosis of the M a y - H e g g l i n a n o m aly has to exclude other giant platelet s y n d r o m e s with a defect in gp I b / I X (Bernard-Soulier s y n d r o m e ) , interstitial nephritis ( E c k s t e i n s y n d r o m e , E p s t e i n synd r o m e , F e c h t n e r s y n d r o m e ) , o t h e r inclusion bodies in the granulocytes (Fechtner s y n d r o m e , Sebastian platelet s y n d r o m e ) , and diseases associated with D 6 h l e bodies (review: [8]). A s the low platelet count in patients with M a y - H e g g l i n a n o m a l y is mostly f o u n d by chance without an indicative family history and the m e g a k a r y o c y t e number of b o n e m a r r o w is n o r m a l or slightly elevated, the patients are often misdiagnosed as having " a u t o - i m m u n e t h r o m b o c y t o p e n i a " . Quantification of platelet-associated I g G is not helpful in that m a n y patients with hereditary t h r o m b o c y t o p e n i a show an elevated platelet associated I g G (unpublished data). In cases with persistent low platelet count of u n k n o w n origin, hereditary types of t h r o m b o c y t o p e n i a should be considered as potential differential diagnosis and verified by family investigation.

Acknowledgements The secretarial expertise of Tanja Ille is gratefully acknowledged. The patients were referred by Prof.Dr.U. Goebel, Department of Paediatrics, University of D%seldorf, Federal Republic of Germany and Dr. K. Wesseler, Department of Paediatrics, County Hospital Detmold, Federal Republic of Germany.

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