16212 Clin Pathol 1994;47:162-165 Acquired pseudo-pseudo Bernard-Soulier

syndrome complicating Gaucher's disease J Clin Pathol: first published as 10.1136/jcp.47.2.162 on 1 February 1994. Downloaded from

H Kelsey, C Christopoulos, AA Gray, S J Machin

Abstract and German Jews). It is characterised by Aims-To investigate the abnormality in often massive splenomegaly with hyper- platelet function in two patients with type splenism and pancytopenia. I Gaucher's disease causing a chronic Pathologically lipid-laden macrophages- bleeding tendency despite normalisation of the Gaucher cells-are found in the red pulp the platelet count after spleen removal. of the spleen, liver sinusoids, and the bone Methods-Routine laboratory methods marrow. Plasma concentrations of glucocere- were used to assess baseline coagulation. broside are raised 2- to 10-fold, and in the Platelet aggregometry was used to assess spleen grossly raised 10-1000 fold.' platelet responses to a range of agonists, Diagnosis has typically been by enzyme assay and abnormalities were further assessed in peripheral blood leucocytes or tissue fibro- in mixing experiments using washed blasts. With the advent of molecular platelets and patients' plasma. methods, diagnosis on the basis of detection Results-Platelets from both patients of the abnormal or missing gene has become with Gaucher's disease failed to aggluti- possible.2 nate to ristocetin, despite normal platelet Until recently management of Gaucher's surface glycoprotein (GP) Ib and plasma disease was supportive with the main thera- von Wiliebrand factor activity. The peutic manoeuvre being removal of the spleen agglutination of normal washed platelets for hypersplenism and excessive bleeding. was abolished by incubation in patient This usually corrects the thrombocytopenia plasma. The inhibitory activity did not and anaemia, but may worsen the bone dis- lie in the IgG fraction of patient plasma, ease. More recently treatment with enzyme and was found to be loosely associated replacement has become possible with man- with the patient platelet surface. nose conjugated glucocerebrosidase. Thrombo- Conclusions-The inhibition of ristocetin cytopenia (often less than 50 x 109/1)

induced platelet agglutination in patients predisposes to a bleeding diathesis, which http://jcp.bmj.com/ with Gaucher's disease causes a pro- may aggravate the anaemia, due to hyper- longed skin bleeding time. This could be splenism. The bleeding tendency may be due to the accumulated glucocerebroside disproportionate to the degree of thrombo- in the plasma coating the platelet mem- cytopenia.1 Hepatic disease may be associated brane. It is suggested that the term with abnormalities in clotting factor syn- pseudo-pseudo Bernard-Soulier syn- thesis,4 causing a prolonged prothrombin drome would be appropriate, as on ini- time. The raised circulating glucocerebroside on September 29, 2021 by guest. Protected copyright. tial screening, the abnormality has the concentration may interfere with the clotting features of Bernard-Soulier syndrome, cascade.5 Here we describe an abnormality in but further investigation shows normal platelet function in two adults with type 1 plasma von Willebrand activity and Gaucher's disease in whom a bleeding ten- platelet surface GP lb concentrations. dency persisted despite spleen removal, rela- The inhibitory activity is not due to a tive normalisation of the platelet count, and platelet specific antibody as is the case in normal liver function tests and coagulation pseudo-Bernard Soulier syndrome. screen.

( Clin Pathol 1994;47:162-165) Methods Department of CASE REPORTS Haematology, University College, Gaucher's disease is the most common of the A 47 year old woman (Ashkenazim) was diag- London lysosomal storage disorders. It is charac- nosed as having Gaucher's disease when she H Kelsey terised by deficiency of the lysosomal enzyme was 14 years old after dental extraction led to C Christopoulos S Machin glucocerebrosidase, resulting in accumulation excessive bleeding. The platelet count at this J of the sphingolipid glucocerebroside in the time was <15 x 109/1. Her spleen was Princess Margaret Hospital, Swindon plasma and cells of the reticuloendothelial removed because of massive enlargement A A Gray system.' It is autosomal recessive and com- when she was 24. Easy bruising and bleeding Correspondence to: prises three types, characterised by the age of continued despite improvement of the Dr H C Kelsey, Department Haematology, onset and degree of involvement of the ner- platelet count, and when she was 37 her left University College Hospital, vous system. Type 1 or chronic non-neurono- hip was replaced. Bleeding was excessive both Gower Street, London most common red cell WC1E 6AU pathic Gaucher's disease is the during and after surgery, requiring a incidence in transfusion and transfusion for Accepted for publication form, with particularly high (1 platelet 7 September 1993 600 to 1 in 2500) among Ashkenazim (Polish haemostasis. Seven years later her right hip Acquired pseudo-pseudo Bernard-Soulier syndrome complicating Gaucher's disease 163

was replaced, bleeding at which was success- Tyrodes buffer, as described before.6 Patient fully prevented by prophylactic platelet trans- and control platelets were then incubated

fusions. As a result of red cell and platelet with patient or control plasma and agglutina- J Clin Pathol: first published as 10.1136/jcp.47.2.162 on 1 February 1994. Downloaded from transfusion she formed several red cell and tion to ristocetin tested immediately and after HLA antibodies. After the second hip opera- 1 hour of incubation at room temperature. tion an anaphylactic transfusion reaction Immunoglobulin G fractions of patient and occurred despite transfusing compatible control sera were isolated by affinity chro- blood. Subsequently an additional red cell matography using a protein G column, eluted antibody was found. Since recovery, three with glycine HC1 buffer (pH 2 8), and then units of autologous blood have been placed in dialysed against phosphate buffered saline long term storage. (pH 7'2) to give a final concentration of 5-10 A 23 year old caucasian woman was found mg/ml. IgG fractions were incubated with to haure Gaucher's disease as a child. She had washed platelets for one hour and cryoprecip- epistaxis and severe menorrhagia as a child itate then added before testing ristocetin and in addition to thrombocytopenia a induced agglutination. platelet function abnormality was detected but could not be further defined. When she was 15 years old her spleen was removed for Results hypersplenism. On recovery the bleeding Both patients had normal platelet counts and problems persisted, with heavy menorrhagia initial clotting screens (table). The bleeding despite a normal platelet count. She devel- time was >15 minutes in both cases. Platelet oped extensive bruising at the time of a road aggregation responses to adenosine diphos- traffic accident when, aged 19 years, she sus- phate, collagen, and arachidonic acid were tained a fractured pelvis. She was referred for normal (table). Ristocetin induced agglutina- investigation of a bleeding tendency when tion was absent at all concentrations tested. surgical correction of the damage sustained This did not correct with procine F VIII, or was contemplated. cryoprecipitate and ristocetin, and there was Blood was collected from the two patients no agglutination on the addition of cryopre- on three separate occasions into EDTA for cipitate alone (representative traces fig 1A). full blood and platelet counts (STK S, Factor VIIIc and vWF:Ag and vWF:RiCoF Coulter Electronics, Florida, USA) and into activities were normal (table). 3*13% trisodium citrate in a ratio of 9 parts Platelet surface glycoproteins Ib and blood to 1 part trisodium citrate, for coagula- IIb/IIIa were present in normal amounts on tion and platelet function studies. Control both patients' platelets. Free platelet bindable plasma and platelets were similarly collected immunoglobulin was detected in both patient from normal male volunteers. Prothrombin sera, and platelet surface immunoglobulin was time (PT), thrombin time (TIT), and acti- raised in the second patient. This immuno-

vated partial thromboplastin time (APPT) globulin reacted with Bemard-Soulier http://jcp.bmj.com/ were performed by routine techniques.6 platelets in both cases (results not shown). Factor VIIIc was assayed by a one stage auto- The response of washed normal platelets to mated method, von Willebrand factor antigen ristocetin was abolished after 1 hour of incu- (vWF:Ag) by ELISA, and ristocetin cofactor bation in patient plasma (fig 1B). This activity (vWF:RiCoF) by platelet agglutina- inhibitory activity was not present in the IgG tion. Von Willebrand multimers were fraction of patient sera (fig 2A). The assessed by sodium dodecyl sulphate gel elec- inhibitory activity was loosely associated with on September 29, 2021 by guest. Protected copyright. trophoresis, as described before6 and platelet patient platelets as washing of patient plate- nucleotides (adenosine triphosphate, adeno- lets restored ristocetin responses when they sine diphosphate; total and releasable) using a were tested immediately after resuspending in luciferase firefly luminescence technique.6 Platelet glycoproteins (GP) Ib and IIb/IIIa, platelet surface immunoglobulin, and free Results ofinitial investigations-full blood count, platelet bindable immunoglobulin were asses- coagulation screen, platelet aggregation and bleeding sed by a fluorescence flow cytometric method.7 time-patient and control values Bleeding time was assessed with a template Parameter Case I Case 2 Normal method and platelet aggregometry6 performed Platelets x 109/1 179 174 150-450 in a four channel aggregometer using the fol- PT (control) 13s (12) 15s (13) 11-15s APTT (control) 39s (36) 39s (43) 30-40s lowing agonists: ristocetin (Lundbeck, Luton, TT (control) lOs (13) 16s (13) 11-15s England) 1-25, 1-0, 0.7, 0-5 mg/ml; ADP F VIIIc 0 95 0-66 0-5-2-0 Co Ltd, vWF Ag 1-35 0-78 0-5-2-0 (Sigma Chemical Poole, Dorset) 5-0, vWF Ricof 1-75 0-85 0-5-2-0 2-5, 1.0, 0-5 umol/l; collagen (Hormon vWF multimers Normal Normal 4 1 0 Platelet nucleotides Normal Normal Chemie, Munich, Germany) 0, ,ug/ml; GP lb Normal Normal and arachidonate (Sigma) 1-0 pM. Porcine GP IIb/IIIa Normal Normal Maidenhead, PSIgG Normal Normal FVIII complex (Porton, Platelet aggregation: Berkshire) and cryoprecipitate were used in Ristocetin 1-25 mg/ml Absent Absent correction studies with ristocetin. 1-0 mg/ml Absent Absent 0-7 mg/ml Absent Absent In mixing experiments washed platelets were 0 5 mg/ml Absent Absent washes in buffer with 10 ADP Normal Normal used (three Tyrodes Collagen Normal Normal mM EDTA and 10 ng/ml prostacyclin ana- Arachidonate Primary Primary logue (ZK36 374 Schering Chemicals) and wave wave then resuspended in 1:4 human albumin with Bleeding time >15 m >15 m 2-10 m 164 Kelsey, Christopoulos, Gray, Machin

Figure 1 (A) Patient A lation, resulting in an apparent deficiency of platelet responses to PRP 200 x 10/I ristocetin alone (1 25 Ristocetin 1-25 mg/ml factor 1X4 and other clotting factors. The in

mglml), porcine FVIII vivo importance of this inhibition was not J Clin Pathol: first published as 10.1136/jcp.47.2.162 on 1 February 1994. Downloaded from (2 IUlml), cryoprecipitate and ctyoprecipitate with clear. In our patients clotting screens were ristocetin. (B) Ristocetin Ristocetin only repeatedly normal. Both patients had pro- 7 _ = = Porcine FVII agglutination response of Cryoprecipitate longed bleeding times and no platelet aggluti- washed controlplatelets 1 minute Cryoprecipitate + ristocetin nation to ristocetin. This was despite normal before and after incubating platelet surface glycoprotein Ib and with Gaucherpatient B plasma plasmafor 1 hour. PRP 200 x 10/i factor VIII-vWF activities. The inhibitory Ristocetin effect resides in the plasma, as shown by mix- 1-25 mg/ml Before ing studies, and is loosely associated with the platelet surface because it can be removed by simple washing, so normalising ristocetin agglutination. Myeloma proteins inhibit platelet adhesion and aggregation-particu- larly adenosine diphosphate induced aggrega- tion,8 probably by coating the platelet surface. 1 hour of incubation A pseudo-Bemard-Soulier syndrome has 1 minute been described9 in which an autoantibody directed against GP Ib, in a patient receiving procainamide, resulted in loss of function. In patient plasma. Reincubation in patient our patients, however, the IgG fraction did plasma for 1 hour resulted in loss of the not inhibit ristocetin induced agglutination, response compared to patient platelets incu- despite the presence of free platelet bindable bated in normal plasma (fig 2B). immunoglobulin in the serum. This latter is not likely to be platelet specific activity, repre- senting HLA antibody, secondary to repeated Discussion exposure to blood products. Furthermore, These two patients with well characterised simple washing is unlikely to remove specifi- Gaucher's disease had a persistent bleeding cally bound IgG, whereas in these experi- tendency despite normalisation of the platelet ments inhibitory activity was easily removed count after spleen removal. In one case this by washing. led to severe bleeding during and after Macromolecules in the circulation, such as surgery, and in the other to persistent menor- hydroxyethyl starch, can induce an acquired rhagia resulting in chronic anaemia. von Willebrand type syndrome with low Abnormalities in clotting factors in Gaucher's plasma F VIII:vWF Ag concentrations and a disease may be caused by impaired synthesis bleeding diathesis.10 Macromolecules, such as secondary to liver disease. In addition, pro- HES and dextran, are also adsorbed on to the http://jcp.bmj.com/ longation of the APTT has been described platelet surface, inducing a thrombasthenic due to interference by plasma sphingolipid in type effect." A similar effect probably occurs the phospholipid dependent phases of coagu- with the accumulated sphingolipid in the plasma of these patients with Gaucher's dis- ease. An inhibitory effect on the phospholipid phases of coagulation has already been Figure 2 (A) Effect of A shown, and binding of glucocerebroside to on September 29, 2021 by guest. Protected copyright. prior incubation ofwashed PRP 200 x 1 0/i the platelet surface may interfere with the platelets with patient and control IgGfractions on Ristocetin 1-25 mg/ml Patient vWF-ristocetin-GP Ib interaction, giving rise ristocetin induced to the platelet function defect described here. agglutination. (B) Effect .Ao We suggest that the term pseudo-pseudo ofwashingpatientplatelets on ristocetin induced Bernard-Soulier syndrome be applied to the agglutination immediately abnormality in platelet function seen in these on remixing with patient patients with Gaucher's disease. Although a plasma and after incubation for 1 hour. plasma factor is responsible for the loss in ris- tocetin induced agglutination, this is not a platelet specific antibody. The effect is proba- 1 minute bly caused by excess sphingolipid directly binding to glycoprotein Ib or to the platelet surface adjacent to the GP on the platelet Ristocetin 1-25 mg/ml surface. Further studies are required to deter- mine the mechanism exactly. Enzyme Immediate replacement treatment in these patients may, by reducing plasma glucocerebroside concen- trations, improve the bleeding tendency.

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