J Clin Pathol 2000;53:863–867 863

Apolipoprotein H, a new mediator in the J Clin Pathol: first published as 10.1136/jcp.53.11.863 on 1 November 2000. Downloaded from inflammatory changes ensuing in jeopardised human myocardium

H W M Niessen, W K Lagrand, HJAMRensink, ChJLMMeijer, L Aarden, C E Hack

Abstract Recently, we have shown co-deposition of Aim—To investigate the presence of mem- complement with the acute phase C brane “flip flop” in ischaemic human reactive protein (CRP) in infarcted sites of myocardium, we assessed depositions of human myocardium.5 The ligand for CRP in H (apoH; â2- infarcted myocardium, however, remains to be 1) in ischaemic myocardium. Serum pro- established. One possibility6 is that binding tein apoH can bind to negatively charged sites for CRP are generated in cells that have and can also inhibit blood undergone so called “flip flop”.7 In normal in vitro. We hypothesised that, cells, various phospholipids are asymmetrically because of its aYnity for phosphatidyl distributed between the inner and outer leaflet serine, apoH might bind to “flip flopped” of the membrane, PS being mainly located in cells and would therefore be useful as a the inner leaflet. In damaged cells (ischaemic, marker for membrane flip flop in vivo. apoptotic, or necrotic cells) phospholipids of Methods—Myocardial tissue specimens the inner and outer leaflet exchange, a were obtained from patients who had died phenomenon known as flip flop,89 leading to within 14 days of acute myocardial infarc- the exposure of PS in the outer leaflet. We tion. hypothesised that because of its aYnity for PS, Results—Immunohistochemical analysis apoH could be used as an in vivo marker for of these specimens revealed that apoH was these “flip flopped” cells. In our present study selectively deposited in infarcted areas of we tested this hypothesis and searched for human myocardium of at least one day’s apoH depositions in the infarcted myocar- duration. Depositions of apoH were not dium, in relation to those of activated comple- found in non-ischaemic myocardial tissue ment. samples obtained from patients who died from other (extracardial) causes. In vitro experiments with the human leukaemia T Patients, materials, and methods PATIENTS

cell line Jurkat, subjected to apoptosis by http://jcp.bmj.com/ etoposide, showed that apoH was bound to Patients, referred to the department of pathol- the membrane of apoptotic cells. How- ogy for necropsy, were included in this study ever, these experiments also indicated that when at necropsy they showed signs of a recently flip flop itself is not suYcient for apoH developed acute myocardial infarction; that is, binding. In addition, Jurkat cells that decreased lactate (LD) staining bound apoH were positive for activated (decolouration) of the aVected myocardium. Department of complement complexes, as was also found Most of the patients had participated in earlier

Pathology, Free studies on the involvement of CRP and comple- on September 29, 2021 by guest. Protected copyright. University Hospital, in the human heart. 5 Conclusions—These results suggest that ment in infarcted myocardium. Our study was PO Box 7057, De approved by the ethics committee of the Free Boelelaaan 1117, 1081 apoH is involved in the inflammatory HV Amsterdam, The processes that occur in ischaemic myo- University Hospital Amsterdam. Netherlands cardium. H W M Niessen (J Clin Pathol 2000;53:863–867) PROCESSING OF TISSUE SPECIMENS ChJLMMeijer Myocardial tissue specimens were obtained from the infarcted as well as from adjacent Department of Keywords: myocardium; apolipoprotein H; Cardiology, Free inflammation; complement sites. These latter sites showed normal LD University Hospital staining patterns and were studied as internal W K Lagrand controls. Before being prepared as cryosec- Apolipoprotein H (apoH; â2-glycoprotein 1) is tions, the tissue specimens were stored at CLB, Sanguin Blood ° a 50 kDa binding serum protein −196 C (liquid N2). The glass slides used for Supply Foundation microscopy were pretreated with 0.1% poly-L- Service, PO Box 9190, that preferentially binds to negatively charged 1006 AD Amsterdam, phospholipids such as phosphatidyl serine lysine (Sigma Chemical Company, St Louis, The Netherlands (PS).12 Because of this property, apoH might Missouri, USA) to enhance the adherence of HJAMRensink inhibit blood coagulation and ADP dependent the frozen tissue sections. L Aarden platelet aggregation, at least in vitro. Its precise C E Hack 3 physiological role remains to be elucidated. Correspondence to: Chonn et al found that the fast clearance of PS We used a monoclonal (C3-9; IgG-1 Dr Niessen containing liposomes by the liver in mice is subclass) against activated complement factor [email protected] mediated via plasma derived apoH, suggesting C3 that has been used previously for immuno- 5 Accepted for publication that this protein might have a role in removing histochemical studies. Cof23, directed against 11 May 2000 PS containing microvesicles.4 apoH, was a gift of Dr Koike (Department of

www.jclinpath.com 864 Niessen, Lagrand, Rensink, et al

Medicine II, Hokkaido University School of with specific antibody solutions was performed J Clin Pathol: first published as 10.1136/jcp.53.11.863 on 1 November 2000. Downloaded from Medicine, Sapporo, Japan). Polyclonal rabbit for 60 minutes at room temperature (C3-9 antihuman apoH/34, was obtained by immu- diluted 1/1000 in PBS-BSA; biotinylated anti- nising rabbits with purified apoH (Department apoH polyclonal antibody diluted 1/500; of AutoImmune Diseases, CLB, Sanguin monoclonal antibody against apoH diluted Blood Supply Foundation, The Netherlands). 1/400). In control experiments, similar incuba- For FACS analysis, fluorescein isothiocy- tions were performed with irrelevant control anate (FITC) conjugated annexin V (Boe- monoclonal antibodies: IgG1 and mouse my- hringer Ingelheim, Heidelberg, Germany) and eloma protein, MOPC (Cappel, Organon phycoerythrin (PE) conjugated streptavidin Teknika, Turnhout, Belgium). (Amersham International, Amersham, UK) The slides incubated with antibodies against were used. complement were washed for 30 minutes with Biotinylation of monoclonal and polyclonal PBS and incubated with horseradish peroxi- antibodies using L-C-biotin-N-hydroxi- dase conjugated rabbit antimouse immuno- succimide ester (Pierce Chemical Co., Rock- globulins (RaM-HRP; Dakopatts A/S), diluted ford, IL) was performed according to the 1/25 in PBS-BSA. The slides incubated with manufacturer’s instructions. biotinylated anti-apoH monoclonal or polyclo- nal antibodies were washed for 30 minutes and FACS ANALYSIS incubated with streptavidin horseradish peroxi- The human leukaemia T cell line Jurkat was dase conjugates (Dakopatts), diluted 1/500 in maintained in Iscove’s modified Dulbecco’s PBS-BSA for one hour. Thereafter, the slides medium (IMDM) (Gibco, Grand Island, New were washed again in PBS and incubated for York, USA) supplemented with 5% (vol/vol) four minutes in 0.5 mg/ml 3,3'- fetal calf serum (FCS), 100 U/ml penicillin diaminobenzidinetetrahydrochloride (DAB; (Gibco), 100 µg/ml streptomycin (Gibco), Sigma) in PBS, pH 7.4, containing 0.01% (vol/

50 µM 2-mercaptoethanol, and 20 µg/ml vol) H2O2. The slides were then washed again, human transferrin (Sigma) (culture medium) counterstained with haematoxylin for 40 sec- ° at 37 C in a humid atmosphere and 5% CO2. onds, dehydrated, cleared, and finally Apoptosis was induced in the Jurkat cells by mounted. incubation with 25 µM etoposide (Sigma) Microscopic criteria10 11 were used to estimate overnight. The cells were then separated from infarct duration in all myocardial tissue speci- the medium by centrifugation at 800 ×g for six mens. Jeopardised fibres were characterised by minutes, resuspended in IMDM (no FCS) in a the intensity of eosinophilic staining of involved 96 well roundbottom plate (100 µl/well, myofibres, loss of nuclei and cross striation, 200 000 cells/well), and washed once with polymorphonuclear neutrophil and lymphocyte IMDM. Thereafter, cells were incubated with infiltration, and fibrosis. However, because serum (or purified apoH) diluted in IMDM morphological judgement is more reliable in (50 µl/well), for 30 minutes at 37°C. After paraffin wax embedded slides, corresponding washing four times with BB (1.19 g Hepes, para n wax embedded tissue slides were also

Y http://jcp.bmj.com/

4.4 g NaCl, 0.19 g KCl, 0.10 g CaCl2, 0.1 g made, to confirm the determination of jeopard-

MgCl2 in 500 ml, pH 7.4) and BA (0.5% (wt/ ised versus non-jeopardised tissue. Two inde- vol) bovine serum albumin (BSA) (Boehringer pendent investigators (HWMN, WKL) each Mannheim), 0.02% (wt/vol) azide), bioti- judged and scored all slides for infarct duration nylated anti-apoH antibodies (5 µg/ml in buVer and anatomical localisation of specific antibody BB-BA, 50 µl/well) or anti-C3 monoclonal as visualised by immunohistochemical staining. antibody (C3-9) were added to the cells for 30 Anatomical localisations examined were myofi- minutes at 4°C. After three washes with BB-BA bre (membrane, cytoplasm, cross striations) on September 29, 2021 by guest. Protected copyright. buVer, a mixture of PE labelled streptavidin and bloodvessel elements. For the final scoring (Becton Dickinson, San Jose, California, USA) results, a consensus was achieved by the two and FITC labelled annexin V (Boehringer investigators. Ingelheim), diluted 1/200 and 1/2000 in BB-BA buVer, respectively, was added to the cells (final volume 50 µl/well). After 20 minutes Results of incubation at 4°C in the dark, the cells were PATIENTS washed three times with BB-BA. Finally, cells Myocardial tissue specimens were obtained were resuspended in 100 µl BB-BA in epics- from 17 patients who had died after acute tubes and analysed on a FACScan (Becton myocardial infartion as confirmed by necropsy Dickinson, San Jose, California, USA). performed within 24 hours after death (table 1). Specimens were obtained from the inf- IMMUNOHISTOCHEMISTRY arcted as well as from the unaVected myocar- Frozen sections (4 µm thick) were mounted on dial tissue. The infarct age, assessed by micro- to glass slides, dried for one hour by exposure to air, and fixed in acetone (“Baker analysed Table 1 Duration of infarctions in the patients included reagent”; Mallinckrodt Baker BV, Deventer, Infarction age Number of patients Holland). The slides were incubated at room temperature for 10 minutes with normal rabbit < 12 hours 3 > 12 to < 24 hours 1 serum (Dakopatts A/S, Glostrup, Denmark) 1–3 days 4 and diluted 1/50 in phosphate buVered saline 3–5 days 2 (PBS) containing 1% (wt/vol) BSA (PBS-BSA) 5–9 days 3 9–14 days 4 after rinsing in PBS. Incubation of the slides

www.jclinpath.com Apolipoprotein H in jeopardised human myocardium 865 J Clin Pathol: first published as 10.1136/jcp.53.11.863 on 1 November 2000. Downloaded from

Figure 3 Immunohistochemical localisation of apolipoprotein H (biotinylated polyclonal antibody) in infarcted myocardium. Specimens were from a patient with infarction of five to nine days’ duration (magnification, ×630).

As shown recently, complement was localised in infarcted sites of human myocardium.5 Apo- H, as detected by the biotinylated monoclonal antibody, was found in the same parts that stained positive for complement (fig 1). Notably, staining for apoH was most intense at the plasma membrane of cardiomyocytes. Cytoplasmatic localisation of apoH was also Figure 1 (A) Immunohistochemical localisation of the found, although staining for apoH in the cyto- activated complement component C3 (monoclonal antibody C3-9) and (B) apolipoprotein H (biotinylated monoclonal plasm was clearly less than that of the plasma antibody) in infarcted myocardium (J, jeopardised; N, membrane. Some staining of cross striations normal). Specimens were from a patient with an infarction was also found (fig 2). In larger infarcts, the of five to nine days’ duration. The frozen tissue sections shown were from the same myocardial infarction site border zone appeared to stain more intensively (magnification, ×100). for apoH than the centre of the infarcted region. scopical criteria,10 11 varied from less than 12 Similar to complement, apoH was not hours to more than 2 weeks. detected in infarctions of less then 24 hours duration. Furthermore, apoH was not found on endothelium, in contrast to complement.5 LOCALISATION OF ACTIVATED COMPLEMENT AND Similar staining results were obtained when a apoH IN INFARCTED MYOCARDIUM biotinylated rabbit polyclonal antibody was http://jcp.bmj.com/ used against apoH (fig 3). Staining of the myocardial tissue specimens with irrelevant control antibodies yielded negative results. In addition, internal controls—specimens taken from non-infarcted sites of the myocardium of the same patient— did not stain for C3 or apoH. Furthermore, myocardial tissue specimens from an immature on September 29, 2021 by guest. Protected copyright. child who died in utero at an amenorrhoea duration of 22 weeks (these specimens were taken to represent a pure, non-ischaemic myo- cardial control) did not stain for C3 or apoH, and neither did an old infarction (> 1 year old). Unfortunately, immunohistochemical stud- ies have the limitation that the well known marker of membrane flip flop, annexin V, can- not be used reliably in tissue slides of the heart. For this reason, we performed in vitro studies using Jurkat cells. These cells were incubated with etoposide overnight, which causes them to die as a result of apoptosis.12 Subsequently, the cells were incubated with anti-apoH or anti- C3. The flip flop phenomenon was assessed by staining for annexin V. The cells that were annexin V negative did not bind apoH (table 2), whereas annexin V positive cells did. Figure 2 Positive staining of myofibre elements Recently Manfredi et al have shown that Jurkat (sarcolemma, (A) cytoplasm, and (B) cross striation cells, positive for annexin V, stained for (arrows)) in infarcted myocardium for apolipoprotein H apoH.13 14 However, when we characterised (biotinylated monoclonal antibody; magnification, ×630). The specimen is from a patient with an infarction of one to these cells in more detail, it appeared that only three days’ duration. cells that also stained with propidium iodide

www.jclinpath.com 866 Niessen, Lagrand, Rensink, et al

Table 2 Binding of annexin V,activated complement, and calised in ischaemic human hearts together J Clin Pathol: first published as 10.1136/jcp.53.11.863 on 1 November 2000. Downloaded from apolipoprotein H to apoptotic cells with complement membrane attack complex (MAC).22 It has been suggested that apolipo- Annexin V (–) Annexin V (+) Serum Serum protein J might be involved in the clearance of damaged and necrotic tissue, together with (–) (+) (–) (+) MAC. A similar role might be played by apoH, Control12134755 especially because the clearance of liposomes Anti-apoH 13 16 66 328 Anti-C3-9 10 13 64 584 by the liver in mice is in part mediated by plasma derived apoH. Interestingly, after inter- Jurkat cells were incubated with 25 µM etoposide overnight. action with serum, liposomes not only become Cells were then analysed for binding of annexin V, activated 4 complement, or apolipoprotein H via fluorecent activated cell positive for apoH but also for complement. In sorter (FACS) analysis. the human myocardium and in in vitro experi- Serum concentration was 2.5%. ments with Jurkat cells, we have shown Results are mean fluorescent intensity of five experiments. Anti-apoH was biotinylated rabbit polyclonal 34. colocalisation of activated complement and apoH. This raises the possibility that apoH, (and thus were beyond the early phase of bound to cells, might trigger or enhance com- apoptosis as characterised by the flip flop phe- plement activation, directly or indirectly via nomenon) bound apoH. This indicates that CRP–ligand complexes. staining for apoH is not discriminative in In conclusion, for the first time we have detecting cells that only underwent flip flop of shown that apoH is localised in infarcted the membrane. However, similar to the human myocardium to areas that also contain immunohistochemical staining results in the CRP and activated complement.5 We therefore heart, apoH positive cells also bound comple- hypothesise that apart from complement, CRP, ment, suggesting a role of both mediators in the and apolipoprotein J, apoH is a new player in inflammatory mediated process of cell death. the inflammatory changes ensuing in infarcted myocardium. Discussion The precise mechanisms contributing to myo- This study was financially supported by the Netherlands Heart cardial cell death in the human myocardium Foundation, grant number 93–119. Dr Niessen is a recipient of the Dr Dekker programme of the Netherlands Heart Founda- after infarction are still not fully understood. tion (D990025). We have hypothesised that in jeopardised myo- cardium the membrane of cardiomyocytes 1 Galli M, Comfurius, Maassen C, et al. Anticardiolipin anti- might become perturbed, thereby becoming a bodies (ACA) directed not to but to a plasma target for acute phase such as serum protein cofactor. Lancet 1990;335:1544–7. 6 2 McNeil HP, Simpson RJ, Chesterman CN, et al. Anti- phospholipase A2 and CRP. This would result phospholipid antibodies are directed against a complex in fixation of CRP to the cells and the antigen that includes a -binding inhibitor of 6 coagulation: beta 2-glycoprotein (apolipoprotein H). Proc subsequent activation of complement. Indeed, Natl Acad Sci U S A 1990;87:4120–4. 3 Sheng Y, Sali A, Herzog H, et al. Site-directed mutagenesis colocalisation of complement and CRP in of recombinant human beta 2-glycoprotein 1 identifies a jeopardised human myocardium was recently cluster of lysine residues that are crucial for phospholipid http://jcp.bmj.com/ demonstrated, supporting this hypothetical binding anti-cardiolipin antibody activity. J Immunol 1996; 5 157:3744–51. mechanism. 4 Chonn A, Semple SC, Cullis PR. Beta 2-glycoprotein I is a Perturbation of the cell membrane involves major protein associated with very rapidly cleared lipo- somes in vivo, suggesting a significant role in the immune an exchange of phospholipids of the inner and clearance of “non-self” particles. J Biol Chem 1995;270: outer leaflets, resulting in flip flop of the mem- 25845–9. 5 Lagrand WK, Niessen HWM, Wolbink GJ, et al. C-Reactive brane. As a consequence of this process, cells protein colocalizes with complement in human hearts dur- expose PS in the outer leaflet. Flip flop of cell ing acute myocardial infarction. Circulation 1997;95:97– 103. on September 29, 2021 by guest. Protected copyright. membranes in vivo is diYcult to demonstrate. 6 Hack CE, Wolbink GJ, Schalkwijk C, et al. A role for secre- It has been shown that in vitro apoH binds to tory phospholipase A2 and C-reactive protein in the 15 removal of injured cells. Immunol Today 1997;18:111–15. PS. Because of this property, apoH deposition 7 Zwaal RF, Schroit AJ. Pathophysiologic implications of in tissues may reflect flip flop of membranes in membrane phospholipid asymmetry in blood cells. Blood 1997;89:1121–32. vivo. In our present study, we indeed found that 8 Zachowski A. Phospholipids in animal eukaryotic apoH is localised to jeopardised human membranes: transverse asymmetry and movement. Bio- chem J 1993;294:1–14. myocardium. Moreover, apoH in particular 9 Higgins CF. Flip-flop: the transmembrane translocation of appeared to bind to the membranes of cardio- . Cell 1994;79:393–5. 10 10.Mallory GK, White PD, Salcedo-Salgar J. The speed of myocytes, supporting its supposed function as healing of myocardial infarction. Am Heart J 1939;18:647– a marker for membrane flip flop. However, in 51. 11 Cotran SC, Kumer V, Robbins LR. The heart. In: Robbins vitro experiments in human leukaemia T cell pathology. Basis of disease, 4th ed. Philadelphia: WB Jurkat cells indicate that flip flop itself is not Saunders 1989:605–14. 12 Martin SJ, Reutelingsperger ChPM, McGahon AJ, et al. suYcient for apoH binding. Distribution of plasma membrane phosphatidylserine is a In addition to binding to PS, apoH might general feature of apoptosis regardless of the initiating 16–18 stimulus: inhibition by overexpression of BCL2 and Ab1. J also bind to cardiolipin. Cardiolipin is a Exp Med 1995;182:1545–56. constituent of the inner19 20 and outer mito- 13 Manfredi AA, Rovere P, Galati G, et al. Apoptotic cell clear- 21 ance in systemic erythematosus. I. Opsonization by chondrial membrane. Thus, cardiolipin antiphospholipid antibodies. Arthritis Rheum 1998;41:205– might also serve as a binding site for apoH. 14. 14 Manfredi AA, Rovere P, Heltai S, et al. Apoptotic cell clear- This could explain the cytosolic localisation of ance in systemic lupus erythematosus. II. Role of apoH that we saw in a few of the infarctions. beta2-glycoprotein I. Arthitis Rheum 1998;41:215–23. 15 Wurm H. Beta-2 glycoprotein-1 (apolipoprotein H) interac- The identity of the ligands for apoH on (apop- tions with phospholipid vesicles. Int J Biochem 198;16:511– totic) cells is currently under investigation. 15. 16 Aubry F, Crickx B, Nicaise P, et al. Anti-beta 2 In addition, another apolipoprotein, apo- glycoprotein-1 antibodies in idiopathic livedo reticularis. liprotein J (clustrin) has been found to be colo- Ann Dermatol Venereol 1995;122:667–70.

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17 Borchman D, Harris EN, Pierangeli SS, et al. Interaction 20 Daum G. Lipids of mitochondria. Biochim Biophys Acta J Clin Pathol: first published as 10.1136/jcp.53.11.863 on 1 November 2000. Downloaded from and molecular structure of cardiolipin and beta-2 1985;822:1–42. glycoprotein-1. Clin Exp Immunol 1995;102:373–8. 21 Hovius R, Lambrechts H, Nicolay K, et al. Improved meth- 18 Pierangeli SS, Harris EN, Davis SA, et al. Beta-2 ods to isolate and subfractionate rat liver mitochondria. glycoprotein-1 enhances cardiolipin binding activity but is Lipid composition of the inner and outer membrane. not the antigen for antiphospholipid antibodies. Br J Hae- 1990; :217–26. matol 1992;82:565–70. Biochim Biophys Acta 1021 19 Peitsch MC, Tschopp J, Kress A, et al. Antibody independ- 22 Vakeva A, Laurila P, Meri S. Co-depostion of with ent activation of the complement system by mitochondria the complement membrane attack complex in myocardial is mediated by cardiolipin. Biochem J 1988;249:495–500. infarction. Immunology 1993;80:177–82.

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elastase, cathepsin G, lysozyme, and (and possibly other commercial brands) of bactericidal/permeability increasing protein ethanol fixed neutrophil slides be aware of Correspondence (BPI). IIF was then repeated on all sera on this phenomenon, and consider repeating any two separate occasions using in house (kindly sera producing such combined “atypical supplied by the Division of Immunology, ANCA” IIF patterns on alternative ethanol Royal Brisbane Hospital) and two commer- fixed neutrophil substrates to clarify their cial (Inova Diagnostics (diVerent batch) and “true” IIF pattern. Furthermore, antigen MPO-ANCA may produce a Medical and Biological Laboratories (MBL, specific ELISA testing for MPO-ANCA and combination of P-ANCA and atypical Nagoya, Japan)) ethanol fixed neutrophil PR3-ANCA should also be performed on all 3 cytoplasmic ANCA indirect slides. The IIF staining patterns and end such sera because combining IIF and ELISA in ANCA testing improves overall diagnostic immunofluorescent patterns on certain point titres were determined by consensus. Table 1 summarised the results. In four of specificity/predictive value compared with ethanol fixed neutrophil substrates 6 the six sera, no reactivity other than MPO- using either test alone. The P-ANCA pattern is defined as perinu- ANCA was detected using the ANCA RCWWONG clear indirect immunofluorescent (IIF) stain- Combi-kit ELISA. Of the other two sera, one K FIELD ing on ethanol fixed normal human also contained -ANCA and the Division of Immunology, Queensland Health Pathology neutrophils.1–3 This pattern is an artefact of other lysozyme-ANCA. Nevertheless, in ad- Services, Level 3, Lions Pathology Building, Princess ethanol fixation, dependent on the redistribu- dition to P-ANCA staining, atypical cytoplas- Alexandra Hospital, Woolloongabba 4102, mic staining was consistently produced by all Queensland, Australia tion of certain cationic neutrophil granule [email protected] proteins (such as myeloperoxidase (MPO), six MPO-ANCA sera on the Inova slides, but not on the MBL or in house slides. These lactoferrin, and lysozyme) around the nega- 1 Lock RJ. Detection of autoantibodies to neutro- tively charged nuclear membrane.12However, findings were reproducible on two diVerent phil cytoplasmic antigens. J Clin Pathol certain MPO-ANCA can produce cytoplas- batches of neutrophil slides from the former 1994;47:4–8. 4 manufacturer. 2 Savige JA, Paspaliaris B, Silvestrini R, et al.A mic rather than perinuclear IIF staining, pos- review of immunofluorescent patterns associ- sibly related to a subpopulation of epitopes on Our small study demonstrates that sera ated with antineutrophil cytoplasmic antibod- MPO that do not redistribute with ethanol containing MPO-ANCA may produce a ies (ANCA) and their diVerentiation from fixation.2 We now report that MPO-ANCA combination of P-ANCA and atypical cyto- other antibodies. J Clin Pathol 1998;51:568–75. 3 Savige J, Gillis D, Benson E, et al. International positive sera may produce a combination of plasmic IIF patterns on certain ethanol fixed consensus statement on testing and reporting P-ANCA and atypical cytoplasmic5 ANCA neutrophil substrates. The recent Inter- of antineutrophil cytoplasmic antibodies IIF patterns on certain ethanol fixed neutro- national Consensus Statement recommends (ANCA). Am J Clin Pathol 1999;111:507–13. phil substrates, potentially leading to interpre- that such combined patterns be reported as 4 Segelmark M, Baslund B, Weislander J. Some 3 patients with antimyeloperoxidase antibodies tative and diagnostic diYculties. “atypical ANCA”. Because atypical ANCA have a cANCA pattern. Clin Exp Immunol Sera from six patients with biopsy con- are not strongly associated with microscopic 1994;96:458–65. firmed microscopic polyangiitis (at diVerent polyangiitis or Wegener’s granulomatosis,3 an 5 Wong RCW, Silvestrini RA, Savige JA, et al. Diagnostic usefulness of classical and atypical stages of disease activity) were selected atypical ANCA IIF report on these sera could cytoplasmic ANCA (antineutrophil cytoplas- because of initial diYculties in the interpret- potentially erroneously lead the requesting mic antibody) immunofluorescence patterns. J ation of their IIF patterns on ethanol fixed clinician away from the correct diagnosis. Clin Pathol. 1999;52:124–8. 6 Hagen EC, Daha MR, Hermans J, et al. neutrophil slides from Inova Diagnostics However, in all six sera, the positive MPO- Diagnostic value of standardized assays for (San Diego, California, USA). All six sera ANCA ELISA result would hopefully redi- anti-neutrophil cytoplasmic antibodies in idio- were MPO-ANCA positive and proteinase rect attention towards a possible diagnosis of pathic systemic vasculitis. EC/BCR project for 3-ANCA (PR3-ANCA) negative by the systemic necrotising vasculitis. ANCA assay standardization. Kidney Int 1998; 53:743–53. corresponding ORGenTec (Mainz, Ger- We have subsequently found that these many) linked immunosorbent assay combined IIF patterns do not occur with all (ELISA). PR3-ANCA positive serum from a MPO-ANCA positive sera on the Inova High prevalence of serum markers of patient with biopsy confirmed Wegener’s slides, and thus speculate that the phenom- coeliac disease in patients with chronic granulomatosis was also tested. To establish enon might be caused by factors in the etha- fatigue syndrome whether other ANCA antigen specificities nol fixation conditions of these slides result- were present, all sera were tested on the ing in the diVerential redistribution of There has been recent interest in the ORGenTec ANCA Combi-kit® ELISA con- diVerent MPO epitopes. Therefore, we rec- possibility that undiagnosed coeliac disease taining proteinase-3, MPO, lactoferrin, ommend that laboratories using this brand (CD) might be the cause of diverse clinical symptoms, most particularly “tired all the Table 1 MPO-ANCA and PR3-ANCA ELISA, ANCA Combi-kit® ELISA, and ANCA IIF time”.1 A recent study reported a prevalence results of three in 100 cases in a primary care environment in which samples were taken IIF staining pattern (titre) on ethanol fixed neutrophils from patients with a range of symptoms and using polyclonal FITC conjugated antihuman IgG signs.2 The second most frequent symptom MPO-ANCA and reported by the endomysial antibody (EMA) PR3-ANCA IgG ANCA Combi-kit IgG In house Sera ELISA (U/ml) ELISA (OD ratio) Inova Diagnostics MBL cytospin positive patients was “being tired all the time”. We decided to examine the prevalence 1 MPO positive (58) MPO (6.4) P (1/160) P (1/160) P (1/160) of EMA in patients attending our tertiary PR3 negative Atypical cytoplasmic (1/160) referral centre with the diagnosis of chronic 2 MPO positive (61) MPO (2.9) P (1/160) P (1/160) P (1/160) fatigue syndrome (CFS). PR3 negative Atypical cytoplasmic (1/10) We tested serum from 100 consecutive 3 MPO positive (8) MPO (1.6) P (1/40) P (1/160) P (1/160) patients (47 men, 53 women; median age, 40 PR3 negative Atypical cytoplasmic (1/40) 4 MPO positive (6) MPO (1.3) P (1/10) P (1/40) P (1/40) years; range, 18–57) referred to our specialist PR3 negative Atypical cytoplasmic (1/40) clinic and satisfying the standard CDC crite- 5 MPO positive (>100) MPO (9.1) P (1/160) P (1/640) P (1/160) ria for a diagnosis of CFS, and from 100 PR3 negative Lysozyme (1.7) Atypical cytoplasmic (1/160) healthy control subjects (45 men, 55 women; 6 MPO positive (36) MPO (5.2) P (1/160) P (1/160) P (1/160) median age, 40 years; range, 18–68) who PR3 negative Lactoferrin (1.1) Atypical cytoplasmic (1/10) were blood donors at the South East Thames 7 MPO negative PR3 (6.44) C (1/40) C (1/40) C (1/40) PR3 positive (53) Blood Transfusion Service. The CFS sam- ples had been stored as part of other studies, ORGenTec MPO-ANCA and PR3-ANCA IgG ELISA: positive, >5 U/ml; negative, <5 U/ml. and were analysed retrospectively. EMA of ORGenTec ANCA Combi-kit® IgG ELISA OD ratio: positive, >1; negative, <1 (only positive results the IgA class were detected by indirect shown). immunofluorescence (IF) using cryostat sec- IIF staining pattern: C, classic granular cytoplasmic IIF staining with central/interlobular accentuation. tions of distal primate oesophagus as sub- P, perinuclear. strate (Binding Site, Birmingham, UK). Posi- Sera 1–6 were from patients with biopsy confirmed microscopic polyangiitis. Serum 7 was from a patient with biopsy confirmed Wegener’s granulomatosis. tive samples were confirmed using an enzyme ANCA, antineutrophil cytoplasmic antibody; ELISA, enzyme linked immunosorbent assay; FITC, linked immunosorbant assay (ELISA) for the fluorescein isothiocyanate; IIF, indirect immunofluorescence; MPO, myeloperoxidase; OD, optical density; detection of antitissue anti- PR3, proteinase 3. bodies3 (Menarini Diagnostics, Wokingham,

www.jclinpath.com 336 Correspondence, Corrections, Calandar of events

UK), being the auto- trinity of coeliac disease. Clin Exp Immunol 01895 274 020; fax +44 01895 274 080; antigen responsible for the IF pattern of 1999;116:258–62. email [email protected]) 4 Wessely S, Hotopf M, Sharpe M. Chronic fatigue EMA. To exclude selective IgA deficiency, and its syndromes. Oxford: Oxford University serum IgA concentrations were measured by Press, 1998. laser nephelometry using specific antisera 5 Watson R, McMillan S, Dickey W, et al. Detec- International Consultation on the tion of undiagnosed celiac disease with atypical according to the manufacturer’s instructions features using antiretulcin and antigliadin anti- Diagnosis of Noninvasive Urothelial (Behring Laser Nephelometer II; Dade Be- bodies. QJMed1992;84:713–18. Neoplasms hring, Dortmund, Germany). 6 Empson M. Celiac disease or chronic fatigue 11–12 May 2001, University of Ancona Two of the 100 CFS samples were positive syndrome—can the current CDC working case definition discriminate? Am J Med 1998;104: School of Medicine, Torrette, Ancona, for EMA using IF, and this was confirmed by 79–80. ELISA, but none of the 100 control samples Italy was positive. None of the subjects had selec- Further details: R Montironi, Ancona Italy tive IgA deficiency. Mean (SD) serum IgA (email [email protected]), DG concentrations among patients with CFS Bostwick, Richmond, VA, USA (email were 2.1 g/litre (0.98). Neither of the positive [email protected]), P-F cases, both women aged 27 and 54, had Correction Bassi, Padua, Italy (email bassipf@ reported symptoms typical of CD, although ux1.unipd.it), M Droller, New York, one had a history of constipation. Routine USA (email michael_droller@ blood tests including serum proteins and full smtplink.mssm.edu), or D Waters, Seattle, blood count were normal, and both had been Niessen HWM, Lagrand WK, Rensink seen by consultant physicians before referral. HJAM, et al. Apolipotrotein H, a new media- WA, USA (email waters@ Both had histories of hypothyroidism, were tor in the inflammatory changes ensuing in vet.vet.purdue.edu) taking long term thyroxine, and were cur- jeopardised human myocardium. J Clin rently euthyroid. Before the diagnosis of CD Pathol 2000;53:863–7. was made retrospectively, both had received C Visser (Department of Cardiology, Free Human Adverse Drug Reactions cognitive behaviour therapy (CBT), a stand- University Hospital, 1007 MB Amsterdam, 30 May 2001, Royal College of Patholo- ard treatment for CFS. In both cases, CBT The Netherlands) was mistakenly ommitted gists, London, UK led to a substantial improvement in the qual- from the list of authors of this paper. The Further details: Michelle Casey, Academic ity of life and physical activity, but neither journal apologises for any inconvenience that Activities Coordinator, 2 Carlton House patient was symptom free at the end of treat- this may have caused. Terrace, London SW1Y 5AF, UK. (Tel ment or at six months follow up. In both +44 020 7451 6700; fax +44 020 7451 cases, CD was subsequently confirmed on 6701; www.rcpath.org) jejunal biopsy after the retrospective identifi- cation. In general, it remains true that although a Calendar of events Professional Standards of Pathologists wide range of physical illnesses can be misdi- in a Modern NHS Pathology Service agnosed as CFS (see Wessely et al for 7 June 2001, Royal College of Patholo- review4), in practice this is uncommon. In particular, if basic physical examination, Full details of events to be included should gists, London, UK investigations, and history are unremarkable, be sent to Maggie Butler,TechnicalEditor Further details: Michelle Casey, Academic Activities Coordinator, 2 Carlton House misdiagnosis of CFS and other physical JCP,The Cedars, 36 Queen Street, Castle Terrace, London SW1Y 5AF, UK. (Tel illnesses is very unusual. Until now there have Hedingham, Essex CO9 3HA, UK; only been two reports concerning three cases +44 020 7451 6700; fax +44 020 7451 of CD being misdiagnosed as CFS.56 email: [email protected] 6701; www.rcpath.org) However, there is now evidence from primary care of a surprisingly high frequency Diagnostic Histopathology of breast of unsuspected positive EMA tests in people Infectious Hazards of Donated Organs with non-specific symptoms and a suggestion Disease 28 June 2001, Royal College of Patholo- that a higher index of suspicion is needed 23–27 April 2001, Hammersmith Hospital gists, London, UK 2 (Imperial School of Medicine), London, when assessing such patients. We now : Michelle Casey, Academic UK Further details extend that observation to our CFS clinic. Activities Coordinator, 2 Carlton House : Wolfson Conference Cen- Indeed, given our prevalence of 2%, and the Further details Terrace, London SW1Y 5AF, UK. (Tel tre, Hammersmith Hospital, Du Cane fact that there is a treatment for CD, we now +44 020 7451 6700; fax +44 020 7451 suggest that screening for CD should be Road, London W12 ONN, UK. (Tel +44 added to the relatively short list of mandatory o20 8383 3117/3227/3245; fax +44 020 6701; www.rcpath.org) investigations in suspected cases of CFS. 8383 2428; email [email protected]) A SKOWERA Recent Advances in Genetics M PEAKMAN Gynecologic and Obstetric Pathology 5 July 2001, Royal College of Pathologists, Department of Immunology, Guy’s King’s and St London, UK Thomas’s School of Medicine, Denmark Hill Campus, 26–29 April 2001, Fairmont Copley Plaza, London SE5 9RS, UK Boston, Massachusetts, USA Further details: Michelle Casey, Academic Activities Coordinator, 2 Carlton House A CLEARE Further details: Department of Continuing E DAVIES Education, Harvard Medical School, 25 Terrace, London SW1Y 5AF, UK. (Tel A DEALE Shattuck Street, Boston, MA 02115, USA. +44 020 7451 6700; fax +44 020 7451 S WESSELY (Tel +1 617 432 1525; fax +1 617 432 6701; www.rcpath.org) Department of Psychological Medicine, Guy’s King’s 1562; email [email protected]) and St Thomas’s School of Medicine BSCC Annual Scientific Meeting 1 Feighery F. Coeliac disease. BMJ 1999;19:236– BSCC London Spring Tutorial: Lung 9–11 September 2001, Majestic Hotel, 9. and Pleural Cavity Fluid Cytology Harrogate, UK 2 Hin H, Bird G, Fisher P, et al. Coeliac disease in primary care: case finding study. BMJ 1999; 27 April 2001, Guy’s Hospital, London, Further details: BSCC OYce, PO Box 352, 318:164–7. UK Uxbridge UB10 9TX, UK. (Tel +44 3 Lock R, Gilmore J, Unsworth D. Anti-tissue transglutaminase, anti-endomysum and anti- Further details: BSCC OYce, PO Box 352, 01895 274020; fax +44 01895 274080; R1-reticulin autoantibodies—the antibody Uxbridge UB10 9TX, UK. (Tel +44 email [email protected])

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