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From www.bloodjournal.org by guest on June 17, 2017. For personal use only. Chronic Hemolytic Agglutinins: A 20-Year Gammopathy With By Robert A male, anemia Anemia 47, developed in association S. Response Evans, Elizabeth chronic hemolytic with hi9h titers of developed globulins. chronic macroglobulin serum titer of cold hemolytic cil with and of the in complement of (C) Cessation of HRONIC titer agglutination agglutinin agglutinin. and Istered There since. a return has have was ANEMIA is been cyanosis reason to From the Veterans Robert 98108. Division, Seattle, © Blood, /0. Chief Medical Service, of Washington C. Gilliland, Department School General of Medical M .D.: Laboratory studies tentative of the Seattle, January Technologist, Bruce Hospital, revised M.D.: University Medical Wash. ogy 1 , /972; S. Evans, ofMedicine, B.S.: Administration June his ab- and to as cold a relatively red cell mucous mem- episodes of patients deof a warm studies of a middle-aged male with a 20-yr history of sympagglutinin syndrome are presented. The slow progression of be attributed to both a gradual increase in cold agglutinin titer and its thermal amplitude. Recent glutinins and complement permit some his hemolytic disease, though obscurities Submitted a production due skin of classify in which intravascular the been sponsymptoms, has de- having kappa agglutinin ac- acrocyanosis in cold levels. appearance immunoglobulin with anemia and branes. Hemoglobinuria from acute red cell destruction may follow chilling although this is not a uniform occurrence. Finally, some velop a chronic hemolytic anemia that persists despite maintenance environment. In this report, toms of the cold his disorder can remissions pretreatment the of macroglobulinemia protein can produce was admin- hemolytic macroglobulin and with cold there normal benign. reduced been macroglobulin Despite monoclonal light chain tivity, and not of the of the following the Chlorambucil toward creased. titer stasis occurred despite During the past 2 yr there has taneous improvement in his and the level of macroglobulin Serum toward is a manifestation in the abnormal to cold discontinued and in chlorambucil leading of anemia infection, and exacerbations and a HEMOLYTIC agglutinins increase small cold hemolytic h#{216} agglutinins appeared macroglobulin. values returned by relapse. There was a second to chlorambucil, but reactivation respiratory protein electrophoresis. The agglutinins was 1 :64,000. The anemia responded to chlorambulevel normal. I 966, anemie component a decrease the C hemolytic M the In and Bruce C. Gilliland followed response agglutinins in 1 966. His symptpm of cold agglutinin syndrome began in 1 951 with cyanosis of the extremities nd face after exposure to cold. In 1 956, hemoglobinuna was first noted after chilling. In 1 962, the cold agglutinin titer was 1 : 1 6,000, and his red cells were coated of Benign to Chlorambucil Baxter, cold complement to Cold History the with Due 1973: Wash. of the interaction of cold agexplanations of the course of mechanism remain.’ Response 98018. accepted February Veterans Administration of edicine. Research, Seattle, Veterans Associate Professor Medicine. Universi;y 9. 1973. Wash. Hospital. 98108. Administration of of Medicine Washington and and Professor Elizabeth Baxter, Hospital, Seattle. Director, School Immunolof Medicine, Wash. 1973 Vol. by Grune 42. No. & Stratton, 3 (September). Inc. 1973 463 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 464 EVANS. to administration of chlorambucil acerbations while occurred was he was observed receiving CASE D.P., a 47-yr-old man, withexposuretocool air red or coffee colored and cleared The He was larged, When first seen urinalyses The with patient’s red except normal, Fourteen He was cord was bone marrow gamma He was was began The 6 mg Fig. the next 6 wk, titer of cold 42, but was the titer patient’s cells that for lymphoma, were was not en- was and continued to The antiserums cells cells also at 50, with were normal. or had margin. Leukocytes an Titration been IE values had red cells. platelets 3 mo. were normal. increase band C showed was Hemolysis cell M-component for hematocrit plasma of serum The for present The the or 1:120 laboratory and lymphoid and normal. was normal. costal of 5’C of specificity electrophoresis fatigue of in in the fast no CH5O units anticomplementary. dose daily. reticulocyte The counts stationary 3 mo were had not decreased After red values the chloride. red were demonstrated. spherocytosis 1:64,000. 2#{176}c. The easy below evidence in a single in acrocyanosis. for 2 wk to 2.5#{176},,. At the of cholorambucil be agglutinated chlorambucil titer of cold during and agglutinins, the then same ensuing rose time, anti-C discontinued, to there medication, by was the serum and serum 30 mo normal was are levels a gradual hematocrit in high during was dilution. the next / \ i; atmosphere nodes platelets (C) Serum electrophoresis serum reticulocytes red no were symptomatology significant remained agglutinins. patient’s in his 4 cm was hematocrit hematocrit while a decrease and feet had been cool negative I positive by he reported but of chlorambucil I. The in the the The and fall in lymph and not 0, C levels. palpable Serum agglutinin were agglutinated 0.65#{176}cof sodium in smears. cold values, in 1966, there The reticulocytes complement in serum hyperplasia, examinations. given was leukocytes group no change now l5#{176},and aspiration complement presented 1965, in March The with were decrease later, region. history 43#{176}() and Serum 37’C at normoblastic on repeated family granules 1:16,000 cells. the spleen solution There There red a further were was hemosiderin and in hypotonic of face, hands, 6 yr, his urine developed abnormalities. content. was months pale, reticulocytes during and washed for ex- palpable. hematocrit c,A(C3)(l:S000-l:lO,0O0). years later, in January occurred, 29,; cells acrocyanosis The physical not hemoglobin agglutinins 0, i positive group The health. notable were the and of cold 1962 because of cyanosis occasions during the past many chilling. good spleen 1962, were titer (C4)and Three in though therapy. warming. without and morphology in of occasions, maintenance GILLILAND REPORT during On two AND disorders. male the liver 1951. had blood a robust twice episodes after always other and normal Two had and seen since after completely patient anemia, was on BAXTER. 1 . I00 80 60 t.t. , CH,0U,.t. --- ,.-.-1-,,-w .-. Fig. 1 . Response of chronic hemolytic anemia due to cold agglutinins to the administration of chlorambucil. Levels of macroglobulin. cold agglutinin, 20 10 ‘ 0 6 rAFAyAjtY4s: 3 4 5 6 7 1966 8 9i011i2 12 34 i967 Chi,,.b.,i 5 67234 S 968 6 7 S 910 and serum hemolytic complement are depicted in relation to hematocnt and hemoglobin. From www.bloodjournal.org by guest on June 17, 2017. For personal use only. CHRONIC 6 mo HEMOLYTIC there was agglutinin a gradual titer had Chlorambucil rise in hematocrit. patient acute was upper fall and ambucil discontinued 3 mo later serum C. Since and 1968, between hematocrit and red between after were cells, and discontinuing During The past 3 yr. greater 100 ml: the of due to and and reinstituted. 7#{176},, . During episodes an to were normal. The rose levels than The to gradually detectable have generally in which with decreased 900 the respiratory formation agglutinin to the developed weeks cold chlor- 35#{176},, as of associated rather increased to bronchitis several were ensuing hematocrit with of elevated. 1gM the to in the Hematocrit destruction serum During a disappearance periods These remained cold with a The fall hematocrit in hematocrit increased the counts The three daily. rapid infection l8%-2l,. have was differential been 8#{176},, . The remission detectable. in reticulocytes there request. been have counts the patient endurance. cold direct level 1:32,000, not there to 4 mg of respiratory a fall to titer mg/l00 varied ml 6 mo chlorambucil. the and nosis to the reticulocyte 1:16,000 patient’s has 40#{176},but and episode with chlorambucil apparently since the associated 30#{176}c and has dropped infections at reduced 1968, leukocyte reticulocytes entered a second C again became an increase in February A second again was to 35#{176},, and the in measurable. The patient and serum Chlorambucil and cleared. was March varied of then l0,, a rise not in the same dose. titers decreased, infection were infection C was again in hematocrit tract to 28#{176},, and serum free ofsymptoms. a decrease 25#{176}c. Reticulocytes was in hematocrit increased, respiratory respiratory 465 was again given Cold agglutinin was again 6 mo there ANEMIA agglutinin reports general A determination of titer antiglobulin was reaction 1:4000, was improvement macroglobulin the hematocrit unchanged. Since was then in symptoms with in December 1970 and 4#{216}0., the less acrocya- was 300 reticulocytes patient has mg/ were refused 6#{176}c. additional determination. MATERIALS The method scribed, and of titration as well as the of the methods cold AND agglutinins used METHODS and in preparation the of serum the complement antiserums levels for has been C globulins, de- (fi,) C4 C3 (lc-,A).2 The cells hemolytic ofa mixed with HCI. of red cells normal concentration cells residual anti-C globulin was with milliliters Fractions were also of were tested phoresis. The serum were concentration Buglio patient’s washed red cells and patient’s red the agglutination out light in our For detection distilled water tration cells of 100 of Kabat The the red addition was of 0.1 N ml of a 20#{176}c suspension incubated for of with were patient’s remaining red serum temperature compared and 0, 1 positive patient’s by the 0.25 and at 37’C of the cells. on Sephadex was leukocytes 30 with for red cells to were tested mm at incubation, a control tested cells Dr. with of l00O agglutination hemolysis by cold for coating buffer at by Tris-HCI 1 positive analysis, red and (Hyland the pH Laboratories). formation 8. cells. Fractions immunoelectro- of rosettes The with the subjects.3 complement-fixing concentrations G antibodies.4 Harboe.5 using diffusion to demonstrate the low G-200 group 0, Ouchterlony by radial used of normal tested by antigamma by with was determined et al. or absence originally antibody of Typing Cold agglutinins 24-hr urine gamma of light from G chains later consumption globulin test to insufficient of the bleedings to cold agglutinin were typed for laboratory. of Bence and then mg/mI munoelectrophoresis. method was the 7.2 or 6.8 mixture at group of of 37’C, sedimented times with milliliter to serum supernatant three fractionated were presence was carried chains the were susceptibility of 1gM by Lo produce adjusted tested for cold agglutinin activity by cellulose acetate electrophoresis, described The of the red agglutinin half serum. method demonstrate was cells in The cold One to temperature washed to normal anti-C pH I .0 ml blood were serum. compared C components to red The The equilibrated added The ofthe as follows: serum. had been was activity determined hemoglobin agglutinin Two of temperature. hemolysis. in the ml was all reagents normal the complement-fixing subject 0.5 After the desired and and normal Jones and Serum and proteinuria, lyophilized. Mayer,6 then The lyophilized examined hemolytic in which for Bence specimen urine Jones C determinations normal values was protein were ranged was from thoroughly dissolved in by done dialyzed water at electrophoresis by a modification 80 to 160 CH U/mI. against a concenand of imthe From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 466 EVANS. SI Cr tagging patient and was used to observe the survival the survival of ofcomplement-coated autologous red cells and ofthe patient BAXTER. AND GILLILAND homologous red cells in a normal subject.7 the in RESULTS The G-200 cold agglutinin activity separation. This material was found produced in the first peak a homogeneous electrophoresis. Ouchterlony analysis of isolated repeated adsorption and elution from red cells antibody but not with anti-A. Bence Jones protein in urine that had been highly concentrated. To determine what per cent of the patient’s of the Sephadex spike on protein cold agglutinin showed reaction could not be serum 1gM was participating cold agglutinins, an aliquot of serum was absorbed repeatedly prepared from group 0, I positive red cells. The concentration measured by radial diffusion, and the cold agglutinin titer was lowing each absorption. moval of cold agglutinin the patient’s serum 1gM Seven activity. at this absorptions were Cold agglutinin particular bleeding using a control activity, showed serum with 2 g of macroglobulin, less than 20 nonspecific absorption The patient’s throughout the washed period hemolysis by cold less than hemolysis The red cells of study. agglutinins hemolysis of normal red cells of the when agglutinated red cells patient washed but were compared of the patient’s cells under the c; red were His necessary activity (Table prepared by with anti-K demonstrated for complete rerepresented 84 of 1). Repeat studies without cold of macroglobulin. agglutinin by anticomplement relatively resistant to normal red cells, as same conditions. red cells.2 compared at 37#{176}C formed as with stroma of 1gM was determined fol- There to rcsettes serums to C was 20%-50% with monocytes and polymorphonuclear leukocytes from a normal donor when incubated at 25#{176}C.The adherence of red cells to leukocytes appeared to be due to C components, since the amount of ‘y G globulin detectable on the red cell surface was not increased above that found on normal red cells.4 Both polymorphonuclear neutrophils and mononuclear cells formed rosettes with the complementcoated red cells ofthe patient. Survival of the patient’s 5’Cr-tagged red cells in his own circulation was served during treatment with chlorambucil. The t survival was 1 1 days at beginning ofchlorambucil 3 mo sion tient’s later. circulation Table The was treatment survival t 13 days. 1 . Relation 15 days group The of Serum No of Absorptions and of normal initial 1M Concentration 0, he was lmg/100 in partial I positive of rapid to Cold cells Agglutinin Titer of Cold Agglutinin Titer 650 25,600 1 510 12.800 2 390 6.400 3 315 3.200 4 245 1.600 5 120 400 6 118 7 100 milliliters of se rum were absorbed with stroma 10 0 from 2 ml of red blood cells. pa- of normal Reciprocal ml) remis- in the destruction ‘yM Before ‘Three phase when obthe From www.bloodjournal.org by guest on June 17, 2017. For personal use only. CHRONIC cells tinins HEMOLYTIC ANEMIA 467 seen in other patients was not observed.’ showed the first with chronic The patient’s a biphasic curve with 90 mm. The remaining an hemolytic red cells initial red cells period were anemia due to cold injected into a normal of rapid removed agglusubject removal of 30% at a gradual rate during with a of 24 days. DISCUSSION The history ofcold ofacrocyanosis agglutinin hemolytic had anemia. component titer ofcold agglutinins in the serum protein duced complement coating red cells were heavily coated to C hemolysis has been shown ment at through the by in vitro components onstrated above that process due the action studies to repeated of cold to be C3) that fixation 1951 at least on required ofC reactions had rather than a partial red agglutination.2 cells, in vitro were introduced own brane red cells, damage consumed red cells. phonuclear existence to the his leukocytes ofsuch The concept that in chronic hemolytic has been a process whose and C4 reported.8’9 of that Further red cell’s pro- resistance comple- takes place studies dem- is a continuing agglutinin and hemolytic red noted the disease for cells circulation. as it was Transfused cells no result at furof released red It was is reasonable by a steady cells, as the and the acute the circulation active. the absence of susceptible to C to deposipatient’s form of memdid not occur.’ cells with the C components by cold has been enhanced by observations red cells of this patient subject. Furthermore, both macrophages and those seen in patients of receptor sites for C3 M serum resistance of his circulating cells our group of patients with this total serum hemolytic comple- when The significance of the coating of red agglutinin as a cause of red cell destruction similar existence cold acquired C components gradually, that results in rapid removal from that 30% of51Cr-tagged circulation of a normal formed rosettes with detectable This abnormal accumulation probable explanation when normal red into levels of chronic his hemolytic anemia, the patient This change may have been depletion postulate that complement was tion on the surface of circulating until and to circulating the abnormal onset at 37#{176}C.The patient’s own and were relatively resistant for between of serum complement is the period of rapid destruction hemolysis increased agglutinin. due to the depletion of serum C, as well as the acquired to complement hemolysis. He is unique in syndrome in having an absence of measurable ment, that to the electrophoresis, the globulins body temperature.’ Following the onset of chronic ther episodes of hemoglobinuria. The lack an initial indicates 15 yr prior of normal red cells with C components (primarily temperatures in present The appeared beginning been were removed it was shown polymorphonuclear red cells on human Others rapidly that his from the red cells leukocytes are coated monocytes with and IgG. The polymor- have to failed confirm the sites.’#{176} the C components on the surface anemia due to cold agglutinins (C4 and C3) has been challenged by Engelfriet that a2D globulin (C3d), an enzymatic cleavage fragment that remains on the red cells in vivo. of the circulating are /lE and red cells globulins et al.” They presented evidence product of C3, is the only C Regardless of the nature of the From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 468 EVANS. adherent complement fraction or fractions, the BAXTER. destruction chronic form of the disease is usually accompanied cytosis, suggesting loss of membrane as a result phagocytic cells as is postulated to occur with IgG of by some of surface antibodies.3 of rosettes by this patient’s red cells and leukocytes for biologically active C components, probably C3b, culating red cells. Chronic hemolytic anemia due to cold agglutinins AND red cells in the degree of spherointeraction with The formation is interpreted on the surface is an GILLILAND as evidence of the cir- uncommon disorder, and so far, there are few descriptions of treatment by measures other than maintaining a warm environment. Unlike hemolytic anemia due to warmreacting autoantibodies of the lgG variety, hemolytic anemia due to cold agglutinins appears to be resistant to steroids. Splenectomy may reduce the hemolytic activity been established.’2 glutinin in vitro these agents glutinins has favorable in some patients, but its value as a general Penicillamine and other mercaptanes by splitting disulfide bonds of the 1gM in the treatment not been established response in two of hemolytic the decade has not agof anemia due to cold since the initial report agof patients.’3”4 Several reports attesting value with alkylating agents, particularly tration of these agents provement in anemia in the marrow. Olesen with chlorambucil.’7 chronic during measure inactivate cold molecule. The value of long-term chlorambucil, has caused a decrease along with a decrease has reported treatment The concentration of treatment have in the M component and an imin the number of lymphoid cells of the cold agglutinin syndrome M component was reduced, and the titer of cold agglutinins was decreased. Dacie satisfactory improvement in three of nine patients while three others had some benefit.’8 Our attempts two other patients were not successful, prior to any evidence of improvement since in the of macroglobulinemia appeared.’5”6 Adminis- and Worlledge given chlorambucil with alkylating have noted therapy, agents in bone marrow depression hemolytic process. occurred Administration of chlorambucil to the patient reported above was followed by significant reduction in levels of 1gM and in the titer of cold agglutinins. The hematocrit rose, and reticulocytes decreased. Total serum complement rose from undetectable levels to approximately 50 of normal during each remission. Reticulocytes were always elevated to 2%-3, even when the hematocrit was within the range of normal. The 51Cr t survival of 15 days during the partial remission was not returned ambucil was levels. being An further evidence to normal. The stopped, as the exacerbation administered of equal in February that the rate of red hemolytic anemia recurred cold agglutinin titer returned severity 1968, following occurred while a respiratory cell destruction had 3 mo after chlorto pretreatment chlorambucil infection cold agglutinin titer was at a relatively low level (1:2000). Reactivation hemolytic anemia occurred again 4 mo later in June 1968, following was when the of the another respiratory infection when cold agglutinin titers were still relatively low. Such exacerbations may be related to an increase in complement formation and activation, as well as greater activity by phagocytic cells. Both factors are probably dependent on the thermal amplitude of the cold agglutinin, including complement deposition on red cells at normal body temperature. It is signifi- From www.bloodjournal.org by guest on June 17, 2017. For personal use only. CHRONIC HEMOLYTIC cant that, complement despite the components Furthermore, serums with The ANEMIA depression were of the cold agglutinin still bound to normal agglutinin isolated from patient resistance to hemolysis to normal cells. was a homogeneous by immuno- 1gM with kappa light chains only. The M component observed acetate electrophoresis appears to have been nearly all cold since it disappeared with repeated absorption of the serum with cell stroma in the cold. after removal of all cold Measurement agglutinin of the patient’s activity showed an The identity of the M component tivity has been well documented been observed globulin with other (rheumatoid While toms, tions the cold been depressed. tion. Finally, the failure of our can to detect there is evidence that followed the E, Bingham M: the and 1gM. agglutinin activity achas anti--y antilipoprotein patient’s G activities.20 M component led to symp- be classed as benign. Serum concentranormal, and bone marrow function has Bence Jones protein benign for the mechanism questions M component cessation in a concentrated evidence for responsible protein and the homeostatic at present but pose important 2 yr that cold antibody on agred before of including 0, and has also been considered The nature of the stimulus ofthe abnormal tion are unknown during activity gammopathy have remained The 24-hr urine sample malignant disease2’ with immunoglobulins, antistreptolysin agglutinin serum 1gM 84 reduction on electrophoresis by others.’9 Similarly, monoclonal factor), his monoclonal of IgG and IgA not our level by chlorambucil, cells at 31 and 37#{176}C. red the patient’s red cells had not lost high titers ofcold agglutinin as compared cold globulin cellulose glutinin, 469 rather than production limiting its producfor future investigadecreased of the cytotoxic spontaneously drug. REFERENCES I . Evans RS, Turner hemolytic The anemia role due of complement Evans anemia mechanism ment due 3. Lo cells coated and sphering by 4. Gilliland detection ment-coated 5. of in RS, J Jandi JH: G: mononuclear cells in JP, Vaughan M, Schubothe Red human Huber RS: 1961, Furth, R, Exclusive cold EA, occurrence hemagglutinins. sensitized Haematol 19:19, FS: of Scand II. & G, MM: 2). Experimental Springfield, Ill., 162 Kr Motulsky AG, Giblett ER, The Br Linscott J WD, monocytes: for the for immunoglobulin third component G. Receptor sites complement: by cold on Binding of autoantibodies. Br J 970 N, Gelfand EW, interaction and and red lgG Jandl between cells. Specificity fragments. J JH, human for Exp lgG Med 1970 CP, von dem haemolytic use Pondman Borne G, complement Immunol MJ, SD: for The examination their tag. H uman Douglas Misset-Groenveld and CA: cell 1968 Engelfriet immune Mayer DM, H, red cells H, 1965 (ed sites and monocytes monocytes subclasses on compleClin Invest Finch red HJ : 162:1281, Rosen The V. a PolIcy receptor 132:1207, Van H, Eberhard complement Science man. JH: J 2:259, Donohue Huber Clin Binding as 10. Abramson Leddy Immunochemistry 7. agglutinins. Viranuvatti 1:249, 1955 Distinct of 1967 isolated Kabat Thomas, The to comple- cell-bound antibody human red cells. Evans J Haematol 6. cells 1970 K, chains cold Cotran BC, Harboe Lind M ‘filler- agglutinins: of red immunoglobulin 158:1582, 49:898, M: Chronic G, chromium Haematol 9. AF, with Biroli of destruction. 1967 Buglio Science Pirzio use II. 8. to cold by lnvest46:l46l, cell E, Bingham of resistance hemolysis agglutinins. 1968 RS, Turner hemolytic cold in red J Clin Invest 47:691, 2. to Chronic Van components in serological 6:721, 1970 Ill. specific Wolters Beckers Loghem anaemias. of KW, AEG, JJ: Preparation antisera and studies. D, Auto- against products, Clin and Exp From www.bloodjournal.org by guest on June 17, 2017. For personal use only. EVANS. 470 12. Evans immune serological NY RS, and Sci 124:422, Ritzmann mercaptanes Med 14. SE, in cold 57:718, therapy in Waldenstrom: Turner H: disease activity. primary it) 1963 the Olesen WC: agglutinin Effect disease. of Dacie hemolytic 19. J Lab cold 1966 cases. macroglobulinemia. of Proc Staff to JA MA JV, Worlledge Christenson WN, Dacie Charlwood PA: containing Identification of Qf an abnormal 3:262, 20. 1957 Potter Med284:831, 21. Hobbs malignant? J M: Auto-immune 6:82, JV, 1969 Croucher Electrophoretic high-litre cause with in Scand SM: Hematol ponents) Response treatment syndrome. Prog sera tinins: chlorambucil macroglobulinemia of four on hemagglutinin GILLILAND 1964 anemias. BEE, The 1:116, AND Chlorambucil agglutinin Haematol Ann H: cold 18. Levin Meet Mayo Clin 36: 135, 1961 16. Clatanoff DV, Meyer 00: 183:126, of 1965 Hematol 3:27, ED: Continuous Report chlorambucil 17. E: Auto- Observations 1961. Schubothe disease. Semin 15. Bayrd M, disease: reactions Acad 13. Clin Bingham hemolytic BAXTER. the Myeloma haemagglu- antibody peak. antibody-like studies cold Br proteins activity. as the J Haematol (M-comN EngI J 1971 JR: Br Med Paraproteins, J 3:699, 1967 benign or From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 1973 42: 463-470 Chronic Hemolytic Anemia Due to Cold Agglutinins: A 20-Year History of Benign Gammopathy With Response to Chlorambucil Robert S. Evans, Elizabeth Baxter and Bruce C. 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