Page 1of9 between this versionandtheVersionrecord. Pleasecitethis articleasdoi:10.1111/trf.14128. through thecopyediting, typesetting, paginationandproofreadingprocess, whichmayleadtodifferences This istheauthormanuscript acceptedforpublicationandhasundergone full peerreviewbuthasnotbeen ColdsComplement: Kids, and Paroxysmal Hemoglo Cold Conflict of Interest: Conflict of None 40References: Manuscript:1590 words [email protected] Email: (734) Phone: 936-0695 MIAnn48109-0054 Arbor, East 1500 MedicalCenter Drive UH BankBlood2F225 University of HospitalsMichigan Associate TransfusionDirector, Medicine Associate Professor, Pathology Cooling L. Laura MD, MS CorrespondingAuthor:

Accepted ArticleCooling, L. Laura Department Universiof Pathology,

This article isprotected by copyright. All rights reserved. Transfusion ty ty of AnnMichigan, Arbor, MI binuria

strong correlation strong betweenand syphilis PCH during developed newly Wasserman for assay (1906) syphilis Paroxysmal hemoglolinuria Paroxysmal cold is relativel a (PCH) case generally is case of PCH attributed Dr. to Dressler a 10-year old 10-yearold a childwith congenitalsyphilis. multiple authors multiple observed childrenappeared that to element in pathophysiologyelement the the disease. of proposed been repetitivethat the association/diss highertemperatures,complement remainsbound with recruitment andwith binding earlycomplement of co by a characterized affinity,weak ‘biphasic’autoan to recapitulate to the disease byincu infirst vitro, which remainsthewhich definitive fortest even PCH toda minutesand close forobservationthe development o attackanRosenbachtest, precipitated by was subme hemoglobinemia inhemoglobinemia ligated the finger considered was aroundfinger placed one and patient’s handthe was exposure. This This exposure. observation two to led testclinical hemoglobinuriainsyphilitic patientsdocument were (alexin) necessary (alexin) forwas hemolysisorthe ‘second the plasma, hemolysinthat was bindto only able re at 37C.warming findings Their that clarified PCH w Historically, Historically, was a condition PCH chronic associate These clinical These two informedtests the development o

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This article isprotected by copyright. All rights reserved. 4 Over the Over severalnext years, 50 cases inte more of Transfusion 3 bating serum patient cellsand red 4C,followed at

in whodescribed1854, intermittenthemoglobinuria s --the s Rosenbachtest andthe Ehrlichtest. tibody that onlybindscells red temperaturelow at yrare autoimmunehemolytic (AIHA). ascaused circulating by “a hemolysin present in ociationantibody during of circulationnormal a is phase’ the disease”. of y. In y. assay, their andDonathLandsteiner attempted dlowcells at temperatures complement andthat theearly20 be particularly susceptible andpresented often immersed inice water.The presenceof ed,episodes with precipitatedoften by cold positive a test. rging the patient’srging infeet the coldforwater 10 mponents. Althoughmponents. antibody dissociates the at fhemoglobinuria.In the Ehrlich ligature a test, w ,Landsteinerand able were others confirm to the complementactivation and has It . d advanced syphilis.with publishedThe first f the Donath-Landsteiner(DL) test (1904), th century. It interesting is century. noteto that 4 Using the andDL-test, the 4 Inthe rmittent 1,2 by It is is It s s key in as

Page 2of9 Page 3of9 non-syphilitic and acute,non-syphilitic non-syphilit 3) transient andinhibitForssmannantigen to antibody-mediated occasion, can occasion, PCH present negative a with DATand p not associated not increaseanin with agglutinin cold could occurin could conditions.other By the 1950s, PCH of the glycosphingolipid of (GSL)globosideas the P-a that the antibodythat in was directedan againstPCH an PCH samples PCH against P male a with male history of prior toweeks 1-3 chromatography. binding direct purifiedtoof globosidePCH-serum a with more severe hemolysis with more theirthan adultcounter who identified anti-Panwho IgG aftertesting against andautoagglutination/rouleaux in50% and17-25% of absence absence autoantibodiesof warm anda detectable bip with hemoglobin with values fallingoften below gm/dL. 6 hematopoieticmalignancies. cases) >70% orgastrointestinal illness: caus other almost is always It post-infectious a complication attack, consistent attack, with elementan extravascular of investigator early wasparoxysmal a enlargement of cases biphasic IgMcases with and IgA antibodies, As publicAs health measures andantibioticseliminate Today, PCHpredominantly is Today, pediatric a disease,ac

ScholarOne, 375GreenbrierDrive,Charlottesville, VA,22901 1 (434)964-4100 Accepted Article 8 Although IgGis anti-P the mostcommon antibody en 1 , P , 2 andpRBC. This article isprotected by copyright. All rights reserved. 1-3 Pediatric patients PCH Pediatric be to tend quite (me young 5 This specificity This refined was by Worlledge andRou 9-12 Transfusion andcarbohydrateother specificities i, (I, HI, Pr P-positive andP-positiveP ic. esinclude vaccination, autoimmunedisorders and followingupperanrespiratory tractinfection (URI titers. presentation. ntigen,Marcus andcolleagues purifiedused globosi wasascategorized 1)chronic chroni syphilitic, 2) hemolysis. ndneutral cell GSLs red by thin layer 1 tigenofthe group P-blood aftersystemtesting si the spleen andsometimes liverfollowing the a PCH In Phillip1963, provided Levine first evidencthe 1-3,17-19 parts. ositiveDL-test. hemolysis. hasic DL- hemolysinDL-hasic in disease. early the cases, respectively.cases, 3 Peripheral bloodfindings Peripheral includespherocytosis 4 Diagnostic findingsDiagnostic areC3+the a DAT, Another Another clinical observationby an made counting6-30%pediatric all for AIHAs. of d syphilis, becameadvanced it clear that PCH 4

7 1-3 A more recent more Apaper demonstrated k red cells. Hemolysis isoften suddenHemolysis and severe, 16,17 Despite recent infection,recent is Despite PCH counteredinPCH, thereare PCH 1,9,17 6 Followingthe identification Red cell-neutrophil Red dian years)age, 5 and ssoin 1965, ). 2,3 1,11-15 On

, , c c 3,16 e x de de

testing to testing red P-positive cells, by hemolysisfollowed fewweeks a later.While man documented episodes of PCH almost I 2 apart.years experience a a experience recoverycomplete within weeks.1-4 workup, inworkup, a pediatric patient.young male studies. response considered is badprognosti a reticulocytopenia was the twice rateapparentin ad PCH, androsetteserythrophagocytosis by neutrophils, wh reticulocyte countsdespite reticulocyte meanhemoglobin a of 6. pool in the pool marrow. PCH likely reflectslikely PCH the suddennessand severity of negative andnegativeP-negative (p) cells. red In both epis many PCH many cases. resistance to earlytoresistance steroid oral andtreatment circ however, gm/dL; secondhis unusuepisodeof PCH was admissions, Duringboth patient developedthe signi whetherautoanti-Phis titer havebeen highemight steroids,IV hours), andIVIG, followed by oral ste Heerythrophagocytosis. did butrequiredrecover a the first days. 2-4the 2 are are infrequentlyencountered (9%). 20,21 In this issue issue In this of TRANSFUSION,Princeetdescribe al It should alsoshouldbe It notedthat both PCH wereepisodes This level of clinical notof is severity level This observedin ScholarOne, 375GreenbrierDrive,Charlottesville, VA,22901 1 (434)964-4100 Accepted Article

1,9,17-19 19

Severe Severe a anemia with lownormal or reticulocyte co 21 In addition, reticulocytesIn addition, be may targetfor a aut In one study,third a children Inone under of (8/25) 6 y This article isprotected by copyright. All rights reserved. 9,18 the authors performed authors DL-test the the a pawith 5 cell 1

19 Unlike most cases pediatricUnlike most of PCH,patient this Transfusion roids for weeks.interestingmany is It speculatto odes, they were abletheywere odes, demonstrateto an autoanti-P. ulatingcell-neutrophil red androsettes neutrophil hemolysis,with depletion the available reticuloof 1,2 c sign, c with rates as mortality in high80% as olde ultpatients (1/7,14%). Inadult AIHA,poor a ggressive RBCggressive transfusion support (27 mL/kgin 24 r re-stimulation.upon ficantanemia hemoglobin with below levels 5 The relative relative reticulocytopeniaThe in observed early 3 (range,3 gm/dL gm/dL).4-9 n both theepisodes, patient URI, prior had a ichusually are citedpathognomonic as findings for y laboratories (including y confineown) my DL- ally severewith ally high outputfailure, cardiac pediatric patients pediatric with PCH, who typically a casetextbook-perfect of PCH, andits by accompanied reticulocytopenia during oantibodiesin PCH untbeenhas documented in ears of age ears hadof low normal or 1 This incidence This of nel that included I-that nel 19 : globoside, : had two e e

cyte cyte r

Page 4of9 Page 5of9 adenovirus. glycoproteins. utilize viruses hostglycosyltransferases with expr paramyxovirus (,paramyxovirus ,respiratory synctial viral viral associated PCH arewith enveloped pox, Epstein Barr, pox, ). influenzabindingglycophorin to and sialylated gly rich inglobo-familyrich GSLs including globoside. antibodies and/or antibodies alteredimmune regulation. autoanti-P haveincludedautoanti-P viralmodification the of Ii, and Pr antigens andIi,Pr are present on . reactive antibodies reactive against virus lipidthe envelop that are expressedthat RBCs, on including , pa globoside. erythrovirus,parvovirus recognizesB19 RBC, erythr associated ever, with --a common febr rafts—membrane enriched microdomains incholesterol epitopes havebeenepitopes described on bacterialmany spec PCH. reticulocytopenia, anemia or with knownonlyone c and particles,secreted viral which their obtain li budding. 25

One puzzle One inPCHthe is prevalence anti-P inth of A third hypothesis bythirdhypothesisA whichinfection i viral might 30-32 26 ScholarOne, 375GreenbrierDrive,Charlottesville, VA,22901 1 (434)964-4100 26,27

As a consequence, there is consequence, a As co-expression of viral 33,34

Accepted Article least one studyAt reportedhas structural alterat Nor is the is possibility cross-reactiveNor antibodie of

This article isprotected by copyright. All rights reserved. 1,2,19 These viruses These infect respiratorytissues of the tr 29 Transfusion 18 Furthermore, Furthermore, enveloped many virusesrequire lipid 22-24 Manyvirusesand bacterial toxins bind glycoconjug pidenvelope from the hostIncell membrane. additi essionhost-type glycosylation of viral on e and/or glycoproteins.e viral colipids. red red cell productionmembrane cross-reactive with of Erythrophagocytosis is notErythrophagocytosisa is contributor major to rainfluenza,HIV,parvovirus andB19some strains o virusesbelonging the to orthomyxovirus(influenza) oblastsand tissuesother via initial binding to aseof hemophagocytosis marrow attributed to parainfluenza), andvirus, herpes families (chicke ilein illness non-envelopedchildren.A ies, andGSLs 26 nduceautoanti-Pan is stimulationofcross- e disease. e Proposedtheories forthe origin of 28 including Somewhatsurprisingly, PCHrarely,if is antigensandhost lipids infected cells on ionsthe red of cell membrane following slimitedto viruses.group-likeBlood − forviral assemblyentry, and H.influenzae 7 Upon review, Upon the most of – a a – common cause of act, arewhich ates on, on, f n , ,

confluent patches, confluent PCH has beenhas treatedPCH with rit azathioprine IVIG, or needed complement at thecomplement C5 stage not be may baeffective due to immaturitytodueN-glycans band of on and 3 other activation. complement Finally, globoside expressi globosideconstitutes 60-70% total of cellthe red susceptible susceptible dueautoanti-P to theto sheer quantity compatibilitynottesting.necessary is It wash to Because appropriate. the PCHhemolysin onlyreacts volume volume overload.Intheserare patients, it be may hemolysis ongoing requiring massive transfusion sup appropriatenesssteroids is inof PCH not clear. upper respiratory upperrespiratory tractinfections has that alsobe Given binding Given the characteristics the antibody a of use a blood a use warmer. bacterial polysaccharides bacterial accountfor may follo PCH expression of globoside expression of on tissuesother andplasma fold higher than fold most othertissues.human . RBC . selected fortransfusion beshould leukoredu Treatment forTreatmentPCH supportive, is including keeping Many patients Many alsoreceive immunosuppressiosteroid Another puzzle isAnother whycomplement-mediated cell dama

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36 resulting highin a density,multivalent f epitope

This article isprotected by copyright. All rights reserved. 26,29 Transfusion 1,3 Moreover, globoside irregularly is Moreover, distributedin RBC RBC orrareprovide P-negative RBC inthesepatient Unusuallyseverehemolysis and/orrecurrent chroni en linked en with PCH. glycolipidandof6% the lipid. total ndthe need chilling,avoidto it common is practic worthwhileconsiderto manual bloodwhole uximab. on on enhanced is red in on youngcells childrenvery of of globoside expressed their on membrane. Overall wing pneumococcalwing vaccination. . redglycoproteins. cell port andsteroids port are at hypertension risk for and attemperatures,low doesinterfere it not with 29 sed on one casereport. In one respect, Inredone uniquely be cells may ced, ABO ced, andcrossmatch-compatible as 11,12,19,38 thepatient andblood warm transfusion as n, although thealthoughn,and efficacy ge is ge limitedto despite RBC widespread Surprisingly,blocks which eculizumab or or antibody bindingand 1,35 Cross-reactive antibodies to Cross-reactive 24,37 39 Patients Patients severe, with

26 26 10

This level is 2-3 is level 2-3 This large, large, e to e s c c . 3 , Page 6of9 Page 7of9 6. 5. 7. 4. 3. 8. 2. 10. 9. coated red red coated cells. exchange, wouldwhich isovolemicpermit red cell tr 1. References 13. 14. 11. 12.

special reference special itsto relationship withbl the P SM,Worlledge Rousso StudiesC. serologyon the of (P.C.H.). Transfusion(P.C.H.). 1963;3:278-280. Celano Levine Falkowski P, F.MJ, specificity The o hemoglobinuria(PCH) withglobosideand forssman gl GA, Schwarting Kundu SK, Marcus ReactionDM. ofant Mackenzie GM.Mackenzie Paroxysmal hemoglobinuria. Medicine1 children. Vox Sang children. Vox 1990;58:281-286. B, SalamaGottsche A, Mueller-Eckhardt Donath-La C. monosialogalactosylglobosideantigens. (LKE) Transf blood red phenotype:P” alloanti-LKEcell andalloa Cooling LR,L, Haverty Dake Aal. D, et hemolytic a ShanbhagJ.Paroxysmal coldSpivak S, hemoglobinuri 2046. hemoglobinuriadue IgAanto Donath-Lansteineranti WhippleNS,Moreau DAB, JM, HankinsJS, Moulds Wang patients with a positive a patients with Donath-LandsteinerTest.H BookerRJ, StampSokol DJ, ParoxysmalR. cold haemo Donath-Landsteinerantibody.Internatl Pediatrics 2 YasutomiH, Hayashi Hayashi T,M, et Paroxysmalal. hemoglobinuria.Transfusion 1986;26:62-64. RS, Shirey K,Park Ness Anal.PM, et anti-ibiphas biphasicanti-IH-specifichemolysin. Sangre 1983;28 VescioCiruzzi Carreras J,LA, JA,J,TeruyaRey Ma 2012;59:953-955. due a anemia to Donath-Landsteinerpositive, IgM an MS, Karafin Shirey NessKing RS, PM, J.KE, Keefer case report. case Rinsho Ketsueki-JapJClinHematol 199 andanti-Ispecificitypossible azathioefficacy of H, Nakamura Watanabe T, Ichimaru T, Hayashida M. Do ScholarOne, 375GreenbrierDrive,Charlottesville, VA,22901 1 (434)964-4100

Accepted Article: 40

This article isprotected by copyright. All rights reserved. Transfusion prine therapyinparoxysmal cold hemoglobinuria: a ansfusionandremoval ofplasma andcomplement- ood group system.groupVoxSang ood 1965;10:293-298. ic hemolysin inic chronic paroxysmal cold rlettaJ.Paroxysmal cold hemoglobinuria by caused f thef antibody inparoxysmal cold hemoglobinuria nti-LKEassociated withrare a LKE-negative, “weak A caseA study childof a chronic hemolyticwith nti-Precognizegalactosylgloboside and ematology 1999;4:137-164. :775-781. 013;55:664-666. 0;31:1548-52. cold hemoglobinuria causedIgM-classan by usion2015;55:115-128. paroxsymalcold haemoglobinuria (P.C.H.), with a. Hematol Hematol a. OncolClinNAm 2015;29:473-478. ti-Iautoantibody. BloodPediatr Cancer ndsteiner autoimmune ndsteiner hemolytic anemiain body. CancerBloodPediatr 2015;62:2044- globinuria: A clinic-pathologicalAglobinuria: study of ycosphingolipids.1979;53:186-192.Blood ibodies causeibodies paroxysmalthat cold nath-Landsteinerantibody of IgM class with 925;8:159-191. WC, Nottage WC, KA. cold Paroxysmal

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anemia due anti-Pr-likeananemia to biphasic hemolysin. T JuddIssit WilkinsonSL, WJ, Johnson PD, TL,Keren pediatric casereport. pediatric Transfusioninpress.2017, SD, Prince Winestone SJ,LE,FriedmanNance Rec DF. 244:1688-90. CL,Lippman Conley SM, AutoimmuneNess P. hemolytic 36. WH, H.Crosby Rappaport Reticulocytopeniain autoim blood group blood P(globoside).antigen Br JHaematol 19 demBorneAE, Von Bos MJ,J,Joustra-Maas etAal. features in features 100 cases.Transfusion 2007;47:50-54. AristaS, Vaglio MC, PerroneAutoimmuneal. MP, et 1998;20:373-375. the review of managementandunusual feapresenting Stevens RF, RF,Wynn Bolton-Maggs PHB, Sche Will K, paroxysmalcold hemoglobinuria. Medicine Lab 2014;4 SN, Slemp Slayten J,Davisson SM, Cipkala DA, Waxma early differentiationearly erythroid inacute leukemia. Gahmberg Andersson CG, LC, Teerenhouvi Vuopio L, P. Cooling groupsBlood Cooling L. ininfection andhost susce parvovirus B19. JB19.Inf parvovirus Dis 1995;172:1198-205. Koerner Cooling LLW, TAW, SJ.Naides Multiple glyco Nayak HuiNayak EK,DP, Barman andbuddingS. Assembly of Immunohematology 2010;26:133-155. Cooling L. Polylactosamines,there’sthan more meet paroxysmalcold hemoglobinuria. JapKetsuiki Rinsho Takeda YoshieM, Kolke K, Hasegawa H, H,Y, Ogata K Biology Biology 2008;18:567-576. VorrotA, A.Imberty Microbialhuman recognition of erythrocyte erythrocyte membraneupon influenza virus attachmen A, Herrmann GrothT, G, Lassmann LadhoffAM, Hilleb 9915. GerlierS, Vincent D, ManieSN. Measles assem virus syncytial virus syncytial hostwith the cell membraneofinfe A, Marty MeangerMillsJ, J, GhildyalShields B, R.

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This article isprotected by copyright. All rights reserved. Transfusion Association proteinof matrix of respiratory Int JInt 1979;24:717-720.Can DF, EA. DF, Steiner Donath-Landsteiner hemolytic ctedVirolArch cells. 2004;149:199-210. ptibility.ClinMicro Rev 2015;28:801-870. ransfusion1986;26:423-425. murineIgMmonoclonal antibody specific for bly within bly membrane rafts. J 2000;74:9911- Virol 86;63:35-46. hemolyticanemiain childhood:serologic cell surface glycoconjugates. OpinCurrStruc sreview a the “Ii”: system.I of the sphingolipidsthe determine tissue tropism of J ClinHematol 1996;37:707-712. omiyama A. HemophagocytosisA. omiyama in childwith a urrentdonath-landsteiner : A Paroxysmal coldAM. haemoglobinuria: A n DA. Two nDA. case studies reviewand a of recht B. StructuralB. recht alterations the human of influenza VirusRes virus. 2004;106:147-65. mune hemolytic mune anemia. Blood 1956;11:929- 5:253-58. Glycophorin A asA Glycophorin cell a surface marker of tures of six tures cases. Clinof Lab Haem t. Bioscience t. Reports 1986;6:45-55. anemiawith reticulocytopenia. 1980 JAMA Page 8of9 Page 9of 7. 9. 0.

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against glycolipidsagainst ofhuman erythrocytes. VoxSang SI.Hakomorireactivities Differential fetal and of hemoglobinuria.Ann Hematol 2011;90:989-90. GP,Opat Gregory S, Quach H.FailShortt Tran H,J, Doi:10.4172/2155-9864.1000163. bloodexchangewhole with rapid improvemenclinical Boxer Cooling G,L, SimonR. Life-threateningautoi of expressing cells of andaffect antibody recognition Takahashi Raska k,M, Czernekova etGlycosylal. L, erythrocyte erythrocyte membranes.Biochim BiophysActa 1983;73 TW,Allieta Tillack M, MoranYoung RE, WW. Localiza refractoryparoxysmalcold hemoglobinuria.Transfus A, S, Lim Koppel Osby M,Garratty Goldfinger G, D. hemagglutinin.Vaccine 2009;27:4325-4336. Influenceproduction: ofhost cell andline s virus RappJ, Schwarzer Hennig E, etGlycanal. R, analys patterns innontypeable patterns Haemophilus influenza lipop SL, Lundstrom Twelkmeyer Sagemark B, NoetMK, al.

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This article isprotected by copyright. All rights reserved. Transfusion adult erythrocyteshuman antiserato directed train the patternglycosylationon of viral ureecluzumab correcttoof paroxysmal cold is in is culture-based cell influenza vaccine . JBiol. Chem 2010;27:20860-20869. mmune hemolytic anemia hemolytic mmune withtreated manual ationpatterns gp120HIV-1 of dependon the type Rituximabas successful therapypatient in a with 1969;16:478-485. tionofglobside andForssman glycolipids on ion2007;47:1902-4. olysaccharide. FEBSolysaccharide. J2007;274:4886-4903. vel globoside-like vel oligosaccharide expression t.DisordJBlood 2013;4:163.Transfus 3:15-24.