A Pattern Approach to Lymph Node Diagnosis

422 Pages·2011·46.172 MB·English

by  Anthony S-Y Leong (auth.)

A Pattern Approach to Lymph Node Diagnosis
A Pattern Approach to Lymph Node
Diagnosis
Anthony S.-Y. Leong,
MB,BS,MD,FRCPA,FRCPath,FCAP,FASCP,
FHKAM(Pathol),HonoraryFHKCPath,HonoraryFRCPT
Director,PathologyLaboratories,SunwayMedicalCenter,PetalingJaya,Malaysia
ProfessorofPathology,JeffreyCheahSchoolofMedicine,MonashUniversity,SunwayCampus,Malaysia
Formerly
MedicalDirector,HunterAreaPathologyService,Newcastle
ProfessorandChair,DisciplineofAnatomicalPathology
UniversityofNewcastle,NewSouthWales,Australia
123
AnthonyS.-Y.Leong,MD
Director
PathologyLaboratories
SunwayMedicalCenter
PetalingJaya
Malaysia
ProfessorofPathology
JeffreyCheahSchoolofMedicine
MonashUniversity
SunwayCampus
Malaysia
[email protected]
ISBN978-1-4419-7175-3 e-ISBN978-1-4419-7176-0
DOI10.1007/978-1-4419-7176-0
SpringerNewYorkDordrechtHeidelbergLondon
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Preface
Thedevelopmentofsophisticatedancillarytechniquesforlymphnodediagnosishasmade
hematopathologyaspecializeddisciplineinmanydevelopedcountries.Immunohistology,
electron microscopy, and molecular diagnostic techniques are expensive procedures and
requirespecializedresourcesandtechnicalskills,commoditiesthatarenotreadilyavailable
in many laboratories other than those in large medical facilities. The general or commu-
nity hospital pathologist is thus still required to make a histological diagnosis based on
microscopic observations of the biopsied lymph node, or at least, to decide if the patho-
logical changes represent a reactive or neoplastic process before referring the material for
an expert opinion. Importantly, as the pathologist to first encounter the excised lymph
node,heorsheisresponsiblefortriageofthespecimen.Thisrequiresfamiliaritynotonly
withthedifferenttechnologiesemployedintheproperexaminationofthesampledmate-
rial but also with the appropriate preparation of the specimen and what each adjunctive
techniquehastooffer.
There are many large and excellent textbooks on lymph node pathology that define
anddescribediseasesoflymphnodesingreatdetail.Thesetaketheconventionalapproach
ofseparatelydescribingeachofthereactiveandneoplasticentitiesthatinvolvethelymph
node and lymphoid tissues, and their accompanying immunophenotypic profiles, and
cytogenetic and molecular characteristics. This approach is contrary to the conventional
method applied in histologic diagnosis which is one of pattern recognition performed
through a systematic assessment of the various anatomical compartments of the tissue for
changes in architecture, expansion of a population of normal cells, or accumulation or
presence of abnormal populations of cells. In the lymph node, such anatomical compart-
ments are not clearly visible. Such a method of assessment is even more important for
the lymph node as anatomical compartmentalization reflects immune and cellular func-
tion.Tothetraineeandgeneral/communitypathologist,thelymphnodemayrepresenta
morass of small and large lymphocytes without clearly visible compartmentalization. This
problem is further compounded by the fact that neoplastic lymphoid cells invariably have
cytologic features similar and even identical to their normal counterparts, so that cellular
density, composition, and compartmentalization become important aspects of the histo-
logicassessment.Reactiveprocessescanproduceexuberantexpansionsofdifferentlymph
node compartments, and the changes are dynamic, adding further to difficulties of diag-
nosis.
Thepatternapproachtolymphnodediagnosishasbeenpreviouslyproposedbutsuch
an approach has not been adopted before in a textbook. The advent of immunohistology
and the availability of a comprehensive range of sensitive antibodies that are immunore-
active in routinely fixed, paraffin-embedded sections make it much easier to adopt such a
diagnosticapproachwhichmoretrulyreflectsthemannerinwhichpathologistsroutinely
examinelymphnodes.Invariably,assomeneoplasmsandmostreactiveconditionsinvolve
more than one compartment and produce more than one pattern of change, there will
be some degree of overlap with this method of diagnostic approach. However, in this
book, this is kept to a minimum and each entity is discussed under its primary pattern
v
vi Preface
of presentation and will be briefly mentioned, especially in differential diagnoses, when it
producesalternativeandless-frequentpatternsofhistologicchange.
Immunohistology is now an established ancillary diagnostic technique that is
employed by most routine laboratories, although the range of antibodies available will
vary.Wewillnotonlydiscusstheminimalrepertoireofantibodiesrequiredforlymphoma
diagnosis but also provide detailed immunoprofiles for complete characterization of each
neoplastic process. Clinical, molecular, and cytogenetic characteristics will also be dis-
cussed as they often aid diagnosis and provide further understanding of the neoplastic
process.Electronmicroscopy,whichislesscontributoryintheareaoflymphomadiagno-
sis,willbetouchedonwhereappropriate.
Itistheprimaryaimofthisbooktodemonstratethatasystematicapproachtolymph
node examination can be achieved through recognition of morphological patterns pro-
ducedbydifferentdiseaseprocesses.Alevelofconfidenceandfamiliaritywithlymphnode
pathology can be attained through the adoption of such an approach that is employed
largely in every other organ system in the body. This book is thus directed at trainee
andgeneral/communitypathologistswhohavefirstcontactwiththeexcisedlymphnode
and are required to make an initial judgment on the morphologic changes, especially if
deciding to refer the specimen on for expert opinion. The classification system employed
throughout this book is that of the 2008 World Health Classification. This book is not
intended to supplant the many excellent texts available on lymph node pathology and is
not intended to provide new information or knowledge. Its purpose is to instruct the
reader in a method of examination that employs the recognition of patterns and colors.
The discussion of each diagnostic entity is accompanied by ample color illustrations that
highlight the diagnostic features. Inaddition, textboxes summarizing the salient features
are provided. This is essentially a “how-to” manual that also integrates current informa-
tion about specific neoplastic entities. References have been deliberately excluded as they
distract from the primary purpose and the reader is referred to the 2008 WHO book for
acomprehensiveandappropriatereferencelistingandtootherselectedreferences.
AnthonyS.-Y.Leong,MD
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
SECTIONI
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Knowledge-BasedDiagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
SystematicExaminationoftheLymphNode . . . . . . . . . . . . . . . . . . . . 7
CellTypeIdentification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
CellSizeandCellularity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Immunohistology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
CytologicalPreparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
ThePatternApproachtoDiagnosis . . . . . . . . . . . . . . . . . . . . . . . . 14
SelectedReading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2. HandlingoftheLymphNodeBiopsy . . . . . . . . . . . . . . . . . . . . . . . 19
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
TheBiopsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
LymphNodeTriage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3. ImmunohistologyandOtherDiagnosticTechniques . . . . . . . . . . . . . . . . 23
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Immunohistology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
BCellMarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
TCellMarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
NaturalKillerandT/NKCellMarkers . . . . . . . . . . . . . . . . . . . . . . . 38
PrecursorB-andTCellMarkers . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Monocyte/MacrophageMarkers . . . . . . . . . . . . . . . . . . . . . . . . . 39
LangerhansCell,FollicularDendriticCell,andInterdigitatingDendritic
ReticulumCellMarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Markersof Reed–SternbergCellsinHodgkinLymphoma . . . . . . . . . . . . . 42
MiscellaneousMarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
MyeloidCellMarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
MastCellMarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
DiagnosticApproachwithImmunohistochemicalStains . . . . . . . . . . . . . . 47
StainingforFollicularDendriticCells . . . . . . . . . . . . . . . . . . . . . . . 47
ImmunohistologicalIdentificationofDiffuseLymphoidInfiltrates . . . . . . . . 49
HistochemicalStains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
FlowCytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
MolecularDiagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
DetectionofClonality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
ImmunoglobulinHeavy-andLight-ChainGenes . . . . . . . . . . . . . . . . . 57
TCellReceptorChainGenes. . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
vii
viii Contents
GenotypeSubgroups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
ConventionalCytogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
FluorescenceInSituHybridization(FISH) . . . . . . . . . . . . . . . . . . . . 60
SouthernBlotAnalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
PolymeraseChainReaction(PCR) . . . . . . . . . . . . . . . . . . . . . . . . . 60
GeneExpressionProfiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
4. AnatomicalandFunctionalCompartments . . . . . . . . . . . . . . . . . . . . . 63
BCellDifferentiationandCorrespondingBCellLymphomas . . . . . . . . . . . 64
TheCortex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
TheLymphoidFollicle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
TCellDifferentiation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
TheParacortex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
High-EndothelialVenules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
MedullaryArea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
ConnectiveTissueFramework . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
SECTIONII
5. NodularLymphoidInfiltrates . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
FollicularPattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
NeoplasticFollicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
FollicularLymphoma(FL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
FollicularHyperplasiaVersusFollicularLymphoma . . . . . . . . . . . . . . . . 91
OtherFollicularPatterns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
MantlePattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
MantleCellLymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
MarginalZonePattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
NodalMarginalZoneLymphoma(MonocytoidBCellLymphoma). . . . . . . . 107
NodularPattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
HeterogenousNodules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
PseudofollicularPattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
HodgkinLymphoma(HL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
ClassicalHodgkinLymphoma(CHL) . . . . . . . . . . . . . . . . . . . . . . . 116
Post-transplantLymphoproliferativeDisorders . . . . . . . . . . . . . . . . . . . 127
ColonizationofFolliclesbyNeoplasticCells . . . . . . . . . . . . . . . . . . . . 131
HomogenousNodules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
ReactiveHyperplasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
FollicularandParacorticalHyperplasia . . . . . . . . . . . . . . . . . . . . . . . 136
Non-specificFollicularHyperplasia . . . . . . . . . . . . . . . . . . . . . . . . . 137
ProgressiveTransformationofGerminalCenters(PTGC) . . . . . . . . . . . . . 137
RheumatoidLymphadenopathy . . . . . . . . . . . . . . . . . . . . . . . . . . 138
ToxoplasmaLymphadenopathy. . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Human Immunodeficiency Virus/Acquired Immunodeficiency Disease
Syndrome(HIV/AIDS)Lymphadenitis . . . . . . . . . . . . . . . . . . . 142
KimuraDisease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
CastlemanDisease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
SyphilisLymphadenopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
KikuchiDisease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Contents ix
SystemicLupusErythematosus . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
InfectiousMononucleosisLymphadenopathy . . . . . . . . . . . . . . . . . . . 158
OtherViralLymphadenopathies . . . . . . . . . . . . . . . . . . . . . . . . . . 161
ParacorticalNodules/Expansion . . . . . . . . . . . . . . . . . . . . . . . . . . 165
DermatopathicLymphadenopathy(DL) . . . . . . . . . . . . . . . . . . . . . . 165
Drug-InducedLymphadenopathy . . . . . . . . . . . . . . . . . . . . . . . . . 166
TheDiagnosticApproachtoNodularInfiltratesinLymphNodes . . . . . . . . . 167
AreTheyFolliclesorNodules? . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
AretheFolliclesReactiveorNeoplastic? . . . . . . . . . . . . . . . . . . . . . . 168
AreFolliclesInfrequentand“Constricted”? . . . . . . . . . . . . . . . . . . . . 168
AretheFolliclesExceptionallyLarge? . . . . . . . . . . . . . . . . . . . . . . . 169
DotheEnlargedGerminalCentersContainAtypicalCells? . . . . . . . . . . . . 169
AretheNodulesHomogenousorHeterogenousinComposition? . . . . . . . . . 169
HomogenousNodules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
HeterogenousNodules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
IfThereIsMarkedFollicularHyperplasia,CanaSpecificDiagnosisBeMade?. . . 170
DotheVagueNodulesRepresentParacorticalNodules? . . . . . . . . . . . . . . 171
6. DiffuseLymphoidInfiltrations . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
BCellNeoplasms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
ChronicLymphocyticLeukemia/SmallLymphocyticLymphoma(CLL/SLL) . . 176
LymphoplasmacyticLymphoma(LPL) . . . . . . . . . . . . . . . . . . . . . . . 186
DiffuseLargeBCellLymphoma(DLBCL) . . . . . . . . . . . . . . . . . . . . 194
ClinicalSubtypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
BurkittLymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
B Cell Lymphoma, Unclassifiable, with Features Intermediate Between
DLBCLandBL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
NodalInvolvementbyPrimaryExtranodalLymphomasandLeukemias . . . . . . 218
TCellandNKCellNeoplasms . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
T-LymphoblasticLeukemia/Lymphoma(PrecursorTCellLymphoblastic
Leukemia/Lymphoma) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
PeripheralTCellLymphoma,NotOtherwiseSpecified(PTCL-NOS) . . . . . . . 223
AngioimmunoblasticTCellLymphoma . . . . . . . . . . . . . . . . . . . . . . 229
AnaplasticLargeCellLymphoma(ALCL),ALK-Positive . . . . . . . . . . . . . 238
Immunohistology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
AnaplasticLargeCellLymphoma,ALK-Negative . . . . . . . . . . . . . . . . . 249
AdultTCellLeukemia/Lymphoma . . . . . . . . . . . . . . . . . . . . . . . . 250
NodalInvolvementbytheCutaneousTCellLymphomas . . . . . . . . . . . . . 255
HistiocyticandDendriticNeoplasms . . . . . . . . . . . . . . . . . . . . . . . . 260
HistiocyticSarcoma. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
LangerhansCellHistiocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
InterdigitatingDendriticCell(IDC)Sarcoma . . . . . . . . . . . . . . . . . . . 272
FollicularDendriticCell(FDC)Sarcoma . . . . . . . . . . . . . . . . . . . . . . 275
HodgkinLymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
DiagnosticApproachtoDiffuseInfiltratesinlymphNodes . . . . . . . . . . . . 279
IstheInfiltrateTrulyDiffuse? . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
DoestheDiffuseInfiltrateInvolvetheNodePartiallyorCompletely? . . . . . . . 280
IstheInfiltrateMonomorphousorPolymorphous?AretheCellsSmallorLarge? . 281
x Contents
7. DefiningMicroscopicFeatures . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
GranulomasandFoamCells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
InfectiveGranulomatousLymphadenitis . . . . . . . . . . . . . . . . . . . . . . 287
MycoticLymphadenitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
ProtozoanandParasiticLymphadenitides . . . . . . . . . . . . . . . . . . . . . 300
Non-infectiveGranulomatousLymphadenitis . . . . . . . . . . . . . . . . . . . 301
Granulomas,Non-necrotizing . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
ForeignBodyandLipidGranulomatousLymphadenitis . . . . . . . . . . . . . . 307
LipidLymphadenopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310
SinusPattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
SinusHistiocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
Rosai–DorfmanDisease(SinusHistiocytosiswithMassiveLymphadenopathy) . . 316
Necrosis,Apoptosis,andInfarction. . . . . . . . . . . . . . . . . . . . . . . . . 317
ClearCells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
MastCellDisease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
HairyCellLeukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
SpindledCells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
InflammatoryMyofibroblasticTumor . . . . . . . . . . . . . . . . . . . . . . . 325
PalisadedMyofibroblastoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
KaposiSarcoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
VascularProminence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
BacillaryAngiomatosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
VascularTransformationofLymphNodeSinuses . . . . . . . . . . . . . . . . . 331
Hemorrhage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
StarrySkyPattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
MottledPattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
Fibrosis/Hyalinization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
SignetRingCells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336
BizarreorMultinucleatedCells . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
ExtramedullaryHematopoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
ProminentEosinophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
ProminentNeutrophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
ProminentPlasmaCells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
InfiltrationofPericapsularFat . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
Extraneous Cells – Pigmented Cells, Epithelial Cells, Foreign Material,
DermatopathicLymphadenitis,Hemosiderin . . . . . . . . . . . . . . . . . 340
InclusionsofBenignExtraneousCells . . . . . . . . . . . . . . . . . . . . . . . 340
8. NodalInvolvementbyLeukemiasandExtranodalLymphomas . . . . . . . . . . 345
NodalInvolvementbyLeukemia . . . . . . . . . . . . . . . . . . . . . . . . . . 345
MyeloidLeukemia/MyeloidSarcoma . . . . . . . . . . . . . . . . . . . . . . . 345
PrimaryMyelofibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
BandTCellProlymphocyticLeukemias . . . . . . . . . . . . . . . . . . . . . . 348
NKCellLymphoproliferativeDisorders . . . . . . . . . . . . . . . . . . . . . . 350
NodalInvolvementbyExtranodalLymphomas . . . . . . . . . . . . . . . . . . 351
SplenicBCellMarginalZoneLymphoma . . . . . . . . . . . . . . . . . . . . . 352
HairyCellLeukemia(HCL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
Heavy-ChainDisease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354