iii

Color Atlas ofHematology

PracticalMicroscopicand ClinicalDiagnosis

HaraldTheml,M.D.

Professor,PrivatePractice

Hematology/Oncology

Munich,Germany

HeinzDiem,M.D.

KlinikumGrosshadern

InstituteofClinicalChemistry

Munich,Germany

TorstenHaferlach,M.D.

Professor,KlinikumGrosshadern

Laboratoryfor LeukemiaDiagnostics

Munich,Germany

2ndrevised edition

262color illustrations

32tables

Thieme

Stuttgart· New York

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tionData isavailable fromthe publisher

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Thisbook isan authorizedrevised

translationof the5th Germanedition

publishedand copyrighted2002 by

ThiemeVerlag, Stuttgart,Germany.

Titleof theGerman edition:

Taschenatlasder Hämatologie

Translator:Ursula Peter-CzichiPhD,

Atlanta,GA, USA

1stGerman edition1983

2ndGerman edition1986

3rdGerman edition1991

4thGerman edition1998

5thGerman edition2002

1stEnglish edition1985

1stFrench edition1985

2ndFrench edition2000

1stIndonesion edition1989

1stItalian edition1984

1stJapanese edition1997

!2004 GeorgThieme Verlag

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Preface

OurCurrent Edition

Althoughthis is the second English edition ofour hematology atlas, this

editionis completely new. As animmediate sign of this change,there are

nowthree authors. Thecompletely updated visualpresentation uses dig-

italimages, andthe contentis organized accordingto themost up-to-date

morphologicalclassification criteria.

In this new edition, ournewly formed team of authors from Munich

(the“Munich Group”) has successfullyshared their knowledge withyou.

HeinzDiem and Torsten Haferlachare nationally recognized as lecturers

ofthe diagnostics curriculum ofthe German Association for Hematology

andOncology.

Goals

Mostphysicians arefundamentally “visuallyoriented.” Apart fromimme-

diatepatient care, the microscopic analysisof blood plays to this prefer-

ence. This explains the delight and level of involvement on the part of

practitionersin the pursuitof morphological analyses.

Specialization notwithstanding, the hematologist wantsto preserve

theopportunity toperform groundbreakingdiagnostics inhematology for

thegeneral practitioner,surgeon,pe diatrician,the MTAtechnician, andall

medical support personnel. New colleagues must also be won to the

cause. Utmost attention to the analysis of hematological changes is es-

sentialfor a timelydiagnosis.

Even before bone marrow cytology, cytochemistry, or immunocyto-

chemistry,information basedon theanalysis of bloodis ofimmediate rel-

evancein thedoctor’s office.It iscentral to thediagnosis ofthe diseasesof

the blood cell systems themselves, which make theirpresence known

throughchanges in bloodcomponents.

Theexhaustive quantitativeand qualitative useof hematological diag-

nostics is crucial. Discussions withcolleagues from all specialties and

teachingexperience with advanced medical students confirmits impor-

tance. In cases wherea diagnosis remains elusive, the awareness of the

next diagnostic step becomes relevant. Then, further investigation

throughbone marrow,lymphnode, ororgan tissuecytology canyield firm

results.This pocket atlas offers the basic knowledgefor the use of these

techniquesas well.

Organization

Reflectingour goals, the inductive organization proceedsfrom simple to

specializeddiagnostics. Bydesign, we subordinatedthe descriptionof the

bone marrowcytology to the diagnostic blood analysis (CBC). However,

wehave respondedto feedback fromreaders ofthe previous editionsand

have included the principles of bone marrow diagnostics and non-

ambiguous clinical bone marrowfindings so that frequent and relevant

diagnosescan be quicklymade, understood, orreplicated.

The nosologyand differential diagnosis of hematological diseases are

presentedto you ina tabular form. Wewanted to offeryou a pocketbook

for everydaywork, not a reference book. Therefore, morphological curi-

osities, oranomalies, are absent in favor ofa practical approach to mor-

phology. The cellular components of organ biopsies and exudates are

brieflydiscussed, mostly asa reminder of theimportance of thesetests.

Theimages are consistently photographedas they normally appearin

microscopy (magnification 100 or 63 with oil immersion lens, oc-

casionally master-detailmagnification objective 10 or 20). Even though

surprising perspectivessometimes result from viewing cells at a higher

magnification,the downsideis that thisby nomeans facilitatesthe recog-

nitionof cells using yourown microscope.

Instructionsfor the Use of this Atlas

Theorganization ofthis atlas supportsa systematicapproach tothe study

of hematology(see Table of Contents). The index offers ways to answer

detailedquestions and access the hematologicalterminology with refer-

encesto the maindescription and further citations.

Thebest wayto becomefamiliar withyour pocketatlas is tofirst havea

cursory lookthrough its entire content. The imagesare accompanied by

shortlegends. Onthe pagesopposite the images you willfind correspond-

ingshort descriptivetexts and tables.This textportion describes cellphe-

nomena anddiscusses in more detail further diagnostic steps as wellas

thediagnostic approach todisease manifestations.

Acknowledgments

Twentyyears ago,Professor HerbertBegemann dedicatedthe forewordto

thefirst edition of thishematology atlas. Heacknowledged that—beyond

cellmorphology—this atlas aimsat the clinicalpicture ofpatients. We are

gratefulfor beingable tocontinue thistradition, andfor theimpulses from

ourteachers and companionsthat make this possible.

Wethank our colleagues:J. Rastetter, W.Kaboth, K. Lennert,H. Löffler,

H. Heimpel, P.M.Reisert, H. Brücher, W. Enne, T. Binder, H.D. Schick,W.

Hiddemann,D. Seidel.

Munich,January 2004 HaraldTheml, Heinz Diem,Torsten Haferlach

Preface

vii

Contents

Physiologyand Pathophysiology of Blood Cells:

Methodsand Test Procedures

.. . .. . . .. . .. . . .. . .. . . .. . .. . . 1

Introductionto the Physiologyand Pathophysiology ofthe

HematopoieticSystem . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . 2

CellSystems .. .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. 2

Principlesof Regulation andDysregulation in theBlood Cell

Seriesand their DiagnosticImplications . .. . .. . . .. . .. . . .. . .. . . .. 7

Procedures,Assays, and NormalValues .. . . .. . .. . . .. . .. . . .. . .. 9

TakingBlood Samples .. .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . . 9

ErythrocyteCount . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . 10

Hemoglobinand Hematocrit Assay . . .. . .. . . .. . .. . . .. . .. . . .. . .. . 10

Calculationof Erythrocyte Parameters . . .. . .. . . .. . .. . . .. . .. . . .. . 10

RedCell Distribution Width(RDW) .. . .. . . .. . .. . . .. . .. . . .. . .. . . 11

ReticulocyteCount .. .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . 11

LeukocyteCount .. . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . . 14

ThrombocyteCount .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . 15

QuantitativeNormal Valuesand Distribution of CellularBlood

Components . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . 15

TheBlood Smear andIts Interpretation (DifferentialBlood Count,

DBC) . . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. 17

Significanceof the AutomatedBlood Count .. . .. . . .. . .. . . .. . .. . . 19

BoneMarrow Biopsy .. .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . . 20

LymphNode Biopsy andTumor Biopsy .. . .. . . .. . .. . . .. . .. . . .. . . 23

Step-by-StepDiagnostic Sequence .. .. . . .. . .. . . .. . .. . . .. . .. . . . 25

NormalCells of the Blood and Hematopoietic Organs . 29

TheIndividual Cells ofHematopoiesis . . .. . .. . . .. . .. . . .. . .. . . . 30

ImmatureRed Cell Precursors:Proerythroblasts and Basophilic

Erythroblasts . . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . . . . . .. . .. . . . 30

MatureRed Blood PrecursorCells: Polychromatic andOrtho-

chromaticErythroblasts (Normoblasts) andReticulocytes . . . .. . . 32

ImmatureWhite Cell Precursors:Myeloblasts and Promyelo-

cytes . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. 34

viii

PartlyMature White CellPrecursors: Myelocytes andMetamyelo-

cytes . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. 36

MatureNeutrophils: Band Cellsand Segmented Neutrophils .. .. . 38

CellDegradation, Special Granulations,and Nuclear Appendages

inNeutrophilic Granulocytes andNuclear Anomalies .. .. . . .. . .. . 40

EosinophilicGranulocytes (Eosinophils) .. . .. . .. . . .. . .. . . .. . .. . . 44

BasophilicGranulocytes (Basophils) .. .. . .. . . .. . .. . . .. . .. . . .. . .. 44

Monocytes .. .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . 46

Lymphocytes(and Plasma Cells) .. . .. . .. . . .. . .. . . .. . .. . . .. . .. . . 48

Megakaryocytesand Thrombocytes .. . .. . .. . . .. . .. . . .. . .. . . .. . . 50

BoneMarrow: Cell Compositionand Principles ofAnalysis . .. . 52

BoneMarrow: Medullary StromaCells . . . .. . .. . . .. . .. . . .. . .. . . . 58

Abnormalitiesof the White Cell Series . .. . .. . . .. . .. . . .. . 61

Predominanceof Mononuclear Roundto Oval Cells .. .. . .. . . .. 63

ReactiveLymphocytosis . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. 66

Examplesof Extreme LymphocyticStimulation: Infectious

Mononucleosis . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . . 68

Diseasesof the LymphaticSystem (Non-Hodgkin Lymphomas) . . . 70

Differentiationof the LymphaticCells and CellSurface Marker

Expressionin Non-Hodgkin LymphomaCells . . .. . .. . . .. . .. . . . 72

ChronicLymphocytic Leukemia (CLL)and Related Diseases .. . . 74

LymphoplasmacyticLymphoma . . .. . .. . . .. . .. . . .. . .. . . .. . .. . 78

FacultativeLeukemic Lymphomas(e.g., Mantle Cell Lymphoma

andFollicular Lymphoma) . . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . . 78

Lymphoma,Usually with Splenomegaly(e.g., Hairy CellLeuke-

miaand Splenic Lymphomawith Villous Lymphocytes) . .. . .. . 80

MonoclonalGammopathy (Hypergammaglobulinemia), Mul-

tipleMyeloma*, Plasma CellMyeloma, Plasmacytoma .. . .. . .. . 82

Variabilityof Plasmacytoma Morphology . . .. . .. . . .. . .. . . .. . .. 84

RelativeLymphocytosis Associatedwith Granulocytopenia

(Neutropenia)and Agranulocytosis . .. . . .. . .. . . .. . .. . . .. . .. . . .. 86

Classificationof Neutropenias andAgranulocytoses . .. . . .. . .. . 86

Monocytosis . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . 88

AcuteLeukemias . . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . . . . . .. . . 90

Morphologicaland Cytochemical CellIdentification . .. . .. . . .. . 91

AcuteMyeloid Leukemias (AML) . . .. . . .. . .. . . .. . .. . . .. . .. . . .. 95

AcuteErythroleukemia (FAB ClassificationType M

) . .. . .. . . .. 100

AcuteMegakaryoblastic Leukemia

(FABClassification Type M

) . . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. 102

AMLwith Dysplasia . . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . 102

HypoplasticAML . . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. 102

Contents

AcuteLymphoblastic Leukemia (ALL) .. . . .. . .. . . .. . .. . . .. . .. . 104

Myelodysplasia(MDS) . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . . 106

Prevalenceof Polynuclear (Segmented)Cells .. . .. . . .. . .. . . .. . 110

Neutrophiliawithout Left Shift . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . 110

ReactiveLeft Shift . . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . 112

ChronicMyeloid Leukemia andMyeloproliferative Syndrome

(ChronicMyeloproliferative Disorders,CMPD) . . .. . . .. . .. . . .. . .. 114

Stepsin the Diagnosisof Chronic MyeloidLeukemia . .. . .. . . .. 116

BlastCrisis in ChronicMyeloid Leukemia .. . .. . . .. . .. . . .. . .. . . 120

Osteomyelosclerosis .. .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . . 122

ElevatedEosinophil and BasophilCounts . . .. . .. . . .. . .. . . .. . .. . . 124

Erythrocyte andThrombocyte Abnormalities .. . .. . . .. . . 127

ClinicallyRelevant Classification Principlefor Anemias: Mean

ErythrocyteHemoglobin Content (MCH) . .. . .. . . .. . .. . . .. . .. . . . 128

HypochromicAnemias .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . 128

IronDeficiency Anemia . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . . . . 128

HypochromicInfectious or ToxicAnemia (Secondary Anemia) .. . 134

BoneMarrow Cytology inthe Diagnosis of HypochromicAne-

mias . . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . 136

HypochromicSideroachrestic Anemias (SometimesNormo-

chromicor Hyperchromic) . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . 137

HypochromicAnemia with Hemolysis . .. . . .. . .. . . .. . .. . . .. . .. . 138

Thalassemias . . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . 138

NormochromicAnemias . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . . 140

NormochromicHemolytic Anemias .. . . .. . .. . . .. . .. . . .. . .. . . .. . 140

HemolyticAnemias with ErythrocyteAnomalies . .. . .. . . .. . .. . . . 144

NormochromicRenal Anemia (SometimesHypochromic or

Hyperchromic) .. .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . 146

BoneMarrow Aplasia . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. 146

PureRed Cell Aplasia(PRCA, Erythroblastopenia) . . .. . .. . . .. . . 146

Aplasiasof All BoneMar rowSeries (Panmyelopathy,Pan-

myelophthisis,Aplastic Anemia) .. .. . . .. . .. . . .. . .. . . .. . .. . . .. 148

BoneMarrow Carcinosis andOther Space-Occupying Processes .. 150

HyperchromicAnemias . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. 152

ErythrocyteInclusions . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . 156

HematologicalDiagnosis of Malaria .. .. . . .. . .. . . .. . .. . . .. . .. . . . 158

Contents

PolycythemiaVera (ErythremicPolycythemia)

andErythrocytosis .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . . 162

ThrombocyteAbnormalities . . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. 164

Thrombocytopenia .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . . 164

ThrombocytopeniasDue to IncreasedDemand

(HighTurnover) . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . 164

ThrombocytopeniasDue to ReducedCell Production .. . . .. . .. . 168

Thrombocytosis(Including Essential Thrombocythemia) .. . . .. . . 170

EssentialThrombocythemia . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . . 170

Cytologyof Organ Biopsies and Exudates .. . .. . . . . . .. . . . 173

LymphNode Cytology .. .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . 174

ReactiveLymph NodeHyperplasia and Lymphogranulomatosis

(HodgkinDisease) . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . 176

Sarcoidosisand Tuberculosis . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . . 180

Non-HodgkinLymphoma . . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . 182

Metastasesof Solid Tumorsin Lymph Nodesor Subcutaneous

Tissue . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. 182

BranchialCysts and BronchoalveolarLavage .. . . .. . .. . . .. . .. . . 184

BranchialCysts . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . 184

Cytologyof the RespiratorySystem, Especially Bronchoalveolar

Lavage .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . . 184

Cytologyof Pleural Effusionsand Ascites . .. . .. . . .. . .. . . .. . .. . 186

Cytologyof Cerebrospinal Fluid .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. 188

References . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . . 190

Index . . .. . . .. . .. . . .. . .. . . .. . .. . . .. . . . . . .. . .. . . .. . . . . . .. . .. . . . 191

Contents

Physiology and Pathophysiology of

Blood Cells: Methods and Test

Procedures

Pluripotent

lymphatic

stem cells

NK cells

T-lymphopoiesis

T-lymphoblasts

T-lymphocytes

B-lymphopoiesis

B-lymphoblasts

B-lymphocytes

Plasma cells

Pluripotent hemato-

Introduction to the Physiology and

Pathophysiology of the Hematopoietic

System

The reason why quantita-

tiveand qualitativediagno-

sis based on the cellular

componentsof the blood is

so important is that blood

cells are easily accessible

indicators of disturbances

in their organsof origin or

degradation—which are

muchless easily accessible.

Thus, disturbances in the

erythrocyte, granulocyte,

and thrombocyte series

allow important conclu-

sions to be drawn about

bonemarrow function, just

asdisturbances of the lym-

phatic cells indicate reac-

tions or disease states of

the specialized lympho-

poietic organs (basically,

the lymph nodes, spleen,

and the diffuse lymphatic

intestinalorgan).

Cell Systems

Allblood cellsderive froma

common stem cell. Under

the influences of local and

humoralfactors, stem cells

differentiate into different

!Fig.1 Modelof cell lineages

inhematopoiesis

Physiologyand Pathophysiology of Blood Cells

Omnipotent

stem cells

Eosinophils

Granulopoiesis

monopoiesis

GranulopoiesisMonopoiesis

Pluripotent myeloid

stem cells

MyeloblastsMonoblasts

Promyelocytes

Myelocytes

Metamyelocytes

Cells with band nuclei

Neutrophilic

granulocytes

with segmented

nuclei

Monocytes

Promonocytes

Eosinophilic

promyelocytes

Eosinophilic

segmented

granulocytes

Macrophages

Erythropoiesis

Proery-

throblasts

Erythroblasts

ErythrocytesErythrocytes

Thrombopoiesis

Mega-

karyoblasts

Mega-

karyocytes

Thrombo-

cytes

Basophils

Basophilic

segmented

granulocytes

Basophilic

promyelocytes

poietic stem cells

cell lines (Fig.1). Erythropoiesisand thrombopoiesisproceed indepen-

dentlyonce the stemcell stage hasbeen passed, whereas monocytopoie-

sisand granulocytopoiesis arequite closely “related.”Lymphocytopoiesis

is themost independent among the remaining cell series. Granulocytes,

monocytes, and lymphocytes arecollectively called leukocytes (white

bloodcells), a term that has been retained since thedays before staining

Introductionto the Physiology and Pathophysiology

Vacuoles

Nucleus (with

delicate reticular

chromatin structure)

Nucleolus

Cytoplasmic granules

Lobed nucleus with

banded chromatin

structure

Coarse chromatin

structure

Cytoplasm

methods were available, when the only distinction that could be made

wasbetween erythrocytes (redblood cells) andthe rest.

All these cells are eukaryotic,that is, they are made up of a nucleus,

sometimeswith visiblenucleoli, surroundedby cytoplasm,which mayin-

cludevarious kinds oforganelles, granulations, and vacuoles.

Despitethe common origin of all the cells, ordinary light microscopy

reveals fundamental and characteristic differences in the nuclear chro-

matin structure in the different cellseries andtheir variousstages of

maturation(Fig.

2).

The developing cells in the granulocyte series (myeloblasts and pro-

myelocytes),for example,show adelicate, fine“net-like” (reticular)struc-

ture. Careful microscopic examination (using finefocus adjustmentto

view dif ferentdepth levels) reveals a detailed nuclear structurethat re-

semblesfine or coarsegravel (Fig.

2a). Withprogressive stages ofnuclear

maturationin this series (myelocytes,metamyelocytes, andband or staff

cells),the chromatincondenses intobands orstreaks, givingthe nucleus—

which at the same time is adoptinga characteristic curved shape—a

spottedand striped pattern(Fig.

2b).

Lymphocytes, on the other hand—particularly in their circulating

forms—always have large, solid-looking nuclei. Like cross-sections

throughgeological slate, homogeneous,dense chromatinbands alternate

withlighter interruptions andfissures (Fig.

2c).

Eachof these cellseries contains precursors thatcan divide (blastpre-

cursors)and matureor almostmature formsthat canno longerdivide; the

morphologicaldifferences betweenthese correspondnot tosteps inmito-

Fig.2 Principlesof

cellstructure with ex-

amplesof dif ferent

nuclearchromatin

structure.a Cell of the

myeloblastto pro-

myelocytetype. b Cell

ofthe myelocyte to

staffor band cell type.

cCell of the lympho-

cytetype with

coarselystructured

chromatin

Physiologyand Pathophysiology of Blood Cells

sis,but result fromcontinuous “maturation processes”of the cellnucleus

and cytoplasm.Once this is understood, it becomes easier not to be too

rigid about morphological distinctions between certain cell stages. The

blasticprecursors usually reside inthe hematopoietic organs(bone mar-

rowand lymph nodes). Since, however,a strict blood–bonemarrow bar-

rierdoes not exist(blasts arekept out ofthe bloodstream essentiallyonly

bytheir limited plasticity,i.e., their inabilityto cross thediffusion barrier

into the bloodstream), it is in principle possible for any cell type to be

found in peripheral blood, and when cell production is increased, the

statisticalfrequency withwhich they crossinto the bloodstreamwill nat-

urallyrise as well. Conventionally, cells are sortedlef tto right from im-

mature tomature, so an increased level of immature cells in the blood-

streamcauses a “leftshift” in thecomposition of acell series—although it

mustbe saidthat onlyin theprecursor stagesof granulopoiesisare thecell

morphologiessufficiently distinct forthis left shift toshow up clearly.

Thedistribution of whiteblood cells outsidetheir places oforigin can-

notbe inferred simply froma drop of capillaryblood. This is becausethe

majority of white cells remainout of circulation, “marginated” in the

epithelial lining of vessel walls or in extravascular spaces, from where

theymay bequickly recruitedback to thebloodstream. Thisphenomenon

explains why white cell counts can vary rapidly without or before any

changehas taken placein the rateof their production.

Cellfunctions. A briefindication of thefunctions ofthe variouscell groups

follows(see Table

1).

Neutrophil granulocytes with segmented nuclei serve mostly to

defendagainst bacteria.Predominantly outsidethe vascularsystem,in “in-

flamed” tissue, they phagocytoseand lysebacteria. The blood merely

transportsthe granulocytes totheir site ofaction.

Thefunction of

eosinophilicgranulocytes is defenseagainst parasites;

theyhave a directcytotoxic action onparasites and theireggs and larvae.

Theyalso playa rolein thedown-regulation ofanaphylactic shockreactions

and autoimmuneresponses, thus controlling the influence of basophilic

cells.

Themain function of

basophilicgranulocytes and their tissue-bound

equivalents(tissue mast cells) is to regulate circulation through the re-

leaseof substancessuch ashistamine, serotonin,and heparin.These tissue

hormonesincrease vascularpermeability atthe siteof variouslocalantigen

activityand thus regulatethe influx of theother inflammatory cells.

Themain function of

monocytesis the defense againstbacteria, fungi,

viruses,and foreign bodies. Defensive activitiestake place mostly outside

thevessels byphagocytosis. Monocytesalso breakdown endogenouscells

(e.g.,erythrocytes) at theend of their lifecycles, and theyare assumed to

performa similar function indefense against tumors. Outsidethe blood-

stream, monocytes develop into histiocytes; macrophages in the

Introductionto the Physiology and Pathophysiology

Table1 Cells in a normal peripheral blood smear and their physiological roles

Celltype Function Count

(%of leuko-

cytes)

Neutrophilicband

granulocytes(band

neutrophil)

Precursorsof segmented cells

thatprovide antibacterial

immuneresponse

0–4%

Neutrophilicsegmented

granulocyte(segmented

neutrophil)

Phagocytosisof bacteria;

migrateinto tissue for this pur-

pose

50–70%

Lymphocytes

(B-and T-lymphocytes,

morphologicallyindistin-

guishable)

B-lymphocytes(20% of

lymphocytes)mature and

formplasma cells ! antibody

production.

T-lymphocytes(70%): cyto-

toxicdefense against viruses,

foreignantigens, and tumors.











20–50%

Monocytes Phagocytosisof bacteria, pro-

tozoa,fungi, foreign bodies.

Transformationin target tissue

2–8%

Eosinophilicgranulocytes Immune defense against para-

sites,immune regulation

1–4%

Basophilicgranulocytes Regulationofthe response to

localinflammatory processes

0–1%

endotheliumof the body cavities; epithelioid cells; foreignbody macro-

phages(including Langhans’ giantcells); and manyother cells.

Lymphocytes are divided into two major basic groupsaccording to

function.

Thymus-dependent T-lymphocytes, which make up about 70% of

lymphocytes,provide localdefense againstantigens fromorganic andinor-

ganicforeign bodies inthe form ofdelayed-type hypersensitivity,as clas-

sicallyexemplified by thetuberculin reaction. T-lymphocytesare divided

into helper cells and suppressor cells.The smallgroup ofNK (natural

killer)cells, whichhave adirect cytotoxicfunction, isclosely relatedto the

T-cellgroup.

Theother group is the bone-marrow-dependent B-lymphocytesor B-

cells,which make up about 20% of lymphocytes.Through their develop-

mentinto immunoglobulin-secretingplasma cells, B-lymphocytesare re-

sponsible forthe entire humoral side of defense againstviruses, bacteria,

andallergens.

Physiologyand Pathophysiology of Blood Cells

Erythrocytesare the oxygen carriers for all oxygen-dependentmeta-

bolicreactions in theorganism. They arethe only bloodcells without nu-

clei,since this allows themto bind and exchangethe greatest numberof

molecules. Theirphysiological biconcave disk shape with a thick rim

providesoptimal plasticity.

Thrombocytesform the aggregatesthat, along with humoralcoagula-

tionfactors, close up vascularlesions. During the aggregation process,in

addition tothe mechanical function, thrombocytic granules also release

factorsthat promote coagulation.

Thrombocytes develop from polyploid megakaryocytesin the bone

marrow. They are the enucleated, fragmented cytoplasmic portionsof

theseprogenitor cells.

Principles of Regulation and Dysregulation in the

Blood Cell Series and their Diagnostic Implications

Quantitativeand qualitative equilibrium between all bloodcells is main-

tained under normal conditions through regulation byhumoral factors,

whichensure abalance betweencell production (mostlyin thebone mar-

row)and cell degradation (mostly inthe spleen, liver, bonemarrow, and

thediffuse reticular tissue).

Compensatoryincreases in cell production are induced by cell lossor in-

creased cell demand. Thiscompensatory process can lead to qualitative

changesin the compositionof the blood,e.g., theoccur renceof nucleated

red cellprecursors compensating for blood loss or increased oxygen re-

quirement,or following deficiency of certainmetabolites (in the restitu-

tion phase, e.g., during iron or vitamin supplementation). Similarly,

during acuteimmune reactions, which lead to an increased demand for

cells,immature leukocyte formsmay appear (“left shift”).

Increasedcell counts in one series can lead to suppressionof cell produc-

tionin another series. The classic example isthe suppression of erythro-

cyteproduction (the pathomechanical details of whichare incompletely

understood)during infectious/toxicreactions, whichaffect thewhite cells

(“infectiousanemia”).

Metabolitedeficiency as a pathogenic stimulus affects the erythrocytese-

riesfirst and most frequently.Although othercell series are alsoaffected,

thisseries, withits highturnover, isthe onemost vulnerableto metabolite

deficiencies. Iron deficiency, for example, rapidly leads to reduced

hemoglobinin the erythrocytes, while vitaminB

and/orfolic acid defi-

ciency will resultin complex disturbances in cell formation. Eventually,

thesedisturbances willstart toshow effectsin theother cellseries as well.

Introductionto the Physiology and Pathophysiology

Toxicinfluences on cell production usually affect allcell series. Theeffects

of toxicchemicals (including alcohol), irradiation, chronic infections, or

tumorload, for example,usually lead toa greater orlesser degree ofsup-

pressionin all theblood cell series,lymphocytes andthrombocytes being

themost resistant. The most extremeresult of toxicef fectsis panmyelo-

phthisis(the synonym “aplastic anemia” ignores thefact that the leuko-

cyteand thrombocyte seriesare usually alsoaffected).

Autoimmuneand allergic processes may selectively affect a singlecell se-

ries.Results of this include “allergic” agranulocytosis,immunohemolytic

anemia, and thrombocytopeniatriggered by either infection or medica-

tion. Autoimmune suppression of the pluripotent stem cells can also

occur,causing panmyelophthisis.

Malignant dedifferentiation can basically occur in cells of any lineage at

anystage wherethe cellsare ableto divide, causingchronic oracute clinical

manifestations.These deviations from normaldifferentiation occur most

frequentlyin the whitecell series, causing “leukemias.”Recent data indi-

catethat in fact inthese cases the remaining cellseries also become dis-

torted,perhaps via generalizedatypical stem cellformation. Erythroblas-

tosis,polycythemia, and essentialthrombocythemia are examples show-

ing that malignant processes can also manifestthemselves primarily in

theerythrocyte or thrombocyteseries.

Malignant “transformations” alwaysaf fect blood cell precursors that

arestill capable ofdividing, andthe result isan accumulation ofidentical,

constantlyself-reproducing blastocytes. Theseare not necessarilyalways

observedin the bloodstream,but can remain inthe bone marrow.That is

why,in “leukemia,” itis often not thenumber of cells, butthe increasing

lackof normal cellsthat is the indicativehematological finding.

Alldisturbances of bonemarrow functionare accompanied byquan-

titative and/or qualitative changes in the composition of bloodcells or

blood proteins. Consequently, in most disorders, careful analysis of

changesin the blood togetherwith clinical findings andother laboratory

dataproduces the same information as bonemarrow cytology. The rela-

tionshipbetween the productionsite (bone marrow)and the destination

(theblood) israrely sofundamentally disturbedthat hematologicalanaly-

sisand humoralparameterswill notsuffice fora diagnosis.This isvirtually

alwaystrue for hypoplastic–anaplastic processes in one or all cell series

with resulting cytopenia but without hematological signs of malignant

cellproliferation.

Physiologyand Pathophysiology of Blood Cells

Procedures, Assays, and Normal Values

TakingBlood Samples

Since cell counts are affected by the state of the blood circulation, the

conditions under which samples are taken should be the same so far as

possibleif comparable values are desired.

Thismeans that bloodshould alwaysb edrawn atabout the sametime of

dayand after at least eight hoursof fasting, since both circadianrhythm

andnutritional statuscan affect thefindings. If strictlycomparable values

are required, there should also be halfan hourof bedrest beforethe

sampleis drawn, butthis is onlypracticable in ahospital setting. Inother

settings(i.e., outpatient clinics), bringingportable instruments to the re-

laxed,seated patient workswell.

A sample of capillary bloodmay be taken when there are no further

teststhat wouldrequire venousaccess fora larger samplevolume. Awell-

perfusedfingertip oran earlobeis ideal;in newbornsor young infants,the

heelis also agood site. Ifthe circulation is poor,the bloodflow can be in-

creased by warming the extremity by immersing it in warm water.

Withoutpressure, the puncture area is swabbed severaltimes with 70%

alcohol,and theskin isthen punctured firmlybut gentlywith asterile dis-

posable lancet.The first droplet of blood is discarded because itmay be

contaminated, and the ensuing blood is drawn into the pipette (see

below). Care should be taken not to exert pressure onthe tissue from

whichthe bloodis being drawn,because thistoo canchange the cellcom-

positionof the sample.

Obviously,if a venous bloodsample is to be takenfor the purposes of

other tests, or if an intravenous injection is going to be performed, the

bloodsample for hematologicalanalysis can betaken from thesame site.

Todo this, the blood is allowed toflow via an intravenous needle into a

speciallyprepared (commercially available)EDTA-treated tube. The tube

isfilled tothe 1-mlmark andthen carefullyshaken severaltimes. Thevery

small amountof EDTA in the tube prevents the blood fromclotting, but

canitself be safelyignored in the quantitativeanalysis.

Procedures,Assays, and Normal Values

Erythrocyte Count

Upto 20years ago,blood cellswere counted“by hand”in anoptical count-

ingchamber. Thismethod hasnow been almostcompletely abandonedin

favorof automated counters that determine thenumber of erythrocytes

bymeasuring the impedance orlight dispersion of EDTAblood (1 ml), or

heparinized capillary blood. Dueto differencesin the hematocrit, the

valuefrom asample taken after(at least15 minutes’)standing orphysical

activitywill be 5–10% higherthan the valuefrom a sampletaken after 15

minutes’bed rest.

Hemoglobin and HematocritAssay

Hemoglobin is oxidized to cyanmethemoglobin bythe additionof cy-

anide, and the cyanmethemoglobin is then determined spectropho-

tometricallyby theautomated counter.The hematocritdescribes theratio

of the volume of erythrocytes to the total blood volume (the SI unit is

withoutdimension, e.g., 0.4).

The EDTA blood is centrifuged in adisposable capillarytube for10

minutes using a high-speed microhematocrit centrifuge (reference

method).The automated hematology counter determines the mean cor-

puscularor cellvolume (MCV,measured infemtoliters, fl)and thenumber

of erythrocytes. It calculates the hematocrit (HCT) usingthe following

formula:

HCT= MCV(fl) !number ofer ythrocytes(10

/µl).

Calculation of Erythrocyte Parameters

Thequality of erythrocytesis characterized bytheir MCV, theirmean cell

hemoglobin content (MCH),and the mean cellular hemoglobin concen-

tration(MCHC).

MCVis measureddirectly using anautomated hemoglobinanalyzer, or

iscalculated as follows:

MCV "

Hematocrit(l/l)

Numberof erythrocytes (10

/µl)

MCH (in picograms per erythrocyte)is calculated using the following

formula:

MCH(pg) "

Hemoglobin(g/l)

Numberof erythrocytes (10

/µl)

Physiologyand Pathophysiology of Blood Cells

MCHCis determined usingthis formula:

MCHC(g/dl) "

Hemoglobinconcentration (g/dl)

Hematocrit(l/l)

Red CellDistribution Width (RDW)

Modernanalyzers also recordthe red celldistribution width (cellvolume

distribution).In normal erythrocytemorphology, this correlateswith the

Price-Jones curve for thecell diameter distribution. Discrepancies are

used diagnostically and indicate the presence of microspherocytes

(smallercells with lightercentral pallor).

ReticulocyteCount

Reticulocytescan be counted using flow cytometry and is based on the

lightabsorb edby stained aggregates of reticulocyteorganelles. The data

arerecorded asthe number ofreticulocytes permill (‰) ofthe totalnum-

berof erythrocytes. Reticulocytescan, ofcourse, be countedin a counting

chamber using a microscope. While this method is not particularly

laborious, it is mostly employed in laboratories that often deal with or

have a special interest in anemia. Reticulocytes are young erythrocytes

immediatelyafter they have extrudedtheir nuclei: they contain, as are-

mainder of aggregated cell organelles, a net-like structure (hence the

name “reticulocyte”)that is not discernible after the usual staining pro-

cedures for leukocytes, but can be observed after vital staining of cells

withbrilliant cresyl blue ornew methylene blue.The staining solution is

mixedin an Eppendorf tubewith an equalvolume of EDTA bloodand in-

cubatedfor 30 minutes.After repeated mixing,a blood smearis prepared

and allowedto dry. The sample is viewed using a microscope equipped

withan oilimmersion lens.The ratioof reticulocytesto erythrocytesis de-

terminedand plotted asreticulocytes per 1000er ythrocytes(per mill).

Normalvalues are listedin Table

2,p. 12.

Procedures,Assays, and Normal Values

Table2 Normal ranges and mean values for blood cell components*

Adults Newborns Toddlers Children

#18 yearsold 1months 2years old 10 yearsold

Leukocytes/µl

or10

/l**

MV 7000 11000 10000 8000

NR 4300–10000

Bandgranulocytes % MV 2 5 3 3

NR 0–5

Segmentedneutrophilic

granulocytes

MV 60 30 30 30

NR 35–85

absolutect./

µl MV 3650 3800 3500 4400

or10

/l** NR 1850–7250

Lymphocytes% MV 30 55 60 40

NR 20–50

absolutect./

µl MV 2500 6000 6300 3100

or10

/l** NR 1500–3500

Monocytes% MV 4 6 5 4

absolutect./

µl NR 2–6

or10

/l** MV 450

NR 70–840

Eosinophilicgranulocytes (%) MV 2 3 2 2

NR 0–4

absolutect./

µl MV 165

or10

/l** NR 0–400

Physiologyand Pathophysiology of Blood Cells

Basophilicgranulocytes (%) MW 0.5 0.5 0.5 0.5

NR 0–1

Male Female

Erythrocytes10

/µl

or10

/l**

MV 5.4 4.8 4.7 4.7 4.8

NR 4.6–5.9 4.2–5.4 3.9–5.9 3.8–5.4 3.8–5.4

Hbg/dl

or10 g/l**

MV 15 13 17 12 14

NR 14–18 12–16 15–18 11–13 12–15

HKT MV 0.45 0.42 44 37 39

NR 0.42–0.48 0.38–0.43

MCH" Hb

(pg) MV 29 33 27 25

NR 26–32

MCV/µm

orfl**

MV 87 91 78 80

NR 77–99

MCHCg/dl

or10 g/l

MV 33 35 33 34

NR 33–36

Erythrocyte,diameter (µm) MV 7.5 8.1 7.3 7.4

Reticulocytes(%) MV 16 24 7.9 7.1 7.6

NR 8–25 8–40

Thrombocytes10

/µl MV 180 155–566 286–509 247–436

NR 140–440

MV" meanvalue, NR " normalrange (range for 95% of the population, reference range), ct. count, ** SI units give the measurements per liter.

* For technical reasons,data may vary considerablybetween laboratories. Itis therefore important also toconsult the referenceranges of the chosenlabora-

tory.

Procedures,Assays, and Normal Values

LeukocyteCount

Leukocytes, unlikeerythrocytes, are completely colorless in their native

state.Another important physicaldifference is the stabilityof leukocytes

in3% acetic acidor saponins;these mediahemolyze erythrocytes(though

not their nucleated precursors). Türk’s solution, used in most counting

methods,employs glacialacetic acidfor hemolysisand crystalviolet (gen-

tianviolet) to lightly stain the leukocytes.A 50-

µlEDTA blood sample is

mixedwith 500

µlTürk’s solution inan Eppendorf tube andincubated at

room temperature for 10 minutes. The suspension isagain mixed and

carefullytransferred to the wellof a prepared counting chamber usinga

pipette or capillary tube. The chamber is allowed to fill from a droplet

placedat one edgeof thewell and placedin a moisture-saturatedincuba-

torfor 10 minutes.With the condenser lowered (or using phase contrast

microscopy),the leukocytesare then counted in atotal offour of thelarge

squaresopposite to each other(1 mm

each).The result is multiplied by

27.5(dilution: 1 +10, volume: 0.4mm

) toyield the leukocyte count per

microliter.Parallel (control)counts show variation ofup to 15%. Thenor-

mal(reference) ranges aregiven in Table

Inan automatedblood cell counter,theer ythrocytesare lysedand cells

witha volumethat exceedsabout 30fl (thresholdvalues varyfor different

instruments) are counted as leukocytes. Any remaining erythroblasts,

hard-to-lyseerythrocytes such as targetcells, giant thrombocytes, orag-

glutinatedthrombocytes are countedalong with the leukocytes,and this

willlead toan overestimateof the leukocytecount. Modernanalyzers can

recognizesuch interference factors and applyinterference algorithms to

obtaina corrected leukocytecount.

Visual leukocyte counts using a counting chambershow a variance of

about 10%; they can be used asa control reference for automatic cell

counts.Rough estimates can also be made by visual assessment of blood

smears:a 40! objectivewill show an averageof two to three cells per field

ofview if the leukocyte count is normal.

Physiologyand Pathophysiology of Blood Cells

Thrombocyte Count

Tocount thrombocytesin acounting chamber,blood mustbe conditioned

with2 %Novocain–Cl solution. Prepreparedcommercial tubes arewidely

used(e.g., Thrombo Plus with2-ml content). EDTA bloodis pipetted into

the tubes, carefullymixed and immediately placed in a counting cham-

ber.The chamberis allowed tostand for 10minutes whilethe cells settle,

after which an area of 1mm

is counted. The result corresponds to the

numberof thrombocytes(the 1+ 100dilution isignored). Inan automated

bloodcell counter,the blood cellsare countedafter they havebeen sorted

bysize. Small cells between 2 and 20fl (thresholds varyfor different in-

struments)are countedas thrombocytes.If giantthrombocytes oraggluti-

natedthrombocytes arepresent, they arenot counted andthe result isan

underestimate. On the other hand, small particles, suchas fragmento-

cytes,or microcytes, will lead to anoverestimate. Modern analyzers can

recognizesuch interference factors and applyinterference algorithms to

obtaina correctedthrombocyte count.If unexpectedresults areproduced,

itis wise tocheck them bydirect reference tothe blood smear.

Unexpected thrombocyte counts should be verified by direct visual

assessment.Using a 100! objective, the field of view normally contains

anaverage of 10thrombocytes. In some instances, “pseudothrombocyto-

penias”are found in automated counts. These are artifacts due to throm-

bocyteaggregation.

Pseudothrombocytopenia (see p. 167) is caused by the aggregation of

thrombocytesin the presenceof EDTA;it does notoccur when heparinor

citrateare used asanticoagulants.

Quantitative Normal Values and Range of

Cellular Blood Components

Determining normal values for blood components is more difficult and

morerisky than onemight expect. Obviously,the values areaffected by a

largenumber of variables, such as age, gender,activity (metabolic load),

circadian rhythm,and nutrition, not to mention the effects of the blood

sampling technique, type andstorage of the blood, and the counting

method.For thisreason, whereavailable, anormal rangeis given,covering

95% of the values found in a clinicallynormal group of probands—from

whichit follows thatone in every20 healthypeople will havevalues out-

sidethe limits ofthis range.Thus, there areareas of overlapbetween nor-

maland pathological data. Data in these borderlineareas must be inter-

preted within a refined reference rangewith data fromprobands who

Procedures,Assays, and Normal Values

Lymphocytes

12 2 4 6 123 5 3 6 9 1 3 5 7 9 11 13

Leukocyte count (10

/l)

Monocytes

Granulocytes

Leukocytes

Hours

Days

Weeks

Months

Years

Fig.3 Mean cell counts at different ages in childhood for leukocytes and their

subfractions(according to Kato)

resembleeach otherand thepatient asclosely as possiblein respectof the

variableslisted above.Due tospace limitations, onlykey agedata arecon-

sideredhere. Figure

3clearly shows that, particularlyfor newborns, tod-

dlers,and young children,particular reference ranges mustbe taken into

account. In addition, the interpretation must also take account of

methodologicalvariation: incell counts, thecoefficient ofvariation (stan-

darddeviation as apercentage of themean value) is usuallyaround 10!

Physiologyand Pathophysiology of Blood Cells

Insum, a healthy distrust for the single data point is the most important

basisfor the interpretation of all data, including those outside the refer-

ence range. For every sample of drawn blood, and every counting

method, at least two or three values should be available before conclu-

sionscan be drawn, unless the clinicalfindings reflect the cytological data.

In addition to this, every laboratory has its own set of reference data to

someextent.

Afterthis account ofthe problems andwide variations betweendifferent

groups,the data inTable

2are presented ina simplified form,with values

roundedup or down forease of comparisonand memorization. Absolute

valuesand the newSI units aregiven where theyare clinically relevant.

The Blood Smearand Its Interpretation

(Differential Blood Count, DBC)

Ablood smear uses capillary or venous EDTA-blood,preferably no more

thanthree hoursold. The slidesmust begrease-free, otherwisecell aggre-

gation andstain precipitation may occur. Unless commercially available

grease-freeslides areused, theslides shouldbe soakedfor severalhours in

asolution of equal parts ofethanol and ether and thenallowed to dry. A

droplet of the blood sample is placed close tothe edge of theslide. A

groundcover glass (spreader slide) is placed infront of the droplet onto

theslide atan angleof about30$. Thecover slideis thenslowly backedinto

theblood droplet.Upon contact, theblood dropletspreads along theedge

ofthe slide(Fig.

4).Without pressure,the coverglass isnow lightly moved

overthe slide.The fasterthe coverglass is moved,and thesteeper angleat

whichit is held,the thinner the smearwill be.

Thequality of the smear technique is crucial for the assessment, because

thecell density at the end of the smear is often twice that at the begin-

ning.

Ina well-prepared smear theblood sample will showa “feathered” edge

where the cover glass left the surface of the slide.The smear must be

thoroughlyair-dried; for goodstaining, at least twohours’ drying time is

needed. The qualityof the preparation will be increased by 10 minutes’

fixation with methanol, and it will then also keepbetter. After drying,

nameand date arepencilled in on theslide.

Procedures,Assays, and Normal Values

Forsafety’s sake, at least one back-up smear should be made from every

samplefrom ever y patient.

Stainingis donewith amixture ofbasic stains(methylene blue,azure) and

acidicstains (eosin),so as toshow complementarysubstances suchas nu-

cleicacids and alkaline granulations.In addition to theseleukocyte com-

ponents, erythrocytes also yield different staining patterns: immature

erythrocytescontain larger residual amounts of RNAand therefore stain

moreheavily withbasophilic stainsthan domature erythrocytes.Pappen-

heim’s panoptic stain contains a balancedmixture ofbasic andacidic

stains: the horizontally stored, air-dried smear is covered with May–

Grünwald staining solution (eosin–methylene blue) for threeminutes,

then about an equal amount ofphosphate buffer,pH =7.3, is carefully

addedand, after afurther three minutes,carefully poured offagain. Next,

theslide iscovered withdiluted Giemsa stain(azure–eosin), whichis pre-

pared byaddition of 1 mlGiemsa stock solution to 1ml neutral distilled

water or phosphate buffer, pH =6.8–7.4. After 15minutes, the Giemsa

stainingsolution is gently rinsed offwith buffer solution and thesmears

areair-dried with thefeathered end slopingupwards.

Theblood smears areinitially viewed with asmaller objective (10!to

20!),which allows the investigator tocheck the cell density andto find

thebest countingarea inthe smear.Experience showsthat thecell projec-

tionis bestabout 1cm fromthe featheredend ofthe smear.At40! magni-

fication,one may expect tosee an averageof two tothree leukocytes per

viewingfield ifthe leukocyte countis normal. It is sometimesuseful to be

able to use this rough estimateto crosscheckimprobable quantitative

values.The detailed analysis ofthe white blood cells isdone using an oil

immersionlens and100! magnification.For this,it is bestto scanthe sec-

Fig.4 Preparationof a blood smear

Physiologyand Pathophysiology of Blood Cells

tionfrom about1 cmto about 3cm fromthe end ofthe smear,moving the

slide to and fro in ameandering movementacross its shortdiameter.

Before(and while)the differential leukocytecount iscarried out, erythro-

cytemorphology andthrombocyte densityshould beassessed. Theresults

ofthe differentialleukocyte count (themorphologies arepresented in the

atlassection, p.30 ff.)may berecorded using manualcounters ormark-up

systems.The more cellsare counted, the morerepresentative the results,

so when pathological deviations are found, it is advisable to count 200

cells.

Tospeed up the staining process, which canseem long and laborious

when arapid diagnosis is required, several quick-stainingsets are avail-

ablecommercially, althoughmost of themdo not permitcomparable fine

analysis.If thestandard stainingsolutions mentioned aboveare tohand, a

quickstain for orientationpurposes can bedone by incubatingthe smear

with May–Grünwald reagent for just one minute and shortening the

Giemsaincubation time to one to twominutes with concentrated “solu-

tion.”

Normalvalues and ranges forthe differential blood countare given in

Table

2,p. 12.

Malariaplasmodia areb estdetermined usinga thick smearin addition

tothe normal blood smear. On a slide,a drop of blood is spread overan

areaof about 2.5cm across.The thick smearis placed inan incubator and

allowedto dryfor atleast 30 minutes.Drying samplesas thicksmears and

thentreating themwith diluteGiemsa stain(as described above)achieves

extensivehemolysis of the erythrocytes and thus an increase in the re-

leasedplasmodia.

Significance of the AutomatedBlood Count

The qualitativeand quantitative blood count techniques described here

mayseem somewhatarchaic giventhe now almostubiquitous automated

cell counters;they are merely intended to show thepossibilities always

readyto be calledon in terms ofindividual analyses carriedout by small,

dedicatedlaboratories.

Theautomated cell count has certainly rationalized blood cell counting.

Dependingon the diagnostic problem and the quality control system of

the individual laboratory, automated counting can even reduce data

rangescompared with “manual” counts.

Afterlysis ofthe erythrocytes, hematologyanalyzers determinethe num-

ber of remainingnucleated cells using a wide range of technologies. All

countersuse cellproperties suchas size,interaction withscatteredlight at

different angles,electrical conductivity, nucleus-to-cytoplasm ratio, and

Procedures,Assays, and Normal Values

the peroxidase reaction, to group individual cell impulses into clusters.

Theseclusters are then quantified andassigned to leukocytepopulations.

Ifonly normalblood cells arepresent, the assignmentof theclusters to

thevarious leukocytepopulations workswell, andthe precision ofthe au-

tomatedcount exceedsthat ofthe manual countof 100cells ina smear by

a factor of 10.If large number of pathological cells are present, such as

blastsor lymphadenoma cells,samples are reliablyrecognized as“patho-

logical,”and asmear canthen be preparedand furtheranalyzed underthe

microscope.

The difficulty arises whensmall populations of pathological cells are

present(e.g., 2% blastspresent afterchemotherapy),or when pathological

cellsare present thatclosely resemblenormal leukocytes (e.g.,small cen-

trocytesin satellitecell lymphoma).These pathologicalconditions are not

alwayspicked upby automatedanalyzers (falsenegative result),no smear

isprepared and studied under the microscope,and the results produced

bythe machinedo not includethe presence ofthese pathological popula-

tions.For this reason,blood samples accompaniedby appropriateclinical

queries(e.g., “lymphadenoma?”“blasts?” “unexplainedanemia?”) should

alwaysbe differentiated andevaluated using amicroscope.

Bone MarrowBiopsy

Occasionally,a disease of theblood cell system cannotbe diagnosed and

classifiedon the basisof theblood count aloneand abone marrow biopsy

isindicated. Insuch casesit ismore importantto performthis biopsycom-

petentlyand produce goodsmears for evaluation than tobe able tointer-

pret the bone marrow cytology yourself.Indications for bone marrow

biopsyare given inTable

In the attempt to avoid complications, the traditional location for bone

marrowbiopsy at the sternum has been abandoned in favor of the supe-

riorpart of the posterior iliac spine (back of the hipbone) (Fig. 5).

Althoughthe bone marrowcytology findings fromthe aspirate are suffi-

cientor even preferablefor mosthematological questions (seeTable

3),it

is regarded as good practice to obtain a sample for

bone marrow his-

tology

atthe same time,since with improvedinstruments the procedure

hasbecome lessstressful, andcomplementary cytologicaland histological

data arethen available from the start. After deeplocal anesthesia of the

dorsalspine and a smallskin incision, a histologycylinder at least 1.5cm

longis obtainedusing a sharphollow needle(Yamshidi). AKlima andRos-

segger cytology needle (Fig.

5) is then placed through the same subcu-

taneouschannel but at a slightly different sitefrom the earlier insertion

pointon the spine and gentlypushed through thecompacta. Themandrel

Physiologyand Pathophysiology of Blood Cells

is pulledout and a 5- to 10-mlsyringe body with 0.5 mlcitrate or EDTA

(heparin is used onlyfor cytogenetics)is attached to the needle. The

patientshould be warned that there willb ea painful drawing sensation

during aspiration, which cannot be avoided.The barrelis thenslowly

pulled,and ifthe procedureis successful,blood fromthe bonemarrowf ills

thesyringe. The syringe bodyis separated from theneedle and the man-

drelreintroduced. The bone marrowaspirate is transferredfrom the syr-

ingeto a Petridish. When the dishis gently shaken,small, pinhead-sized

bonemarrow spiculeswill beseen lyingon thebottom. Asmear, similarto

ablood smear,can beprepared ona slidedirectly fromthe remainingcon-

tentsof thesyringe. Ifthe firstaspirate hasobtained material,the needleis

removedand a lightcompression bandage isapplied.

Ifthe aspiratefor cytologycontains nobone marrowfragments (“punc-

tio sicca,”dry tap), an attempt may be madeto obtain a cytology smear

fromthe (as yet unfixed) histology cylinderby rolling it carefully on the

slide,but this seldom produces optimal results.

The preparation of the precious bone marrow material demands

special care. One ortwo bone marrow spicules are pushed to the outer

edgeof thePetri dish,using the mandrelfrom thesternal needle,a needle,

or awooden rod with a beveled tip, and transferred toa fat-free micro-

scopyslide, onwhich theyaregently pushedto andfro bythe needlealong

the length of the slide ina meanderingline. Thishelps theanalyzing

Fig.5 Bone marrow biopsy from the superior par t of the posterior iliac spine

(backof the hipbone)

Procedures,Assays, and Normal Values

technician to make a differentiatial count. Itshould benoted that too

muchblood inthe bone marrowsample willimpede thesemiquantitative

analysis.In additionto thistype ofsmear, squashpreparations shouldalso

beprepared from the bone marrowmaterial for selective staining.To do

this,a few smallpieces of bonemarrow are placedon a slideand covered

bya second slide. Thetwo slides arelightly pressed and slidagainst each

other,then separated (seeFig.

6).

Thesmears areallowed to air-dryand someare incubatedwith panop-

ticPappenheim stainingsolution (seeprevious text).Smears being sentto

a diagnostic laboratory (wrapped individually and shipped as fragile

goods)are better left unstained.Fresh smears ofperipheral blood should

accompanythe shipmentof eachset ofsamples. (Forprinciples ofanalysis

andnormal values see p.52 ff., forindications for bone marrow cytology

andhistology see p.27f f.)

Fig.6 Squashpreparation and meandering smear for the cytological analysis of

bonemarrow spicules

Physiologyand Pathophysiology of Blood Cells

LymphNode Biopsy and Tumor Biopsy

These procedures, less invasivethan bone marrowbiopsy, are a simple

andoften diagnosticallysufficient methodfor lymphnode enlargementor

other intumescences. The unanesthetized, disinfected skin issterilized

andpulled taut overthe node. A no. 1 needle ona syringe withgood suc-

tionis pushed throughthe skin intothe lymph nodetissue (Fig.

7).Tissue

is aspirated from several locations, changing the angle of the nee dle

slightlyafter each collection, andsuction maintained while the needleis

withdrawnintothe subcutis. Aspirationceases andthe syringeis removed

without suction.The biopsy harvest, which is in the needle, is extruded

ontoa microscopyslide andsmeared outwithout forceor pressureusing a

coverglass (spreader slide). Staining is done as described previouslyfor

bloodsmears.

Procedures,Assays, and Normal Values

Skin puncture

Aspiration

Collecting

aspirates from

different lymph

node locations

Detaching the

syringe body,

equalizing the

pressure difference

Removal of the

syringe body and

cannula

Pulling back the

syringe barrel

Pushing the

biopsy material

onto a slide

Fig.7 Procedurefor

lymphnode biopsy

Physiologyand Pathophysiology of Blood Cells

Step-by-Step Diagnostic Sequence

Onthe basis ofwhat has been saidso far, thefollowing guidelines forthe

diagnosticworkup of hematologicalchanges may beformulated:

1. The firststep isquantitativedetermination ofleukocytes (L),erythrocytes

(E)and thrombocytes (T).Because the normalrange can varyso widely

inindividual cases (Table

2),the following ruleof thumb shouldbe ob-

served:

Acomplete bloodcount (CBC) shouldbe includedin thebaseline data,

likeblood pressure.

2. All quantitative changes in L+ E+ Tcall for a careful evaluation of the

differential blood count (DBC). Since clinical findings determine

whethera DBC isindicated, it may besaid that:

Adifferential blood count isindicated:

➤

Byall unexplained clinicalsymptoms, especially

➤

Enlargedlymph nodes orsplenomegaly

➤

Significantchanges in anyof

– Hemoglobincontentor number of erythrocytes

– Leukocytecount

– Thrombocytecount

Theonly initial assumption hereis that mononuclear cellswith unseg-

mentednuclei can be distinguished from polynuclear cells. Whilethis

nomenclaturemay notconform toideal standards,it iswell established

and, moreover, of such practicalimportance as a fundamental dis-

tinguishing criterion thatit is worth retaining. A marked majority of

mononuclearcells over the polynuclearsegmented granulocytes is an

unambiguousearly finding.

Thenext step has to be takenwith far more care anddiscernment:

classifyingthe mononuclear cells accordingto their possible origin,lym-

phatic cells,monocytes, or various immature blastic elements, which

otherwise only occur in bone marrow. The aim of the images in the

Atlassection ofthis bookis tofacilitate thispart ofthe differential diag-

nosis.To agreat extent,the possibleorigins ofmononuclear cellscan be

distinguished;however,the limits ofmorphology andthe vulnerability

to artifacts are also apparent, leaving the door wide open to further

Step-by-StepDiagnostic Sequence

diagnostic steps (specialist morphological studies, cytochemistry,

immunocytochemistry.A predominanceof mononuclear cellelements

has thesame critical significance in the differential diagnosis of both

leukocytosesand leukopenias.

3. After theevaluation of the leukocytes,assessment of erythrocyte mor-

phologyis a necessary part of everyblood smear evaluation. It is,nat-

urally, particularly important in cases showing disturbances in the

erythrocytecount or thehemoglobin.

4. After careful consideration of the results obtained so far and the

patient'sclinical record, thelast step isthe analysis of thecell composi-

tion of the bone marrow.Quite often,suspected diagnoses are con-

firmedthrough humoraltests such aselectrophoresis, orthrough cyto-

chemical tests such as alkaline phosphatase, myeloperoxidase,non-

specificesterase, esterase, oriron tests.

Bone marrow analysis is indicated whenclinical findings andblood

analysis leave doubts in the diagnostic assessment, for example in

casesof:

➤

Leukocytopenia

➤

Thrombocytopenia

➤

Undefinedanemia

➤

Tricytopenia,or

➤

Monoclonalhypergammaglobulinemia

A bone marrow analysis may beindicated in order to evaluate the

spreading of a lymphadenoma or tumor, unless the bloodstream al-

readyshows the presenceof pathological cells.

5. Bone marrow histologyis also rarely indicated (even morerarely than

thebone marrow cytology). Examplesof the decision-making process

between bone marrow cytologyand histology (biopsy) are shown in

Table

Oftenonly histologicalanalysis canshow structuralchanges or focalin-

filtrationof the bonemarrow.

This is particularly true of the frequently fiber-rich chronic myelo-

proliferativediseases, suchas polycythemia verarubra, myelofibrosis–

osteomyelosclerosis(MF-OMS), essential thrombocythemia(ET), and

chronic myeloid leukemia (CML) as well as malignant lymphoma

without hematological involvement(Hodgkin disease or blastic non-

Hodgkinlymphoma) and tumorinfiltration.

Physiologyand Pathophysiologyof BloodCells

Table3 Indicationsfora differentialblood count(DBC), bonemarrowaspiration, andbiopsy

Indications Procedures

Allclinicallyunclear situations:

➤

Enlargedlymphnodes orspleen

➤

Changesinthe simpleCBC (peniasor

cytoses)

Differentialbloodcount (DBC)

➤

Whenadiagnosis cannotbe madebased on

clinicalfindingsand analysisof peripheral

blooddifferentialblood analysis,cytochem-

istry,phenotyping,molecular geneticsor

FISH

Bonemarrowanalysis

Bonemarrowaspirate

(Morphology,phenotyp-

ing,cytogenetics,FISH,

moleculargenetics)

Bonemarrow

trephinebiopsy

(Morphology,immuno-

histology)

➤

Punctiosicca("dr ytap") not possible +

➤

Aplasticbonemarrow notpossible +

➤

Suspicionofmyelodysplastic syndrome + (+)(e.g.hypoplasia)

➤

Pancytopenia + +

➤

Anemia,isolated + –

➤

Granulocytopenia,isolated + –

➤

Thrombocytopenia,isolated(except ITP) + –

➤

SuspectedITP (+) Fortherapy failure –

➤

SuspectedOMF/OMS –(BCR-ABLin peripheral

bloodissuf ficient)

➤

SuspectedPV –(BCR-ABLin peripheral

bloodissuf ficient)

–

➤

SuspectedET –(BCR-ABL inperipheral

bloodissuf ficient)

➤

SuspectedCML –(BCR-ABLinperipheral

bloodissuf ficient)

(+)Conditionsfor the

analysis

➤

CMPE(BCR-ABLnegative) (+)Differentialdiagnoses +

➤

SuspectedAL/AL + (+)Not intypicalcases

➤

Suspectedbonemarrow metastases – +

➤

Monoclonalhypergammaglobulinemia + +

➤

NHL(exceptions,see below) + +

➤

TypicalCLL + (+)Prognostic factor

➤

Follicularlymphoma (+) Todistinguishvs

otherNHL

➤

Hodgkindisease – +

Furtherindicationsfor abone marrowanalysis are:

➤

Unexplainedhypercalcemia + +

➤

Inexplicableincreaseof boneAP – +

➤

Obvious,unexplainedabnormalities – +

➤

Hyperparathyroidism – +

➤

Pagetdisease – +

➤

Osteomalacia – +

➤

Renalosteopathy – +

➤

Gauchersyndrome – +

+Recommended,– notrecommended, (+)conditionallyrecommended, ITP idiopathicthrombocytopenia,

OMF/OMS osteomyelofibrosis/osteomyelosclerosis,PV polycythemiavera, ET essentialthrombocythemia,

CMPD chronicmyeloproliferativedisorders, AL acuteleukemia,NHL non-Hodgkin lymphoma,CLL chronic

lymphocyticleukemia,FISH fluorescence insitu hybridization,AP alkalinephosphatase.

Step-by-StepDiagnostic Sequence

Normal Cells of the Blood and

Hematopoietic Organs

The Individual Cells of Hematopoiesis

Immature RedCell Precursor s:Proer ythroblasts and

Basophilic Erythroblasts

Proerythroblasts are the earliest, least mature cellsin theerythrocyte-

forming series (erythropoiesis). Proerythroblasts are characterized by

their size (about 20

µm), and by having a very dense nuclear structure

with a narrow layer of cytoplasm, homogeneous in appearance, with a

lighterzone at the center;they stain deep blue afterRomanowsky stain-

ing. These attributes allow proerythroblasts to be distinguished from

myeloblasts(p. 35)and thusto beassigned to theerythrocyte series.After

mitosis, their daughter cells display similarcharacteristics except that

theyhave smallernuclei. Daughtercells are calledbasophilic erythroblasts

(formerlyalso called macroblasts). Theirnuclei are smaller and thechro-

matinis more coarselystructured.

Thematuration of cells in theerythrocyte series isclosely linkedto the

activity of macrophages (transformed monocytes),which phagocytose

nucleiexpelled from normoblasts and iron fromsenescent erythrocytes,

andpass these cellcomponents on to developingerythrocytes.

Diagnostic Implications. Proerythrocytes exist in circulating blood only

under pathological conditions (extramedullary hematopoiesis; break-

downof the blood–bone marrowbarrier by tumor metastases, p. 150; or

erythroleukemia,p. 100).Inthese situations,basophilic erythroblastsmay

also occur; only exceptionally in the course of a strong postanemia re-

generationwill avery few ofthese be releasedinto theblood stream (e.g.,

in thecompensation phase after severe hemorrhage or as a response to

vitamindeficiency, see p.152).

NormalCells ofthe Bloodand Hematopoietic Organs

a b

Normallyer ythropoiesistakes place only in thebone marrow

Fig.8 Early erythropoiesis. aThe earliest recognizable red cellprecursor is the

largedark proerythroblastwith looselyarranged nuclearchromatin (1). Beloware

twoor thochromaticerythroblasts (2), on the righta metamyelocyte (3). b Pro-

erythroblast(1). c Proerythroblast (1) nextto a myeloblast(2) (see p.34); lower

regionof imageshows a promyelocyte(3). Towardthe upperleft area metamye-

locyte(4) and asegmented neutrophilic granulocyte(5).

!Fig.9 Nucleated erythrocyte precursors. a Two basophilic er ythroblasts with

condensedchromatin structure (1) anda polychromatic erythroblast withan al-

mosthomogeneous nucleus(2). bThe erythropoiesisin thebone marrowis often

organized around a macrophage with a very wide, light cytoplasmic layer (1).

Groupedaround it are polychromaticerythroblasts of variable size. Erythroblast

mitosis (2). c Polychromatic erythroblast (1) and orthochromatic erythroblast

(normoblast)(2).

Mature RedBlood Precursor Cells: Polychromatic

and Orthochromatic Erythroblasts (Normoblasts)

and Reticulocytes

Theresults of mitosis of erythroblasts arecalled normoblasts. This name

coverstwo cell typeswith relatively denseround nuclei andgrayish pink

stainedcytoplasm. The immaturecells in which thecytoplasm displays a

grayishblue hue, which are still able todivide, are nowcalled “polychro-

matic erythroblasts,” while the cells in which the cytoplasm is already

takingon apink hue, whichcontain a lotof hemoglobinand are nolonger

ableto divide,are called “orthochromaticerythroblasts.” Thenuclei ofthe

lattergradually condenseinto smallblack sphereswithout structuraldefi-

nition that eventually are expelled from the cells. The now enucleated

youngerythrocytes containcopious ribosomes thatprecipitate into retic-

ular (“net-like”) structures after special staining (see p.11), hence their

name,reticulocytes.

Toavoid confusing erythroblasts andlymphoblasts (Fig.

9d), note the

completely rounded, very dense normoblast nuclei andhomogeneous,

unstructuredcytoplasm of theerythroblasts.

DiagnosticImplications. Polychromaticand orthochromatic erythroblasts

may be released into the bloodstream whenever hematopoiesis is acti-

vated,e.g., in the compensation or treatment stage after hemorrhageor

iron or vitamin deficiency. They are alwayspresent when turnoverof

bloodcells is chronicallyincreased (hemolysis). Onceincreased blood re-

generationhas been excluded,the presence of erythroblastsin the blood

shouldprompt considerationof two otherdisorders: extramedullarypro-

duction of blood cells in myeloproliferative diseases (p. 114),and bone

marrow carcinosis with destruction of the blood–bone marrow barrier

(p.154).In thesame situations,the reticulocytecounts (afterspecial stain-

ing)are elevated abovethe average of25‰ for menand 40‰ for women,

respectively,and canreach extremes ofseveral hundred permill.

NormalCells ofthe Bloodand Hematopoietic Organs

During increased turnover, nucleated red cell precursors may

migrateinto the peripheral blood

Fig.9 d Thedensity of the nuclearchromatin is similar in lymphocytes(1) and

erythroblasts(2), but inthe erythroblastthe cytoplasm iswider and similar in co-

lorto apolychromatic erythrocyte(3). eNormal redblood cellfindings with slight

variancein sizeof theer ythrocytes.A lymphocyte(1) anda fewthrombocytes (2)

areseen. Theerythrocytes areslightly smallerthan thenucleus ofthe lymphocyte

nucleus.

Immature White CellPrecur sors:Myeloblasts and

Promyelocytes

Myeloblastsare theleast maturecells inthe granulocytelineage. Mononu-

clear,round-to-ovoid cells, they may be distinguished from proerythro-

blasts by the finer, “grainy” reticularstructure of their nuclei and the

faintlybasophilic cytoplasm. Onfirst impression,they may looklike large

oreven small lymphocytes(micromyeloblasts), butthe delicate structure

oftheir nucleialways givesthem awayas myeloblasts.In someareas, con-

densed chromatinmay start to look like nucleoli.Sporadically, the cyto-

plasmcontains azurophilic granules.

Promyelocytesare the productof myeloblast division, and usually grow

largerthan theirprogenitor cells.During maturation, theirnuclei showan

increasingly coarse chromatin structure. The nucleus is eccentric; the

lighterzone overits bay-likeindentation correspondsto theGolgi appara-

tus. The wide layer of basophilic cytoplasm contains copious large

azurophilic granules containing peroxidases, hydrolases, and other

enzymes.These granulationsalso existscatteredall aroundthe nucleus,as

maybe seen by focusingon different planes of thepreparation using the

micrometeradjustment on themicroscope.

DiagnosticImplications. Ordinarily, both celltypes are encountered only

inthe bone marrow, where theyare the most actively dividingcells and

mainprogenitors of granulocytes. Intimes of increased granulocyte pro-

duction, promyelocytesand (in rare cases) myeloblasts maybe released

intothe blood stream(pathological left shift,see p. 112).Understrong re-

generation pressure from the erythrocyte series, too—e.g., during the

compensation phase following various anemias—immature white cell

precursors,like the red cellprecursors, may be sweptinto the peripheral

blood.Bone marrow involvementby tumormetastases also increasesthe

permeabilityof the blood–bone marrow barrier for immature white cell

precursors(for an overview,see p.112ff.).

In some acute forms of leukemia, myeloblasts (and also, rarely, pro-

myelocytes)dominate the bloodanalysis (p.97).

NormalCells ofthe Bloodand Hematopoietic Organs

Round cells with “grainy” reticular chromatin structure are

blasts,not lymphocytes

Fig.10 Granulocyteprecursors. a The leastmature precursor in granulopoiesis

isthe myeloblast,which isreleased intothe bloodstream only underpathological

conditions.A largemyeloblast is shownwith afine reticularnuclear structure and

anarrow layerof slightlybasophilic cytoplasmwithout granules.b Myeloblastand

neutrophilic granulocytes with segmented nuclei (blood smear from a patient

with AML). c Myeloblast (1), which shows the start of azurophilic granulation

(arrow),and apromyelocyte (2) withcopious largeazurophilic granules, typically

in a perinuclear location. d Large promyelocyte (1),myelocyte (2), metamyelo-

cyte(3), and polychromaticerythroblast (4).

Partly Mature White CellPrecursor s:Myelocytes and

Metamyelocytes

Myelocytesare the directproduct of promyelocytemitosis andare always

clearly smaller than their progenitors. The ovoid nuclei have a banded

structure;the cytoplasmis becominglighter withmaturation andin some

casesacquiring a pink tinge. A special typeof granules, which no longer

stain red likethe granules in promyelocytes (“specific granules,” perox-

idase-negative),are evenly distributedin the cytoplasm. Myelocytemor-

phology iswide-ranging because myelocytes actually coverthree differ-

entvarieties of dividingcells.

Metamyelocytes(young granulocytes) are theproduct of the final myelo-

cytedivision andshow further maturationof thenucleus withan increas-

ingnumber of stripes andpoints of density thatgive the nucleia spotted

appearance.The nucleislowly takeon akidney beanshape and havesome

plasticity.Metamyelocytes are unable todivide. From this stage on,only

furthermaturation of the nucleus occurs bycontraction, so that the dis-

tinctions (between metamyelocytes, band neutrophils, andsegmented

neutrophils)are merelyconventional, althoughthey do relateto thevary-

ing“maturation” of thesecell forms.

Diagnostic Implications. Like theirprecursors, myelocytes and metamy-

elocytes normally appearin the peripheral blood only during increased

cellproduction in response tostress or triggers,especially infections (for

anoverview of possibletriggers, see p.112).Under these conditions, they

are,however, moreabundant than myeloblastsor promyelocytes.

NormalCells ofthe Bloodand Hematopoietic Organs

Myelocytesand metamyelocytes also occur inthe blood stream

insevere reactive disease

Fig11 Myelocytes and metamyelocytes. a Early myelocyte. The chromatin

structureis denser than that ofpromyelocytes. The granules donot lie over the

nucleus(as canbe seenby turningthe finefocus adjustmentof the microscopeto

andfro). Theblood smearis fromacase ofsepsis, hencethe intensivegranulation.

bSlightly activated myelocyte(the cytoplasmis still relativelybasophilic). c Typi-

calmyelocyte (1) close to asegmented neutrophil (2). d Thismetamyelocyte is

distinguishedfrom a myelocyteby incipient lobeformation.

Mature Neutrophils: BandCells and Segmented

Neutrophils

Band cells (band neutrophils) represent the further development of

metamyelocytes.Distinguishing between the differentcell types is often

difficult.The term“band cell” shouldbe used whenall nuclearsections of

thenucleus are approximately thesame width (the “bands”). Thebegin-

ningsof segmentation may be visible,but the indentations should never

cutmore than two-thirdsof the wayacross the nucleus.

Segmentedneutrophils representthe finalstage in thelineage thatstarted

with myeloblasts, forming gradually, without any clear transition or

furthercell divisions,by increasing contractionof their nuclei.Finally, the

nuclearsegments areconnected onlyby narrowchromatin bridges,which

should be no thicker than one-third of the averagediameter of the nu-

cleus.The chromatin ineach segment formscoarse bands,or patches and

isdenser than the chromatinin band neutrophils.

The cytoplasm of segmentedneutrophilic granulocytes varies after

stainingfrom nearlycolorless tosoft pinkor violet.The abundantgranules

areoften barely visibledots.

Thenumber ofsegments increaseswith the age of thecells. Thefollow-

ing approximate values aretaken to represent a normal distribution:

10–30% havetwo segments, 40–50% have three segments, 10–20% have

foursegments, and 0–5% of the nuclei have fivesegments. A left shift to

smallernumbers of segmentsis a discreetsymptom ofreactive activation

of this cell series. Aright shift to higher numbers of segments (over-

segmentation) usually accompanies vitamin B

and folic acid deficien-

cies.

DiagnosticImplications. Banded neutrophilic granulocytes(band neutro-

phils)may occurin smallnumbers (upto 2%)in anormal bloodcount. This

isof no diagnosticsignificance. A higherproportion than2 %may indicate

aleft shift andconstitute the firstsign of areactive condition(p. 113).The

diagnostic value of segmented neutrophilic granulocytes (segmented

neutrophils) is that normal values are the most sensitivediagnostic in-

dicatorof normally functioninghematopoiesis (and,especially, of normal

cellular defenseagainst bacteria). An increase in segmented neutrophils

withouta qualitativeleft shiftis notevidence ofan alterationin bonemar-

row function, because under certain conditions stored cells may be re-

leased into the peripheral blood (for causes, see p. 111).In conjunction

with qualitative changes (left shif t, toxic granulations), however,

granulocytosisdoes infact indicatebone marrowactivationthat mayhave

a variety of triggers (pp.110f.), and if the absolutenumber hasfallen

belowthe lower limitof the normalrange (Table

2,p. 12),a bone marrow

defector increased celldeath must be considered.

NormalCells ofthe Bloodand Hematopoietic Organs

Advancing nuclear contraction and segmentation:continuous

transformationfrom metamyelocyte to band cell andthen seg-

mentedneutrophilic granulocyte

Fig.12 Neutrophils (neutrophilic granulocytes). a Transitional form betweena

metamyelocyteand aband cell.b Copiousgranulation ina bandcell (1)(toxicgra-

nulation) next to band cells (2) with Döhle bodies (arrows). c Two band cells.

d Band cells can also occur as aggregates. e Segmented neutrophilic granulo-

cytes. f Segmented neutrophilic granulocyte after the peroxidase reaction.

gSegmented neutrophilicgranulocyte afteralkaline leukocytephosphatase (ALP)

staining.

Cell Degradation, Special Granulations, and Nuclear

Appendages in NeutrophilicGranulocytes and Nuclear

Anomalies

Toxic granulation is the term used when the normally faint stippled

granulesin segmentedneutrophils stainan intensereddish violet, usually

againsta background of slightlybasophilic cytoplasm; unlikethe normal

granules,they stain particularlywell inan acidic pH(5.4). This phenome-

non is a consequence of activity against bacteria or proteins and is ob-

served in serious infections, toxic or drug effects, or autoimmune

processes (e.g., chronic polyarthritis). At the same time, cytoplasmic

vacuolesare often found, representingthe end stage ofphagocytosis (es-

peciallyin cases ofsepsis), as areDöhle bodies: smallround bodies ofba-

sophiliccytoplasm that have been describedparticularly in scarlet fever,

butmay be present in all serious infectionsand toxic conditions. A defi-

ciency or complete absence of granulation in neutrophils is asign of

severedisturbance of the maturation process (e.g., in myelodysplasiaor

acute leukemia). The Pelger anomaly, named after its first describer,is a

hereditary segmentation anomalyof granulocytes that results in round,

rod-shaped,or bisegmentednuclei. The sameappearance asa nonheredi-

tarycondition (pseudo-Pelger formation,also called Pel–Ebsteinfever, or

[cyclic]Murchison syndrome) indicatesa severe infectiousor toxic stress

response or incipient myelodysplasia; it also may accompany manifest

leukemia.

NormalCells ofthe Bloodand Hematopoietic Organs

c d

Note the granulations, inclusions, and appendages in segmen-

tedneutrophilic granulocytes

Fig.13 Variations ofsegmented neutrophilic granulocytes.a Reactivestate with

toxicgranulation of the neutrophilicgranulocytes, more visibly expressedin the

cellon thelef t(1) thanthe cell onthe right (2)(compare withnonactivated cells,

p.39). b Sepsis with toxic granulation,cytoplasmic vacuoles, and Döhle bodies

(arrows) inband cells (1) and a monocyte (2). c Pseudo-Pelgercell looking like

sunglasses(toxic or myelodysplasticcause). d Döhle-likebasophilic inclusion(ar-

row)without toxic granulation. Togetherwith giant thrombocytes thissuggests

May–Hegglinanomaly. continued !

Nuclearappendages, which must not to be mistakenfor small segments,

areminute (lessthan the sizeof athrombocyte) chromatinbodies thatre-

mainconnected to themain partof the nucleusvia a thinbridge and con-

sequentlylook like a drumstick,sessile nodule, or small tennis racket. Of

these,only the drumstickform correspondsto the X-chromosome,which

hasbecome sequesteredduring theprocess ofsegmentation. Aproportion

of 1–5% circulating granulocyteswith drumsticks (at least 6 out of 500)

suggestsfemale gender; however,b ecausethe drumstick form is easyto

confuse with the other (insignificant) formsof nuclear appendage, care

shouldbe taken beforejumping to conclusions.

Rarely, degrading forms of granulocytes, shortly before cytolysis or

apoptosis,may befound inthe blood(they aremore frequentinexudates).

Inthese, thesegments ofthe nucleusare clearlylosing connection, andthe

chromatin structure of the individual segments, which are becoming

round,becomes dense andhomogeneous.

DiagnosticImplications. Toxicgranulation indicatesbacterial, chemical,or

metabolicstress. Pseudo-Pelger granulocytes are observedin cases of in-

fectious–toxicstress conditions, myelodysplasia,and leukemia.

The use of nuclear appendages to determine gender has lost signifi-

cancein favor ofgenetic testing.

NormalCells ofthe Bloodand Hematopoietic Organs

g h

Note the granulations, inclusions, and appendages in segmen-

tedneutrophilic granulocytes

Fig.13 continued. e Hypersegmented neutrophilic granulocyte (six or more

segments). There is an accumulation of these cells in megaloblasticanemia. f

Drumstick(arrow 1) as anappendage with a thinfilament bridge to thenucleus

(associated with the X-chromosome), adjoined by a thrombocyte (arrow 2). g

Very largegranulocyte from a blood sample taken after chemotherapy. hSeg-

mentedneutrophilic granulocyteduring degradation,often seen asan artifactaf-

terprolonged sample storage(more than eighthours).

Eosinophilic Granulocytes(Eosinophils)

Eosinophilsarise from the same stemcell population as neutrophils and

maturein parallel withthem. The earliest pointat which eosinophilscan

be morphologically defined in the bone marrow is at the promyelocyte

stage.Promyelocytes containlarge granules thatstain blue–red; notuntil

theyreach the metamyelocytestage dothese become adense population

of increasingly round, golden-red granules filling the cytoplasm. The

Charcot–Leydencrystals foundbetween groupsof eosinophilsin exudates

and secretions have the same chemicalcomposition as theeosinophil

granules.

Thenuclei of matureeosinophils usually haveonly two segments.

Diagnostic Implications. In line with theirfunction (seep. 5) (reaction

against parasites and regulation of the immune response, especially

defenseagainst foreign proteins), an increaseof eosinophils above400/

µl

should be seen as indicating the presenceof parasitosis, allergies,and

manyother conditions (p.124).

Basophilic Granulocytes(Basophils)

Like eosinophils, basophils (basophilic granulocytes) mature in parallel

withcells ofthe neutrophillineage. Theearliest stageat whichthey canbe

identified isthe promyelocyte stage,at which large, black–violet stained

granulesare visible.In mature basophils,which arerelatively small,these

granulesof tenoverlie the two compact nuclear segments likeblackber-

ries. However,they easily dissolve in water, leaving behind faintly pink

stainedvacuoles.

Closerelations ofbasophilic granulocytes aretissue basophils ortissue

mast cells—but these are never foundin blood. Tissuebasophils have a

roundnucleus underneath largebasophilic granules.

Diagnostic Implications. Inline with their role in anaphylactic reactions

(p.5), elevatedbasophil countsare seenabove allin hypersensitivityreac-

tions of various kinds. Basophils are also increased in chronicmyelo-

proliferativebone marrow diseases,especially chronic myeloidleukemia

(pp.117,120).

NormalCells ofthe Bloodand Hematopoietic Organs

Roundgranules fillingthe cytoplasm: eosinophilicand basophilic

granulocytes

Fig.14 Eosinophilic and basophilic granulocytes.a–c Eosinophilic granulocytes

withcorpuscular, orange-stained granules.d In contrast,the granules ofneutro-

philicgranulocytes arenot roundbut morebud-shaped. eBasophilic granulocyte.

Thegranules are corpuscular likethose of theeosinophilic granulocyte but stain

deepblue toviolet. f Veryprominent largegranules ina basophilic granulocytein

chronicmyeloproliferative disease.

Monocytes

Monocytesare produced in the bone marrow;their line of development

branchesoff at avery early stagefrom that ofthe granulocytic series(see

flowchart p.

3),but does notcontain anydistinct, specific precursorsthat

canb esecurely identified with diagnostic significance ineveryday mor-

phological studies. Owing totheir great motility and adhesiveness, ma-

turemonocytes are morphologically probably the most diversifiedof all

cells.Measuring anywhere between20 and 40

µmin size, their constant

characteristicis anovoid nucleus,usually irregularin outline,with invagi-

nations and often pseudopodia-like cytoplasmic processes. The fine,

“busy” structure of their nuclearchromatin allowsthem tobe distin-

guished frommyelocytes, whose chromatin has a patchy, streakystruc-

ture,and alsofrom lymphocytes, whichhave dense,homogeneous nuclei.

The basophilic cytoplasmic layer varies in width, stains a grayish color,

andcontains a scatteredpopulation of veryfine reddish granulesthat are

atthe very limitof the eye’sresolution. These characteristicsvary greatly

withthe sizeof themonocyte, whichin turnis dependenton thethickness

of the smear. Where the smear is thin, especially at the feathered end,

monocytesare abundant,relatively largeand looselystructured, andtheir

cytoplasmstains lightgray–blue (“dovegray”). In thick,dense partsof the

smear,some monocytes look more like lymphocytes: only a certain nu-

clearindentation and the “thundercloud”gray–blue staining of thecyto-

plasmmay still markthem out.

DiagnosticImplications. Inline withtheir function (seep. 5)as phagocytic

defense cells, an elevation of the monocyte population above 7% and

above850/

µl indicatesan immune defense reaction; only when a sharp

rise inmonocyte counts is accompanied by a dropin absolute counts in

theother cellseries is monocyticleukemia suggested (p.101).Phagocyto-

sisof erythrocytes and white blood cells(hemophagocytosis) may occur

insome virus infectionsand autoimmune diseases.

➤

Monocytosisin casesof infection: alwayspresent atthe endof acutein-

fections;chronic especially in

– Endocarditislenta, listeriosis, brucellosis,tuberculosis

➤

Monocytosisin cases ofa non-infectious response, e.g.,

– Collagenosis,Crohn disease, ulcerativecolitis

➤

Monocytosesin/as neoplasia, e.g.,

– Paraneoplastic in cases of disseminating tumors, bronchial carci-

noma, breast carcinoma, Hodgkin disease, myelodysplasias (es-

peciallyCMML, pp. 107 f)and acute monocyticleukemia (p. 101)

NormalCells ofthe Bloodand Hematopoietic Organs

g h

Monocytes show the greatest morphological variation among

bloodcells

Fig.15 Monocytes.a–c Range ofappearances oftypical monocytes withlobed,

nucleus,gray–blue stained cytoplasm andfine granulation. d Phagocyticmono-

cytewith plasmavacuoles. e Monocyte(1) tothe right ofa lymphocyte withazu-

rophilicgranules (2). fMonocyte (1)with nucleus resemblingthat of aband neu-

trophil,but its cytoplasm stainstypically gray–blue. Lymphocyte(2). g A mono-

cytethat has phagocytosedtwo erythrocytes and harborsthem in itswide cyto-

plasm (arrows) (sample taken after bone marrow transplantation). hEsterase

staining,a typical markerenzyme for cellsof the monocytelineage.

Lymphocytes(and Plasma Cells)

Lymphocytesare produced everywhere,particularly in the lymphnodes,

spleen,bone marrow, andthe lymphatic islandsof the intestinalmucosa,

under the influence of the thymus (T-lymphocytes, about 80 %), or the

bonemarrow (B-lymphocytes,about20 %).A smallfraction ofthe lympho-

cytesare NK cells (natural killer cells). Immatureprecursor cells (lymph

nodecytology, p.177)are practicallynever released intothe bloodand are

thereforeof no practicaldiagnostic significance. The cellsencountered in

circulatingblood are mostly “small”lymphocytes with ovalor round nu-

clei6–9

µmin diameter. Their chromatinmay be described asdense and

coarse. Detailed analysis under themicroscope, usingthe micrometer

screwto viewthe chromatinin different planes,reveals notthe patch-like

or bandedstructure of myeloblast chromatin, or the “busy”structure of

monocytechromatin, but slate-like formationswith homogeneous chro-

matin and intermittent narrow, lighter layers that resemble geological

break lines. Nucleoliare rarely seen. The cytoplasm wraps quite closely

aroundthe nucleusand is slightlybasophilic. Onlya fewlymphocytes dis-

play theviolet stained stippling of granules; about 5% of small lympho-

cytes and about3 % of large ones. The familyof large lymphocytes with

granulationconsists mostly of NK cells. Animportant point is that small

lymphocytes—whichcannot be identified as T- orB-lymphocytes on the

basisof morphology—are notfunctional endforms, but undergotransfor-

mationin responseto specificimmunological stimuli. Thefinal stageof B-

lymphocyte maturation (in bone marrow and lymph nodes)is plasma

cells, whose nuclei often show radial bars, and whose basophilic cyto-

plasmlayer is alwayswide. Intermediate forms(“plasmacytoid” lympho-

cytes)also exist.

DiagnosticImplications. Valuesbetween 1500and 4000/

µland about35 %

reflectnormal outputof thelymphatic system.Elevated absolutelympho-

cytecounts, often along withcell transformation, are observedpredomi-

nantlyin viralinfections (pp.67,69) orin diseasesof the lymphaticsystem

(p.75ff.). Relative increases atthe expense of otherblood cell series may

be a manifestation of toxicor aplastic processes (agranulocytosis, p. 87;

aplasticanemia, p. 148), becausethese irregularities are rare inthe lym-

phatic series.A spontaneous decrease in lymphocyte counts is normally

seenonly in veryrare congenital diseases (agammaglobulinemia[Bruton

disease], DiGeorge disease [chromosome 22q11 deletion syndrome]).

Somesystemic diseasesalso lead tolow lymphocytecounts (Hodgkindis-

ease,active AIDS).

Matureplasma cells arerarely found in blood(plasma cell leukemiais

extremelyrare). Plasma-cell-like (“plasmacytoid”) lymphocytes occur in

viralinfections or systemicdiseases (see p.68 f.and p. 74f.).

NormalCells ofthe Bloodand Hematopoietic Organs

Lymphocytesare small round cells with dense nuclei and some

variationin their appearance

Fig.16 Lymphocytesa–c Rangeof appearanceof normallymphocytes (someof

them adjacent to segmented neutrophilic granulocytes). d In neonates, some

lymphocytesfrom aneonate showirregularly shaped nucleiwith notchesor hints

ofsegmentation. eA fewlarger lymphocyteswith granulesmay occur ina normal

person.f Occasionally, andwithout any recognizabletrigger, the cytoplasmmay

widen.g A smear takenafter infection maycontain a fewplasma cells, the final,

morphologicallyfully developed cellsin theB-lymphocyte series (forfurther acti-

vatedlymphocyte forms, seep. 67).

Megakaryocytes and Thrombocytes

Megakaryocytes can enter the bloodstream only inhighly pathological

myeloproliferative disease or acute leukemia. They are shown here in

order to demonstrate thrombocyte differentiation. Megakaryocytes re-

sidein the bone marrowand have giant,extremely hyperploid nuclei(16

timesthe normalnumber ofchromosome setson average),whichbuild up

byendomitosis. Humoralfactors regulate theincrease of megakaryocytes

andthe release ofthrombocytes when moreare needed (e.g.,bleeding or

increasedthrombocyte degradation).Cytoplasm with granulesis pinched

offfrom megakaryocytesto formthrombocytes.The residualnaked mega-

karyocytenuclei are phagocytosed.

Only mature thrombocytes occur in blood. About 1–4

µm in size and

anuclear,their light bluestained cytoplasm andits processes givethem a

star-like appearance, with fine reddish blue granules near the center.

Youngthrombocytes are larger and more “spread out;” older ones look

likepyknotic dots.

DiagnosticImplications. Ina blood smear,there arenormally 8–15throm-

bocytesper viewfield using a100! objective;they mayappear dispersed

orin groups. Tosomeone quicklyscreening a smear,they will givea good

indication ofany increase or strong decrease inthe count, which can be

usefulfor early diagnosisof acute thrombocytopenias(p. 164f.).

Small megakaryocyte nuclei are found in the bloodstream only in

severemyeloproliferative disorders(p. 171).

NormalCells ofthe Bloodand Hematopoietic Organs

c d

Megakaryocytes are never present in a normal peripheral blood

smear.Thrombocytes are seen in everyfield view

Fig.17 Megakar yocytesandthrombocytes. aMegakaryocytes ina bonemarrow

smear.The widecytoplasm displays fine,cloudy granulationas a signof incipient

thrombocyte budding. b Normaldensity of thrombocytes among the erythro-

cytes,with little variation in thrombocytesize. c and d Peripheral bloodsmears

with aggregations ofthrombocytes. When such aggregates are seen against a

backgroundof apparent thrombocytopenia,the phenomenon iscalled “pseudo-

thrombocytopenia”and is usually an effect ofthe anticoagulant EDTA (seealso

p.167).

Bone Marrow: Cell Composition

and Principles of Analysis

Asindicated above,and aswill beshown below,almost alldisorders ofthe

hematopoietic system can b e diagnosed using clinical findings, blood

analysis,and humoraldata. There isno mysteryabout bone marrowdiag-

nostics.The basic categoriesare summarized hereto give anunderstand-

ingof howspecific diagnostic informationis achieved;photomicrographs

will show the appearance of specific diseases. Once the individualcell

types,as givenin thepreceding pages,are recognized,it becomes possible

to interpret the bone marrow smears that accompany the variousdis-

eases,and to follow further,analogous diagnostic steps. As afirst step in

theanalysis ofa bonemarrow tissuesmear or squashpreparation, various

areasin several preparationsare broadlysurveyed. This isfollowed byin-

dividualanalysis ofat least 200 cellsfrom tworepresentative areas. Table

4shows the meannormal values andtheir wide ranges.

Acombination ofestimation andquantitative analysisis used,based on

thefollowing criteria:

Cell Density. This parameter isvery susceptible to artifacts. Figure

showsroughly the normal cell density.A lower count may bedue to the

mannerin whichthe samplewasobtained orto thesmearing procedure.A

bonemarrow smeartypically showsareas whereconnective tissueadipo-

cyteswith large vacuolespredominate. Only ifthese adipocytes areasare

presentis itsafe toassume that thesmear containsbone marrowmaterial

andthat an apparentdeficit of bone marrowcells is real.

Increasedcell density: e.g., in all strong regenerationor compensation

processes, and in cases of leukemiaand myeloproliferative syndromes

(exceptosteomyelosclerosis).

Decreasedcell density: e.g.,in aplastic processesand myelofibrosis.

NormalCells ofthe Bloodand Hematopoietic Organs

Table4 Cell composition inthe bonemarrow: normalvalues (%)

Median

values

(J.Boll)

Medianvalues andnormal

range

(K.Rohr)

Redcell series

– Proerythrocytes 1

– Macroblasts(basophilic

erythroblasts)

3 3.5 0.5–7.5

– Normoblasts(poly- and

orthochromicerythro-

blasts)

16 19 (7–40)

Neutrophilseries

– Myeloblasts 2.7 1 (0.5–5)

– Promyelocytes 9.5 3 (0–7.5)

– Myelocytes 14 15 (5–25)

– Metamyelocytes 10.5 15 (5–20)

– Bandneutrophils 9.8 15 (5–25)

– Segmentedneutrophils 17.5 7 (0.5–15)

Smallcell series

– Eosinophilicgranulocytes 5 3 (1–7)

– Basophilicgranulocytes 1 0.5 (0–1)

– Monocytes 2 2 (0.5–3)

– Lymphocytes 6 7.5 (2.5–15)

– Plasmacells 1.5 1 (0.5–3)

Megakaryocytes

Celldensities varywidely, 0.5–2per viewfield during screeningat lowmagni-

fication.

Ratios of RedCell Series to White Cell Series. In the final analysis, bone

marrowcytology allows aquantitative assessment onlyin relative terms.

Theimportant ratio ofred precursor cellsto white cellsis 1: 2 formen

and1 :3 for women.

Shiftstowards erythropoiesisare seen inall regenerativeanemias (hemor-

rhagic anemia, iron deficiency anemia, vitamin deficiency anemia, and

hemolysis), pseudopolycythemia (Gaisböck syndrome), and poly-

cythemia,also in rare pseudo-regenerative disorders,such as sideroach-

resticanemia and myelodysplasias. Shifts towardgranulopoiesis are seen

inall reactive processes (infections, tumordefense) and in all malignant

processes of the white cell series (chronic myeloid leukemia, acute

leukoses).

BoneMarrow: CellComposition andPrinciples of Analysis

Distributionand CellQuality inErythropoiesis. Inerythropoiesis polychro-

matic erythroblasts normally predominate. Proerythroblasts and ba-

sophilicerythroblasts only makeup a smallportion (Table

4).Here, too, a

leftshift indicates an increasein immature cell typesand a right shift an

increase in orthochromatic erythroblasts. Qualitatively,vitamin B

and

folicacid deficiencylead toa typical loosening-upof thenuclear structure

in proerythroblasts and tonuclear segmentation and break-up in the

erythroblasts(megaloblastic erythropoiesis).

A leftshif tis seen in regenerative anemias excepthemolysis. Atypical

proerythroblastspredominate in megaloblasticanemia and erythremia.

Aright shift isseen in hemolyticconditions (nests ofnormoblasts, ery-

throns).

Distributionand CellQuality in Granulopoiesis.The sameprinciple is valid

asfor erythropoiesis:the more maturethe cells,the greaterproportion of

theseries they make up. Aleft shift indicates a greaterthan normal pro-

portionof immature cells anda right shift agreater than normal propor-

tionof maturecells (Table

4).Strong reactiveconditions maylead todisso-

ciationsin thematuration process,e.g., thenucleus showsthe structureof

amyelocyte while thecytoplasm is stillstrongly basophilic. In malignan-

cies, the pictureis dominated by blasts, which may often be difficult to

identifywith any certainty.

Aleft shift is observed inall reactive processes andat the start ofneo-

plastic transformation (smoldering anemia, refractory anemia with

excessblasts [RAEB]). In acute leukemias, undifferentiated and partially

maturedblasts may predominate. In agranulocytosis, promyelocytesare

mostabundant.

Aright shift is diagnosticallyirrelevant.

Cytochemistry. To distinguish between reactive processesand chronic

myeloidleukemia, leukocyte alkalinephosphatase is determinedin fresh

smearsof blood.To distinguishbetween differenttypes ofacute leukemia,

theperoxidase and esterasereactions are carriedout (pp. 97and 99),and

ironstaining is performed(p. 109)if myelodysplasia issuspected.

Cytogenetic Analysis. This procedure will take the diagnosis forwardin

casesof leukemia andsome lymphadenomas. Thefresh material mustbe

heparinizedbefore shipment, preferablyafter discussionwith a specialist

laboratory.

NormalCells ofthe Bloodand Hematopoietic Organs

The bone marrow contains a mixture of all thehematopoietic

cells

Fig.18 Bonemarrow cytology.a Bonemarrow cytologyof normalcell densityin

ayoung adult(smear from abone marrowspicule shownat the lowerright; mag-

nification!100). b Moreadipocytes with largevacuoles are present inthis bone

marrow preparation withnormal hematopoietic cell densities; usually found in

olderpatients. c Normal bonemarrow cytology (magnification!400). Even this

overviewshows clearly thater ythropoiesis(dense, black, roundnuclei) accounts

foronly about one-thirdof all thecells.

Qualitativeand QuantitativeAssessment of theRemaining Cells.Lympho-

cytecounts may be slightlyraised in reactive processes,but a significant

increasesuggests a disease ofthe lymphatic system.The exact classifica-

tion of these disease follows the criteria of lymphocyte morphology

(Fig.

16).If elevatedlymphocyte counts arefound only inone preparation

orwithin a circumscribed area, physiologicallymph follicles in the bone

marroware likely tobe the source.In a borderlinecase, the histologyand

analysisof lymphocyte surfacemarkers yield moredefinitive data.

Plasma cellcounts are also slightly elevated in reactiveprocesses and

very elevated in plasmacytoma. Reactive increaseof lymphocytesand

plasmacells with concomitant low counts inthe other series is often an

indicationof panmyelopathy (aplasticanemia).

Raisedeosinophil and monocytecounts in bone marrowhave the same

diagnosticsignificance as in blood(p. 44).

Megakaryocytecounts arereduced under theef fectsof alltoxic stimuli

onbone marrow. Countsincrease after bleeding, inessential thrombocy-

topenia(Werlhof syndrome), and inmyeloproliferative diseases (chronic

myeloidleukemia, polycythemia,and essential thrombocythemia).

Iron Stainingof Erythropoietic Cells. Perls’ Prussian blue (also known as

Perls’acid ferrocyanidereaction) showsthe presenceof ferritinin 20–40%

of all normoblasts, in the form of one to four small granules. The iron-

containing cells are called sideroblasts. Greater numbers of ferritin

granules in normoblasts indicate a disorder of iron utilization (side-

roachresia,especially in myelodysplasia), particularly when the granules

forma ring aroundthe nucleus(ring sideroblasts). Perls’Prussian bluere-

actionalso stains thediffuse iron precipitates inmacrophages (Fig.

19b).

Underexogenous iron deficiency conditions theproportion of sidero-

blasts andiron-storing macrophages is reduced. However, if theshif tin

ironutilization is due to infectiousand/or toxic conditions, theiron con-

tent in normoblasts is reduced while the macrophagesare loaded with

ironto thepoint ofsaturation. Inhemolytic conditions,the ironcontent of

normoblastsis normal; itis elevated onlyin essential orsymptomatic re-

fractoryanemia (including megaloblasticanemia).

NormalCells ofthe Bloodand Hematopoietic Organs

c d

Notjust the individual cell,but its relative proportion isrelevant

inbone marrow diagnostics

Fig.19 Normalbone marrow findings.a Normal bone marrow:megakaryocyte

(1),erythroblasts (2),and myelocyte(3). b Ironstaining inthe bonemarrow cyto-

logy:iron-storing macrophage.c Normalbone marrowwith slightpreponderance

ofgranulocytopoiesis, e.g.,promyelocyte (1), myelocyte(2), metamyelocyte(3),

andband granulocyte (4). dNormal bone marrow withslight preponderance of

erythropoiesis,e.g., basophilic erythroblast (1),polychromatic erythroblasts (2),

and orthochromatic erythroblast (3). Compare(dif ferentialdiagnosis) with the

plasmacell (4) withits eccentric nucleus.

Bone Marrow: Medullary Stroma Cells

➤

Fibroblastic reticularcells form a firm but elastic matrix in which the

blood-formingcells reside, and are thereforerarely found in the bone

marrow aspirate or cytological smear. When present, they are most

likely to appear as densecell groups with long fiber-forming cyto-

plasmic processes and small nuclei. Iron staining showsthem up as a

groupof reticular cells which,like macrophages, have thepotential to

store iron.If they become the prominent cell population in the bone

marrow,an aplastic ortoxic medullary disordermust be considered.

➤

Reticular histiocytes (not yet active in phagocytosis)are identical to

phagocytic macrophages and are the main storagecells for tissue-

boundiron. Because of theirsmall nuclei and easy-flowingcytoplasm,

theyare noticeable afterpanoptic staining onlywhen they containob-

viousentities such aslipids or pigments.

➤

Osteoblastsare large cellswith wide, eccentric nuclei.They differ from

plasmacells inthat thecytoplasm has noperinuclear lighterspace (cell

center)and stains a cloudy grayish-blue. As theyare normally rare in

bonemarrow, increasedpresence ofosteoblasts in themarrow mayin-

dicatemetastasizing tumor cells(from another location).

➤

Osteoclasts are multinucleated syncytia with wide layers of grayish-

blue stained cytoplasm, which often displays delicate azurophilic

granulation.They are normally extremely rarein aspirates, and when

theyare found it is usually under the same conditionsas osteoblasts.

Theyare distinguished frommegakaryocytes by theirround and regu-

larnuclei and bytheir lack of thrombocytebuds.

Fromthe above,it willbe seen thatbone marrowfindings can beassessed

on the basis of a knowledge of the cells elements described above

(p.32ff.), takingaccount of theeight categories given on p. 52f. It also

showsthat a diagnosis from bone marrow aspirates can safely be made

onlyin conjunction withclinical findings, bloodchemistry, andthe quali-

tativeand quantitative bloodvalues. For thisreason, a table ofdiagnostic

steps takingaccount of these categories is provided at the beginning of

eachof the followingchapters.

NormalCells ofthe Bloodand Hematopoietic Organs

Cells from the bone marrowstroma neveroccur in theblood

stream

Fig.20 Bone marrow stroma.a Spindle-shaped fibroblasts form the structural

frameworkof the bone marrow (shownhere: aplastic hematopoiesis after ther-

apyfor multiple myeloma). bA macrophage has phagocytosed residualnuclear

material (here after chemotherapy foracute leukemia). c Bone marrow osteo-

blastsare rarelyfound inthe cytologicalassessment. Thefeatures thatdistinguish

osteoblasts from plasma cellsare their more loosely structured nuclei and the

cloudy,“busy” basophiliccytoplasm. d Osteoclastsare multinucleatedgiant cells

withwide, spreading cytoplasm.

Abnormalities of the White Cell Series

A veryold-fashioned, intuitive way of classifying CBCsis to divide them

into thosein which round to oval (mononuclear) cells predominateand

thosein whichsegmented (polynuclear) cellspredominate. However,this

old methoddoes allow the relevant differential diagnosis to be inferred

froma cursory screeningof a slide.

DifferentialDiagnostic Notes

Differentialdiagnosis when roundto oval cellspredominate

➤

Reactivelymphocytoses and monocytoses,pp. 67f., 89

➤

Lymphaticsystem diseases (lymphomas, lymphocytic leukemia),

p.70

➤

Acuteleukemias, including episodes ofchronic myeloid leukemia

(CML),p. 96ff.

➤

Low counts of cells with segmented nuclei (agranulocytosis–

aplasticanemia–myelodysplasia), pp.86, 106, 148

Differentialdiagnosis when segmentedcells predominate

➤

Reactiveprocesses, p.112ff.

➤

Chronicmyeloid leukemia, p.114ff.

➤

Osteomyelosclerosis,p. 122

➤

Polycythemiavera, p.162 ff.

Abnormalitiesof theWhite CellSeries

Predominance of Mononuclear Round

to Oval Cells

(Table5)

Thecell counts (per microliter) showa wide range ofvalues and depend

ona varietyof conditionseven innormal blood.Any diseasemay therefore

result in higher (leukocytosis) or lower cell counts (leukopenia). The

assessment of cell counts requiresthe knowledge of normal values and

theirranges (spreads) (Table

2,p. 12). Themost important diagnostic in-

dicator therefore is not the cell count but the cell type.In a first assess-

ment,mononuclear (roundto oval)and polynuclear (segmented)cells are

compared.This is the first stepin the differential diagnosis of themulti-

facetedspectrum of bloodcells.

Absoluteor relativepredominance ofmononuclear cells pointsto ade-

finedset of diagnostic probabilities. Thedifferential diagnostic notes op-

positecan help witha first orientation.

Consequently,the most importantstep is todistinguish between lym-

phaticcells andmyeloid blasts.The nuclearmorphology makes itpossible

todistinguish between the two celltypes. In lymphatic cellsthe nucleus

usuallydisplays dense coarse chromatin withslate-like architecture and

lighterzones between verydense ones. Incontrast, the immaturecells in

myeloidleukemias contain nuclei with a more delicate reticular, some-

timessand-like chromatin structurewith a finer,more irregular pattern.

Predominanceof MononuclearRound toOval Cells

Table5 Diagnostic work-upfor abnormalitiesin the whitecell serieswith mono-

nuclearcells predominating

Clinicalfindings Hb MCH Leukocytes Segmented

cells

Lympho-

cytes(%)

Othercells

Fever,lymphnodes,

possiblyexanthema

n n ↓/n ↓ ↑

Stimulated

forms

–

Fever,severelym-

phoma,possibly

spleen↑

n n ↑ ↓ ↑ Large blastic

stimulatedcells

(DD:lympho-

blasts)

Slowlyprogressing

lymphnodeenlarge-

mentinseveral loca-

tions

↓/n ↓/n ↑ ↓ ↑↑ –

Nightsweat,slowly

progressinglym-

phoma(!spleen),

possiblyfever

↓ ↓ n/↑ ↓ ↑ Plasmacytoid

cells,possibly

rouleauxforma-

tionofthe

erythrocytes

Slowlyprogressing

lymphnodeenlarge-

mentinone ora few

locations

↓ n/↓ n/↑/↓ n/↓ Possibly↑ Possiblycells

withgrooves

Isolatedsevere

splenomegaly

↓ ↓ ↓/↑ ↓ Hairy cells

Fever,angina n n ↓ ↓ ↑ Monocytes ↑

Diffusegeneral

symptoms

↓ ↓ ↑ ↓ n Monocytes↑

Paleskin,fever

(signsofhemor-

rhage)

↓ ↓ ↑/n/↓ ↓ ↓ Atypicalround

cellspredomi-

nate

Fatigue,night

sweat,possibly

bonepain

↓ ↓ n/↓ n n Possibly rouleaux

formationsof

erythrocytes

Diagnosticsteps proceedfrom left toright. | Thenext stepis usually unnecessary;!optional

step,may notprogress thediagnosis; →thenext stepis obligatory.

Abnormalitiesof theWhite CellSeries

Thrombo-

cytes

Electro-

phoresis

Tentative

diagnosis

Evidence/advanced

diagnostics

Bonemar-

row

Ref.page

n n/

↑ Virusinfec-

tion,

e.g.,rubeola

Clinicaldevelopments,

possiblyserological

tests

p.66

↑ Mono-

nucleosis

Mononucleosisquick

test,EBVserology

(cytomegalytiter?)

p.68

n/↓ n/

↓ Leukemic

non-Hodgkin

lymphoma,

esp.CLL

Markeranalysisof

peripherallymphocytes

issufficient intypical

diseasepresentations;

lymphnode histology

fortreatment decisions,

ifnecessary

Always

involvedin

CLL,not

alwaysin

otherlym-

phomas

p.74

n/↓ Possibly

↑

Immunocy-

toma(incl.

Walden-

strömdis-

ease)

Immunoelectrophore-

sis;markeranalysis for

lymphocytesin blood

andbone marrow;

lymphnode histologyin

caseof doubt

Usually

involved

p.78

n/↓ n Non-Hodgkin

lymphoma,

e.g.follicular

lymphoma

Lymphnodehistology

Forthepur-

poseof stag-

ing,possibly

positive

p.78

↓ n Hairycell

leukemia

Cellsurface markers

Always

involved,dis-

creteinthe

beginning

p.80

n n Agranulo-

cytosis

→ Maturation

arrest

p.86

n Possibly

↑

Reactive

monocytosis

inchronic

infectionor

tumor

Determinedisease

origin

p.88

↓ n Acute

leukemia

Cytochemistry,marker

analysis,possibly cyto-

genetics

→

Markerfor

blasts

p.90ff

n/↓ Sharp

peaks↑

Multiple

myeloma

Immunoelectrophore-

sis,skeletalX-ray

→

Bonemarrow

contains

plasmacells

p.82

Predominanceof MononuclearRound toOval Cells

ReactiveLymphocytosis

Lymphaticcells showwide variabilityand transformeasily. Thisis usually

seen asenlarged nuclei, a moderately loose, coarsechromatin structure,

anda marked wideningof the basophilic cytoplasmiclayer.

Clinicalfindings, whichinclude acute feversymptoms, enlargedlymph

nodes, and sometimesexanthema, help to identify a lymphatic reactive

state. Unlike the case in acute leukemias, erythrocyteand thrombocyte

counts are not significantly reduced. Although the granulocyte count is

relativelyreduced, itsabsolute value(per microliter)rarely fallsbelow the

lowerlimit of normalvalues.

Morphologicallynormal lymphocytespredominate inthe bloodanalyses in

thefollowing diseases:

➤

Whoopingcough (pertussis) withclear leukocytosisand total lympho-

cyte counts up to 20000 and even50 000/

µl; occasionally, slightly

plasmacytoiddifferentiation.

➤

Infectious lymphocytosis,a pediatric infectious disease with fever of

shortduration. Lymphocyte countsmay increase to" 50000/

µl.

➤

Chickenpox,measles, and brucellosis, in which a less well-developed

relativelymphocytosis isfound, and thecounts remain withinthe nor-

malrange.

➤

Hyperthyroidismand Addisondisease, which showrelative lymphocy-

tosis.

➤

Constitutionalrelative lymphocytosis, which canreach up to 60% and

occurswithout apparent reason(mostly in asthenicteenagers).

➤

Absolutegranulocytopenias with relativelymphocytosis (p.86).

➤

Chroniclymphocytic leukemia (CLL), whichis always accompaniedby

absoluteand relative lymphocytosis,usually with highcell counts.

Transformed, “stimulated” lymphocytes (“virocytes”) predominate in the

CBCin the followingdiseases with reactivesymptoms:

➤

Lymphomatous toxoplasmosis does not usually involve significant

leukocytosis.Slightly plasmacytoid cellforms are found.

➤

Inrubella infections thetotal leukocyte countis normal orlow, and the

lymphocytosisis onlyrelatively developed. The cellmorphology ranges

frombasophilic plasmacytoid cellsto typical plasma cells.

➤

In hepatitis the totalleukocyte andlymphocyte countsare normal.

However,the lymphocytes often clearly show plasmacytoid transfor-

mation.

➤

Themost extreme lymphocytetransformation is observedin mononu-

cleosis (Epstein–Barrvirus [EBV] or cytomegalovirus [CMV] infection)

(p.68).

Abnormalitiesof theWhite CellSeries

During lymphatic reactive states, variable cells with dense,

roundnuclei (e.g., virocytes) dominate the CBC

Fig.21 Lymphaticreactive states.a–e Widevariability of thelymphatic cellsin a

lymphotropicinfection (inthis case cytomegalovirusinfection). Someof the cells

mayresemble myelocytes, but theirchromatin is always denser thanmyelocyte

chromatin.

Examplesof Extreme Lymphocytic Stimulation:

InfectiousMononucleosis

Epstein–Barrvirus infectionshould be consideredwhen, aftera prodromal

fever of unknown origin, there are signs of enlarged lymph nodesand

developingangina, andthe bloodanalysis showspredominantly mononu-

clearcells anda slightly,or moderately,elevated leukocyte count.Varying

proportionsof the mononuclear cells(at least 20%) may berather exten-

sively transformed round cells (Pfeiffer cells, virocytes). Immunological

markersare necessaryto ascertain thatthese arestimulated lymphocytes

(mostly T-lymphocytes) defending the B-lymphocyte stem population

againstthe virus attack. The nuclei of these stimulated lymphocytesare

two- to three-fold larger than thoseof normallymphocytes and their

chromatin has changed from a dense and coarsestructure to alooser,

moreirregular organization. The cytoplasmis always relativelywide and

moreor less basophilic with vacuoles. Granules areabsent. A small pro-

portion of cells appear plasmacytoid. In thecourse ofthe disease, the

degree of transformation and the proportionsof the different cell mor-

phologies changealmost daily. A slight left shift andelevated monocyte

countare often foundin the granulocyte series.

Acuteleukemia isoften consideredin thedifferential diagnosisin addi-

tionto other viral conditions, because thetransformed lymphocytes can

resemblethe blasts found in leukemia. Absence of a quantitativereduc-

tionof hematopoiesisin allthe bloodcell series,however,makes leukemia

unlikely,as dothe variety andspeed of changein the cellmorphology. Fi-

nally,serological tests (EBV antigen test, testfor antibodies, and, if indi-

cated,quick tests) canadd clarification.

Whereserological testsare negative,the causeof the symptomsis usu-

allycytomegalovirus rather thanEBV.

Characteristicsof Infectious Mononucleosis

Ageof onset: Schoolage

Clinicalfindings: Enlarged lymph nodes(rapid onset), inflammations

ofthroat and possiblyspleen !

CBC:Leukocytes !, stimulated lymph nodes,partially lymphoblasts

(hematocritand thrombocytes arenormal)

Furtherdiagnostics: EBV serological test (IgM+), transaminases usu-

ally!

Differentialdiagnosis: Lymphomas (usually withoutfever), leukemia

(usually Hb ", thrombocytes ") Persistent disease (more than 3

weeks): possibly test for blood cell markers, lymph node cytology

(#histology)

Course,treatment: Spontaneous recovery within 2–4weeks; general

palliation

Abnormalitiesof theWhite CellSeries

Extreme transformationof lymphocytes leads to blast-like cells:

mononucleosis

Fig.22 Lymphocytesduring viral infection. a“Blastic,” lymphatic reactive form

(Pfeiffercell), in addition toless reactive virocytes inEpstein–Barr virus (EBV) in-

fection.This phase withblastic cells lastsonly a fewdays. b Virocyte(1) with ho-

mogeneousdeep blue stained cytoplasmin EBV infection, inaddition to normal

lymphocyte (2) and monocyte (3). c Virus infection can also leadto elevated

counts of large granulated lymphocytes (LGL) (1). Monocyte (2). d Severe

lymphatic stress reaction with granulated lymphocytes. A lymphoma must be

consideredif this findingpersists.

Table6a Classificationof non-Hodgkin:comparison ofthe relevantclasses inthe Kieland WHOclassifica-

tions

WHO Kiel Clinical

Characteristics

Marker*

Precursorlymphomas/leukemias

➤

Precursor-B-lymphoblastic

lymphoma

B-lymphoblastic

lymphoma

Aggressive Tdt,

CD19,22,

79a

➤

PrecursorB-cellactive lympho-

blasticleukemia

B-ALL

MatureB-celllymphoma

➤

Chroniclymphocyticleukemia

– contains thelymphoplasma-

cytoidimmunocytoma

B-CLL

Lymphoplasmacytoid

immunocytoma

Usuallyindolent

CD5,19,

20,23

tris12or

13q-,

11q-,6q-,

p53

➤

B-cellprolymphocyticleukemia Aggressive

Diseases of theLymphatic System

(Non-Hodgkin Lymphomas)

Malignantdiseases ofthe lymphaticsystem arefurther classifiedas Hodg-

kinand non-Hodgkin lymphomas (NHL). NHLwill be discussed here be-

causemost NHLs canbe diagnosed on theblood analysis.

➤

Non-Hodgkinlymphomas arise mostlyfrom small orblastic B-cells.

➤

Small-cellNHL cellsare usually leukemicand relativelyindolent, ofthe

typeof chronic lymphocyticleukemia and itsvariants.

➤

Blastic NHL(precursor lymphoma) is not usually leukemic. Anexcep-

tionis thelymphoblastic lymphoma, whichtakes itscourse as anacute

lymphocyticleukemia.

➤

Plasmacytomais an osteotropic B-cell lymphoma that releasesnot its

cellsbut their products(immunoglobulins) into the bloodstream.

➤

Malignant lymphogranulomatosis (Hodgkin disease) cannotbe diag-

nosed on the basis of blood analysis. It is therefore discussed under

lymphnode cytology (p.176).

The modern pathological classification of lymphomas is based on mor-

phologyand cell immunology. Theupdated classification system accord-

ingto Lennert(Kiel) has beenadopted inGermany and wasrecently mod-

ifiedto reflectthe WHOclassification. Thedefinitions ofstages I–IVin NHL

agreewith the AnnArbor classification for Hodgkin’sdisease.

Abnormalitiesof theWhite CellSeries

Table6a Continued

WHO Kiel Clinical

Characteristics

Marker*

➤

Lymphoplasmacyticleukemia Lymphoplasmacytic

immunocytoma

SometimesIgM

paraprotein

(Waldenström

disease)

–/+CD5

➤

Mantlecelllymphoma Centrocytic lymphoma Usually aggres-

sive

–CD23,

CD5

t(11;14)

bcl-1

rearr.

➤

Marginalzonelymphoma

– Nodal

– Extranodal inmucosa(MALT)

– Lienal (splenic)

Monocytoidlymphoma

MALT(mucosa-assoc.

lymphoidtissue)lym-

phoma

Lymphomawith

splenomegaly

Usuallyaggres-

sive

Oftenindolent

–CD5,

CD23

➤

Follicularlymphoma

– Grade 1,2

– Grade 3(a,b)

Centroblastic/centro-

cyticlymphoma;

centroblasticlymphoma

(a),secondary (b),follic-

ular

Usuallyindolent

Highlymalignant

–CD5,

!CD10

t(14;18)

bcl2

➤

Hairycell leukemia Hairycellleukemia Usuallyindolent –CD103,

CD11c,

CD25

t(2;8)

t(8;14)

➤

Plasmacellmyeloma (plasma-

cytoma)

– Monoclonal hypergamma-

globulinemia(GUS)

– Solitary boneplasmacytoma

– Extraosseous boneplasmacy-

toma

– Primary amyloidosis

– Heavy-chain disease

[Plasmacytomaisnot

includedinthe Kiel

classification]

CD138

Primarylarge-cell lymphoma

➤

Diffuselarge-cell B-cell

lymphoma

– Centroblastic

– Immunoblastic

– Large-cell anaplastic

Centroblastic

Immunoblastic

Large-cellanaplastic

Extremelymalig-

nant

Extremelymalig-

nant

Extremelymalig-

nant

CD20,

79a,19,

➤

Burkittlymphoma Burkitt lymphoma Extremelymalig-

nant

t(2;8)

t(8;14)

myc

* Citedmarkersare positive,absent markersare indicatedwitha minussign.

Predominanceof MononuclearRound toOval Cells

Table6b T-celllymphomas(since T-celllymphomasmake uponly10% ofallNHLs,this tablegivesjust abrief

characterization;formarkers seeTable7)

WHO

(!Kiel)

Clinicalcharacteris-

tics

Classification Manifest ation

➤

T-prolymphocyticleukemia

➤

Largegranularlymphocyte leukemia(LGL)

➤

T-celllymphoblasticleukemia

Leukemic

Aggressive

Sometimesindolent

Aggressive

➤

Sézarysyndrome, mycosisfungoides Cutaneous Chronic,progressive

➤

AngioimmunoblasticT-celllymphoma (AILD) Nodaland ENT Usuallyaggressive

➤

LymphoblasticT-celllymphoma

➤

T-cellzonelymphoma (nonspecificperipheral

lymphoma)

➤

Lennertlymphoma withmultifocalepithelioid

cells

➤

Large-cellanaplasticlymphoma (ki1)

Nodal

Aggressive

Sometimesslowly

progressive

Sometimesslowly

progressive

Aggressive

Differentiation ofthe Lymphatic Cells andCell Surface

MarkerExpression in Non-Hodgkin Lymphoma Cells

Non-Hodgkinlymphoma cellsderive monoclonallyfrom specificstages in

theB- or T-celldif ferentiation, and their surfacemarkers reflect this. The

surface markersare identified in immunocytological tests (Table

7) car-

riedout on heparinized bloodor bone marrowspicules.

Theblastic lymphomas will notbe discussed further in thecontext of

diagnosticsbased on bloodcell morphology.The findings inthe primarily

leukemic forms of the disease, such aslymphoblastic lymphoma, re-

semblethose for ALL (p. 104).Other blastic lymphomas can usuallyonly

bediagnosed onthe basisof lymphnode tissue(Fig.

65).Of course,despite

allthe progress inthe analysis ofblood cell differentiation, oftenanalysis

ofhistological slidesin conjunction withthe bloodanalysis isrequired for

aconfident diagnosis.

Abnormalitiesof theWhite CellSeries

Table7 Cell surface markersof lymphaticcells inleukemic, low-grade malignantnon-Hodgkin lymphoma

Marker B-CLL B-PLL HCL FL MCL SLVL T-CLL GL SS T-PLL ATLL

SIg (+) ++ ++ ++ ++ ++ – – – – –

CD2 – – – – – – + + + + +

CD3 – – – – – – + – + + +

CD4 – – – – – – – – + +/– +

CD5 ++ – – – + – – – + + +

CD7 – – – – – – – – +/– + –/+

CD8 – – – – – – + +/– +/– +/– +/–

CD19/20/24 ++ ++ ++ + + ++ – – – – –

CD22 +/– ++ ++ + + ++ – – – – –

CD10 – – – +/– – – – – – – –

CD25 – – ++ – – +/– – – – – +

CD56 – – – – – – – + – – –

CD103 – – ++ – – – – – – – –

CLL chroniclymphocyticleukemia;PLL prolymphocytic leukemia;HCL hairycell leukemia;FL follicularlymphoma; MCL mantlecelllymphoma; SLVLsplenic lymphomawithvillous lymph-

ocytes;LGL largegranular lymphocyteleukemia; SS Sézarysyndrome; ATLLadult T-celllymphoma.

Predominanceof MononuclearRound toOval Cells

ChronicLymphocytic Leukemia (CLL) and RelatedDiseases

A chronic lymphadenoma,or chronic lymphocytic leukemia, can some-

timesbe clinically diagnosedwith some certainty.An exampleis the case

ofa patient(usually older) withclearly enlargedlymph nodes andsignifi-

cantlymphocytosis (in 60%of the cases thisis greaterthan 20 000/

µland

in20 %of the cases it isgreater than 100000/

µl)in the absence ofsymp-

tomsthat point to a reactivedisorder. The lymphoma cells arerelatively

small,and thenuclear chromatin iscoarse and dense.The narrowlayer of

slightlybasophilic cytoplasm doesnot contain granules.Shadows around

thenucleus are an artifact producedby chromatin fragmentation during

preparation(Gumprecht’s nuclear shadow).In order toconfirm the diag-

nosis,the B-cell markerson circulatinglymphocytes should becharacter-

ized to show that the cells are indeed monoclonal. The transformed

lymphocytesare dispersed at varying celldensities throughout the bone

marrow and the lymph nodes. A slowly progressing hypogammaglobu-

linemiais another importantindicator of a B-cellmaturation disorder.

Transition to a diffuse large-cell B-lymphoma (Richter syndrome) is

rare: B-prolymphocyticleukemia (B-PLL) displays unique symptoms. At

least 55% of the lymphocytes in circulating blood have large central

vacuoles.When 15–55% ofthe cells areprolymphocytes, the diagnosisof

atypical CLL, or transitional CLL/PLL is confirmed. In some CLL-like dis-

eases,the layer of cytoplasmis slightly wider. B-CLLwas defined as lym-

phoplasmacytoid immunocytoma in the Kiel classification. According to

theWHO classification,it is aB-CLL variation(compare this withlympho-

plasmacytic leukemia, p.78). CLL of the T-lymphocytesis rare. The cells

shownuclei witheither invaginations orwell-defined nucleoli(T-prolym-

phocytic leukemia). The leukemic phase of cutaneousT-cell lymphoma

(CTCL)is known as Sézarysyndrome. The cell elementsin this syndrome

andT-PLL are similar.

!Fig.23 CLL. a Extensive proliferation of lymphocytes with densely structured

nucleiand little variation in CLL.Nuclear shadows are frequently seen, asign of

thefragility of thecells (magnification #400).b Lymphocytes inCLL with typical

coarsechromatin structureand smallcytoplasmic layer(enlargement ofa section

from 23 a, magnification #1000); only discreet nucleoli may occur. c Slightly

eccentric enlargement of the cytoplasmin the lymphoplasmacytoid variant of

CLL.

Abnormalitiesof theWhite CellSeries

Monotonous proliferation of small lymphocytes suggests chro-

niclymphocytic leukemia (CLL)

Fig.23 d Proliferationof atypical large lymphocytes (1) with irregularly struc-

turednucleus, well-defined nucleolus, andwide cytoplasm (atypicalCLL or tran-

sitional form CLL/PLL). e Bone marrow cytology in CLL: There is always strong

proliferationof the typical small lymphocytes, which areusually spread out dif-

fusely.

Table8 Staging of CLLaccording toRai (1975)

Stage Identifyingcriteria/definition

(Lowrisk) 0 Lymphocytosis" 15000/µl

Bonemarrow infiltration" 40%

(Intermediate

risk)

Lymphocytosisand lymphadenopathy

Lymphocytosisand hepatomegalyand/or spleno-

megaly(with orwithout lymphadenopathy)

(Highrisk) III Lymphocytosis andanemia (Hb $ 11.0 g/dl)(with or

withoutlymphadenopathy and/ororganomegaly)

IV Lymphocytosisand thrombopenia($ 100000/

µl)

(withor withoutanemia, lymphadenopathy,or

organomegaly)

Table9 Staging of CLLaccording toBinet (1981)

Stage Identifyingcriteria/definition

A Hb "10.0 g/dl,normal thrombocyte count

$3 regionswith enlargedlymph nodes

B Hb"10.0 g/dl,normal thrombocytecount

"3 regionswith enlargedlymph nodes

C Hb$10.0 g/dland/or thrombocytecount $100 000/µl

independentof thenumber ofaffected locations

Characteristicsof CLL

Ageof onset: Matureadulthood

Clinical presentation:Gradual enlargement of all lymph nodes, usu-

allymoderately enlargedspleen, slowonset ofanemia andincreasing

susceptibilityto infections, laterthrombocytopenia

CBC:In all casesabsolute lymphocytosis; inthe course of thedisease

Hb", thrombocytes ",immunoglobulin "

Furtherdiagnostics: Lymphocytesurface markers(see pp.68f f.);bone

marrow (alwaysinfiltrated); lymph node histology further clarifies

thediagnosis

Differential diagnosis: (a) Related lymphomas: marker analysis,

lymphnode histology;(b) acute leukemia:cell surfacemarker analy-

sis,cytochemistry, cytogenetics (pp. 88ff.)

Course, therapy:Individually varying, usually fairly indolent course;

inadvanced stagesor fast progressingdisease: moderatechemother-

apy(cell surface marker,see Table

Abnormalitiesof theWhite CellSeries

Atypicallymphocytes are not part of B-CLL

Fig.24 Lymphomaofthe B-celland T-celllineages. aPrevalenceof largelympho-

cyteswithclearlydefined nucleoliandwide cytoplasm:prolymphocyticleukemiaof

theB-cellseries (B-PLL).b Thepresenceof largeblasticcells (arrow)inCLL suggesta

raretransformation (Richter syndrome). c The rare Sézary syndrome(T-cell lym-

phomaof theskin) ischaracterized by irregular,indented lymphocytes. d Prolym-

phocytic leukemiaof the T-cell series (T-PLL) with indented nuclei and nucleoli

(rare).e Bonemarrowin lymphoplasmacyticimmunocytoma: focalor diffuselym-

phocyteinfiltration (e.g.,1), plasmacytoid lymphocytes(e.g., 2) andplasma cells

(e.g.,3). Red cellprecursors predominate (e.g.,basophilic erythroblasts, arrow).

Thepathological staging forCLL is alwaysAnn Arbor stageIV because the

bone marrowis affected. In the classifications of disease activity by Rai

andBinet (analogousto that forleukemic immunocytoma), thetransition

betweenstages is smooth(Tables

8and 9).

LymphoplasmacyticLymphoma

TheCBC showslymphocytes withrelatively widelayers of cytoplasm.The

bone marrow contains a mixture of lymphocytes, plasmacytic lympho-

cytes,and plasma cells.In up to30 %of cases paraproteinis secreted, pre-

dominantly monoclonal IgM. This constitutes the classicWaldenström

syndrome(Waldenström macroglobulinemia). The differential diagnosis

maycall for exclusion ofthe rare plasma cell leukemia(see p. 82)and of

lymphoplasmacytoidimmunocytoma, whichis closely relatedto CLL(see

p.74).

Characteristics

➤

Lymphoplasmacytoidimmunocytoma: This is a special form of B-

CLLin which usuallyonly afew precursors migrate into theblood-

stream (alesser degree of malignancy). A diagnosis may only be

possibleon the basis ofbone marrow orlymph node analysis.

➤

Lymphoplasmacytic lymphoma: Few precursors migrate into the

bloodstream (i.e.,bone marrow or lymph node analysis is some-

times necessary). There is often secretion of IgM paraprotein,

whichcan lead tohyperviscosity.

Further diagnostics:Marker analyses in circulating cells, lymphnode cy-

tology,bone marrowcytology andhistology, andimmunoelectrophoresis.

Plasmacytoma cells migrate into the circulating blood in appreciable

numbersin only1–2% ofall casesof plasmacell leukemia.Therefore, para-

proteinsmust be analyzedin bone marrowaspirates (p. 82).

FacultativeLeukemic Lymphomas

(e.g.,Mantle Cell Lymphoma and FollicularLymphoma)

Inall casesof non-Hodgkinlymphoma, thetransformed cells maymigrate

intothe blood stream. This is usually observedin mantle cell lymphoma:

Thecells are typicallyof medium size. Onclose examination, theirnuclei

showloosely structuredchromatinand theyare lobed withsmall indenta-

tions(cleaved cells).Either initially,or,more commonly,during thecourse

of the disease, a portion of cells becomes largerwith relatively enlarged

nuclei(diameter 8–12

µm).These largercells are variably“blastoid.” Lym-

phoid cells also migrate into the blood in stage IV follicular lymphoma.

Abnormalitiesof theWhite CellSeries

Deep nuclear indentation suggests follicular lymphoma or

mantlecell lymphoma

Fig.25 Mantle celllymphoma. a Fine, densechromatin and smallindentations

ofthe nuclei suggest migrationof leukemic mantlecell lymphoma cells intothe

bloodstream. b Denser chromatinand sharp indentations suggest migrationof

follicularlymphoma cells intothe blood stream.c Diffuse infiltrationof the bone

marrow with polygonal, in some cases indented lymphatic cells in mantle cell

lymphoma.Bone marrow involvement in follicularlymphoma can often only be

demonstratedby histological andcytogenetic studies.

“Monocytoid” cells with a wide layerof only faintly staining cytoplasm

occur in blood in marginal zone lymphadenoma(differential diagnosis:

lymphoplasmacyticimmunocytoma).

Lymphoma,Usually with Splenomegaly

(e.g.,Hair yCell Leukemia and Splenic Lymphoma

withVillous Lymphocytes)

Hairy cellleukemia (HCL). In cases of slowly progressivegeneral malaise

withisolated splenomegaly and pancytopeniarevealed by CBC(leukocy-

topenia,anemia, andthrombocytopenia), thepredominating mononuclear

cellsdeserve particular attention. Thenucleus is oval, oftenkidney bean-

shaped, and shows a delicate, elaborate chromatin structure. The cyto-

plasm is basophilicand stains slightly gray. Long, very thin cytoplasmic

processes givethe cells the hairy appearance that gave rise to the term

“hairycell leukemia” used in the internationalliterature. The disease af-

fectsthe spleen, liver,and bone marrow.Severe lymphoma is usuallyab-

sent. Aside from the typical hairy cells with their long,thin processes,

thereare alsocells witha smoothplasmamembrane, similarto cellsin im-

munocytoma. A variant shows well-defined nucleoli (HCL-V,hairy cell

leukemiavariant). A bone marrow aspirateoften does not yieldmaterial

foran analysis(“punctio sicca”or“empty tap”)because themarrow isvery

fibrous.Apart from the bone marrowhistology, advanced cell diagnostics

aretherefore very important, in particularin the determination of blood

cellsurface markers (immunophenotyping). Thisanalysis reveals CD 103

and11c as specific markersand has largelyreplaced the test fortartrate-

resistantacid phosphatase.

Spleniclymphoma with villous lymphocytes(SLVL). This lymphaticsystem

diseasemostly affects the spleen. There islittle involvement of thebone

marrowand noinvolvement ofthe lymph nodes.The blood containslym-

phatic cells, which resemble hairy cells. However,the “hairs,” i.e., cyto-

plasmicprocesses, arethicker and mostlyrestricted toone area atthe cell

pole,and the CD 103marker isabsent.

Splenomegalymay develop in all non-Hodgkin lymphomas.In hairy cell

leukemia, the rare splenic lymphadenoma with villous lymphocytes

(SLVL)and marginal zonelymphadenoma may beseen. These mostly af-

fectthe spleen.

Abnormalitiesof theWhite CellSeries

Cytoplasmicprocesses the main feature ofhair ycell leukemia

Fig.26 Hairy cell leukemiaand splenic lymphoma. a and bOvaloid nuclei and

finely“fraying” cytoplasm arecharacteristics of cellsin hairy cellleukemia (HCL).

cOccasionally, thehairy cellprocesses appear merelyfuzzy.d and eWhen thecy-

toplasmicprocesses lookthickerand muchless likehair, diagnosisof therare sple-

niclymphoma withvillous lymphocytes(SLVL) mustbe considered.Here, too,the

nextdiagnostic step isanalysis of cellsur facemarkers.

Table10 Differential diagnosisof monoclonal hypergammaglobulinemia

Type Characteristics

Benigndisorders

➤

Essentialhypergammaglobuline-

mia= MGUS(monoclonal gam-

mopathyof unknownsignificance)

Usuallyin advancedage

$10%, plasmacells foundin the

bonemarrow, noprogression,

normalpolyclonal Ig

➤

Symptomatichypergammaglobu-

linemia,secondary to

– Infections

– Tumors

– Autoimmunedisease

Allages (otherwiseas above)

Malignantdiseases

➤

Plasmocytoma

(usuallyIgG, Aor light-chain

[BenceJones protein],rarely IgM,

D,E) 90% disseminated(multiple

myeloma),5% solitary,5 %

extramedullary(like alymphoma

orENT tumor)

– Osteolysisor X-raywith severe

osteoporosis

– Plasmocytosisof thebone mar-

row" 10%

– Monoclonalgammaglobulin in

serum/urinewith progression

➤

Lymphoma

e.g., immunocytoma,CLL

(potentiallyall lymphomasof the

B-cellseries)

– Enlargedlymph nodes

– Usuallyblood lymphocytosis

– Monoclonalimmunoglobulin,

usuallyIgM

MonoclonalGammopathy (Hypergammaglobulinemia),

MultipleMyeloma*, Plasma Cell Myeloma, Plasmacytoma

* The currentWHO classification suggests “multiplemyeloma” (MM) forgeneralized

plasmacytomaand “plasmacytoma”only for therare solitaryor nonosseous formof

plasmacytoma.

Plasmacytoma is the result of malignant transformation of the most

matureB-lymphocytes (Fig.

1,p. 2). Forthis reason thediagnostics of this

diseasewill be discussed here,even though migration ofits specific cells

intothe blood stream(plasma cell leukemia) isextremely rare(1–2 %).

Immunoelectrophoresis ofserum and urine is performed when elec-

trophoresisshows verydiscrete gammaglobulin, orglobulin, fractions, or

whenhypogammaglobulinemia isfound (inlight-chain plasmacytoma).A

wide rangeof possibilities arises for the differential diagnosis of mono-

clonaltransformed cells (Table

10).

Thepresence ofmore than 10%of plasma cells,or atypical plasmacells

inthe bone marrow,is an important diagnosticfactor in the diagnosisof

plasmacytoma.For more criteria,see p. 84.

Abnormalitiesof theWhite CellSeries

Plasmacytomacannot be diagnosed without bone marrowana-

lysis

Fig.27 Reactive plasmacytosis and plasmacytoma. a Bonemarrow cytology

withclear reactive features in thegranulocyte series: strong granulationof pro-

myelocytes(1) and myelocytes(2), eosinophilia(3), and plasmacell proliferation

(4):reactive plasmacytosis (magnification #630). b Extensive(about 50%) infil-

tration ofthe bone marrow of mostly well-differentiated plasma cells:multiple

myeloma(magnification #400).

Table11 Staging ofplasmacytomas accordingto Salmonand Durie

StageI

Allthe followingare present:

– Hb" 10g/dl

– Serumcalcium is normal

– X-rayshows normal bonestruc-

tureor solitaryskeletal plasma-

cytoma

– IgG$ 5g/dl*

IgA$ 3g/dl*

Lightchains inthe urine*

$4 g/24h

StageII

Findingsfulfill neitherstage Inor

stageIII criteria

StageIII

Oneor moreof thefollowing are

present:

– Hb$ 8.5g/dl

– Serumcalcium is elevated

– X-rayshows advanced bonelesions

– IgG" 7g/dl*

IgA" 5g/dl*

Lightchains inthe urine:

"12g/24 h

* Monoclonalin eachcase.

Variabilityof Plasmacytoma Morphology

Itis not easy to visually distinguishmalignant cells from normal plasma

cells.Like lymphocytes,normal plasmacells havea denselystructured nu-

cleus. Plasma cell nuclei with radial chromatin organization, known as

“wheel-spokenuclei,” are mostlyseen during histologicalanalysis.

Thefollowing attributes suggesta malignantcharacter of plasmacells:

thecells are unusuallylarge (Fig.

28c), theycontain crystalline inclusions

orprotein inclusions(“Russell bodies”) (Fig.

28b), orthey havemore than

onenucleus (Fig.

28c).

Inthe differential diagnosis,they must bedistinguished from hemato-

poieticprecursor cells(Fig.

28d), osteoblasts(Fig. 20c),and blasts inacute

leukemias(see Fig.

31,p. 97).

Bonemarrow involvement may befocal or in rare caseseven solitary.

Asidefrom cytological tests, bone mar rowhistology assays aretherefore

indicated. Sometimes,the biopsy must be obtained from a clearly iden-

tifiedosteolytic region.

Althoughplasmacytomas progressslowly, staging criteriaare available

(stagingaccording to Salmonand Durie) (Table

11).

Therapymay be put on hold instage I. Smoldering indolent myeloma

can be left for a considerable time without the introduction of therapy

stress. However, chemotherapy is indicated once the myeloma has

exceededthe stage 1criteria.

Abnormalitiesof theWhite CellSeries

Atypiasand differential diagnoses of multiple myeloma

Fig.28 Atypical cells in multiple myeloma. a Extensive infiltration ofthe bone

marrowby looselystructured, slightly dedifferentiatedplasma cellswith wide cy-

toplasm in multiple myeloma.b In multiple myeloma, vacuolated cytoplasmic

proteinprecipitates (Russell bodies)may be seenin plasma cells butare without

diagnosticsignificance. c Binuclear plasma cellsare frequently observed inmul-

tiplemyeloma (1). Mitoticred cell precursor(2). d Differentialdiagnosis: red cell

precursorcells can sometimes look like plasma cells.Proerythroblast (1) and ba-

sophilicerythroblast (2).

Abnormalitiesof theWhite CellSeries

RelativeLymphocytosis Associated with

Granulocytopenia (Neutropenia)and Agranulocytosis

Neutropenia is def ined by a decrease in the number of neutrophilic

granulocyteswith segmented nuclei to less than 1500/

µl (1.5#10

/l). A

neutrophilcount of less than50 0/

µl(0.5 #10

/l)constitutes agranulocy-

tosis.Absolute granulocytopeniaswith benign causedevelop intorelative

lymphocytoses.

Inthe most common clinical picture,drug-induce dacute agranulocy-

tosis,the bone marrow iseither poor incells or lacks granulopoieticpre-

cursorcells (aplastic state), or showsa “maturation block” at the myelo-

blast–promyelocytestage. The differential diagnosis ofpure agranulocy-

tosisversus aplasias ofseveral cell linesis outlined on page146.

Classification ofNeutropenias and Agranulocytoses

1. Drug-induced:

a) Dose-independent—acute agranulocytosis. Causedby hypersensi-

tivity reactions: for example to pyrazolone,antirheumatic drugs

(anti-inflammatoryagents), antibiotics, orthyrostatic drugs.

b) Relativelydose-dependent—subacute agranulocytosis (observedfor

carbamazepine [Tegretol], e.g. antidepressants, and cytostatic

drugs).

c) Dose-dependent—cytostatic drugs,immunosuppressants.

2. Infection-induced:

a) E.g., EBV,hepatitis, typhus, brucellosis.

3. Autoimmuneneutropenia:

a) With antibody determination of T-cell orNK-cell autoimmune re-

sponse

b) Incases ofsystemic lupuserythematosus (SLE),Pneumocystis carinii

pneumonia(PCP), Felty syndrome

c) In cases ofselective hypoplasiaof thegranulocytopoiesis (“pure

whitecell aplasia”)

4. Congenitaland familial neutropenias:

Variouspediatric forms; sometimesnot expressed untiladulthood,

e.g.cyclical neutropenia

5. Secondary neutropeniain bone marrowdisease:

Myelodysplasticsyndromes (MDS), e.g.,acute leukemia, plasmacy-

toma,pernicious anemia

Bonemarrow diagnosisis indicated incases of unexplainedagra-

nulocytosis

Fig.29 Thebone marrow inagranulocytosis. aIn the earlyphase ofagranulocy-

tosisthe bone marrow shows onlyred cell precursor cells (e.g., 1),plasma cells

(2),and lymphocytes (3); inthis sample a myeloblast—asign of regeneration—is

alreadypresent (4). b Bonemarrow in agranulocytosisduring the promyelocytic

phase,showing almostexclusively promyelocytes(e.g., 1);increased eosinophilic

granulocytes(2) are alsopresent.

Table12 Possible causesof monocytosis

Infection

Nonspecificmonocytosis occursin

manybacterial infectionsduring

recuperationfrom orin thechronic

phaseof:

– Mononucleosis(aside fromstimu-

latedlymphocytes thereare also

monocytes)

– Listeriosis

– Acuteviral hepatitis

– Parotitisepidemica (mumps)

– Chickenpox

– Recurrentfever

– Syphilis

– Tuberculosis

– Endocarditislenta

– Brucellosis(Bang disease)

– Variolavera (smallpox)

– Rockymountain spottedfever

– Malaria

– Paratyphoidfever

– Kala-azar

– Thypusfever

– Trypanosomiasis(African sleeping

sickness)

Chronicreactive conditionof the

immunesystem, e.g., in:

– Autoimmunediseases

– Chronicdermatoses

– Regionalileitis

– Sarcoidosis

Paraneoplasm

(asattempted immunedefense),

e.g., in:

– Largesolid tumors

– Lymphogranulomatosis

Neoplasm

– Chronicmyelomonocytic

leukemia(CMML, p.107)

– Acutemonocytic leukemia

(p.100)

– Acutemyelomonocytic leukemia

(p.98)

Abnormalitiesof theWhite CellSeries

Monocytosis

Ifmononuclear cells standout in showingan unusuallyelaborate nuclear

structurewith ridges and lobes and awider cytoplasmic layer with very

delicategranules (for characteristics see p.46, for cell function, seep. 6),

andthis is inthe context ofrelative ("10%) or absolutemonocytosis (cell

count "900/

µl), a series of possible triggers must be considered (Table

12).If the morphology does notclearly identify the cells as monocytes,

thenesterase assays should be donein a hematological laboratory using

unstainedsmears.

a b

Conspicuously large numbers of monocytesusually indicatea

reactive disorder.Only a monotonous predominance of mono-

cytessuggests leukemia

Fig.30 Reactivemonocytosis and monocyticleukemia. aReactive and neoplas-

ticmonocytes aremorphologically indistinguishable; heretwo relativelyconden-

sedmonocytes in reactive monocytosisare shown. b Whenever monocytes are

foundexclusively, amalignant etiologyis likely:in this caseAML M

accordingto

theFAB classification (seep. 100).Auer bodies (arrow). cMonocytes of different

degreesof maturity,segmented neutrophilicgranulocytes (1),and asmall myelo-

blast(2) in chronicmyelomonocytic leukemia (CMML,see p.107).

Table13 Overview ofall forms ofleukemia

Typeof leukemia Classification/clinical findings

Acuteleukemias (AL#AML,ALL)

– MyeloidM

0-7

(incl.monocytic, ery-

throid,megakaryoblastic leukemias)

Alwaysacute disease,often with

feverand tendencyto hemorrhage

– Lymphocytic There maybe lymphoma andthy-

musinfiltrates

Chronicmyeloid leukemia(CML)

– Persistentleukemic diseasesof the

myeloproliferativesystem (p.114)

Chronicdisease, usuallywith

splenomegaly

Chroniclymphocytic leukemia(CLL)

andother leukemiclymphomas

– B-CLL(90 %),T-CLL (p.74f.)

– Prolymphocyticleukemia (p.77)

– Hairycell leukemia(p.80)

– Chroniclymphocytic leukemia,

(rarely)prolymphocytic

leukemiaand hairycell leukemia

areprimary leukemiclym-

phomaswith achronic course.

Allother non-Hodgkinlym-

phomascan developsecondary

leukemicdisease

Chronicmyelomonocytic leukemia

(CMML)

– Leukemicform ofmyelodysplasia

(p.106)isclassified between

myelodysplasiaand myeloproliferative

diseases

Subacutedisease withtransitions

intoacute formsof myeloid

leukemia(secondary AMLfollow-

ingMDS)

Abnormalitiesof theWhite CellSeries

Acute Leukemias

Acuteleukemias aredescribed inthis placefor morphologicalreasons, be-

cause they involve a predominance of mononuclear cells (p.63). Al-

though—orperhaps because—theterm “leukemia” isrelatively imprecise,

anoverview seems required(Table

13).

Thecellular phenomenoncommon tothe differentforms ofleukemia is

the rapidly progressive reduction in numbers of mature granulocytes,

thrombocytes, and erythrocytes. Simultaneously, the leukocyte count

usuallyincreases due tothe occurrence of atypical round cells.

Predominanceof MononuclearRound toOval Cells

Notethat in about one-fourthof all leukemiastotal leukocyte counts are

normal,or evenreduced, and theatypical round cellsaffect only therela-

tive(differential) blood analysis(“aleukemic leukemia”).

Inall formsof leukemia,the morefluffy layeredareas ofa smearshow that

the nuclearchromatin structure is not dense and coarse, as in a normal

lymphocytenucleus (p. 49),but more delicately structuredand irregular,

often“sand-like”. Acareful blood cellanalysis should b ecarried out—per-

haps withthe assistance of a specialist laboratory—beforebone marrow

analysisis performed.

In most cases, the highleukocyte countfacilitates thediagnosis of

leukemia.Apart from theleukemia-specific blast cells,a variable number

of segmentedneutrophilic granulocytes may also remain, depending on

thedisease progressionat the timeof diagnosis. Thisgap in thecell series

betweenblasts and maturecells is called “leukemichiatus.” It isfound in

ALL but not in reactive responses or chronicmyeloid leukemia, which

show a continuous left shift. Morphological or differential diagnosis of

acuteleukemia isfollowed bythe diagnostic work-upthat continueswith

cytochemicaltests. Immunologicalidentification ofleukemia cells isalways

indicated(Table

14).

Diagnosticwork-up when acuteleukemia is suspected:

CBC,cytochemistr y,bone marrow. Collection of materialto identify cell

surfacemarkers, cytogenetics, moleculargenetics.

Morphologicaland Cytochemical Cell Identification

After af irst-linediagnosis of acute leukemia has been arrived at on the

basisof thecell morphology (seeabove), thediagnosis must berefined by

cytochemical testing of blood cells or bone marrow (onfresh smears).

Table

14showsa leukemiaclassification based onboth morphologicaland

cytochemical criteria. The table shows that a peroxidase test allows

leukemias tobe classified as peroxidase-positive (myeloid or monocytic)

orperoxidase-negative (lymphoblastic). Thenext step isthe immunologi-

calclassification based on cellmarkers.

Inroutine clinical hematology,the FAB classification(Table

14)will be

withus for a fewmore years.

Table14 Classification of the acute nonlymphatic leukemias by morphology, cytochemistry, and

immunology

FABtype* Peroxidase PAS α-Naph-

thyl-

acetat-

esterase

Naph-

thyl-

ASD-

esterase

Immuno-

phenotype

AMLwithminimal

markerdifferentia-

tion,undifferen-

tiatedblasts

withoutgranules;

distinguishedfrom

andALLonly by

immunopheno-

typing

$3% % % % CD13 "or

CD33" or

MPO"

CD79a #

andcyCD3 #

andcyCD22 #

andCD61/

CD41# and

CD14#

AMLwithdistinct

markerdifferentiation

(butwithoutmorpho-

logicaldifferentia-

tion);sporadicdis-

cretecytoplasmic

granulationpossible

&3% Negative

tofine

gran.

% % MPO" and

CD13/CD33/

CD65s"/#

andCD14 #

AMLwithmorpho-

logicallymature

cells;"10 %ofthe

blastscontainver y

smallgranules

"3% Negative

tofine

gran.

% % MPO" and

CD13/CD33/

CD65s/

CD15"/#

andCD14 #

Acutepromyelo-

cyticleukemia;the

predominantpro-

myelocytescontain

copiousgranules,

somecontainAuer

bodies;variantM

containsfew

granules;peripheral

bilobalblasts

!100% % % % MPO" and

CD13" and

CD33"and

normallyCD

34#and

HLA-DR#

Acutemyelomono-

cyticleukemia;

30–80%of bone

marrowblastsare

myeloblasts,pro-

myelocytes,and

myelocytes;20%

aremonocytes;vari-

antM

eosinophilia;

additionalimma-

tureeosinophils

withdarkgranules

&3% % +

"20%

+ Mixed M1/

M2and M5

Abnormalitiesof theWhite CellSeries

Table14 Continued

FABtype* Peroxidase PAS α-Naph-

thyl-

acetat-

eesterase

Naph-

thyl-

ASD-

esterase

Immuno-

phenotype

a) Acute mono-

blasticleuke-

mia;monoblasts

predominantin

thebloodand

bonemarrow

! % +++

"80%

+++ CD 13/CD33/

CD65/CD14/

CD64"/#

andHLA-DR

b) Acute mono-

cyticleukemia;

monocytesin

theprocessof

maturationpre-

dominante

! % +++ +++

Acuteerythroid

leukemia;50% of

bonemarrowblasts

areerythropoietic,

30%myeloblasts

% % % Erythroblasts

GlyA" and

CD36"

Myeloblasts

MPO/CD

13/CD33/CD

65s"/#and

CD14#

Acutemegakaryo-

cyticleukemia;very

polymorphic,some-

timesvacuolated

blasts,somewith

cytoplasticblebs,

sometimesaggre-

gatedwiththrom-

bocytes

% ! ! ! CD 13/CD33

#/"undCD

41"oder CD

61"

* FrenchAmerican BritishClassification (FAB)1976/85

** Abiphenotypic leukemiamustbe considereda possibilityifseveral additionallymphoblas-

ticmarkers arepresent

PAS Periodicacid-Schiff reaction

Predominanceof MononuclearRound toOval Cells

Table15 WHOclassification ofAML

AMLwith specific

cytogenetictrans-

locations

– With t(8;21)(q22;q22), AML1/ETO

– Acute promyelocyticleukemia(AML M3with t(15;17)(q22;

q11-12)andvariants, PML/RAR-

– With abnormalbone marroweosinophils and(inv16)

(p13;q22)orto t(16;16)(p13;q22); CBF

β/MYH11

– With 11q23(MLL)anomalies

AMLwith dysplasiain

morethan 1cell line

(2or 3cell lines

affected)*

– With precedingmyelodysplastic/myeloproliferativesyn-

drome

– Without precedingmyelodysplastic syndrome

Therapy-inducedAML

undMDS

– After treatmentwith alkylatingagents

– After treatmentwith epipodophyllotoxin

– Other triggers

AMLthat doesnot fit

anyof theothercat-

egories

– AML, minimaldifferentiation

– AML withoutcell maturation

– AML withcell maturation

– Acute myelomonocyticleukemia

– Acute monocyticleukemia

– Acute erythroidleukemia

– Acute megakaryoblasticleukemia

– Acute panmyelosiswithmyelofibrosis

– Myelosarcoma/chloroma

– Acute biphenotypicleukemia**

* Thedysplasia mustbeevident inat least50% ofthebone marrowcells andin2–3 celllines.

** Biphenotypicleukemias shouldbe classifiedaccording totheir immunophenotypes.They

aregrouped betweenacute lymphocyticand acutemyeloidleukemias.

Abnormalitiesof theWhite CellSeries

Chromosome analysis provides important information. In practice,

however,the diagnosis ofacute leukemia is still basedon morphological

criteria.

However,where the possibilities of modern therapeutic and prognostic

methodsare fullyaccessible, new laboratoryprocedures basedon genetic

and molecular biological testingprocedures form part of the diagnostic

work-upof AML. The current WHO classification takes account of these

newmethods, placinggenetic, morphological, and anamnestic findingsin

ahierarchical order (Table

15).

According tothe new WHO classification, blasts account for more than

20% of cells in acute myeloid leukemia (in contradistinction tomyelo-

dysplasias).

Predominanceof MononuclearRound toOval Cells

AcuteMyeloid Leukemias (AML)

Morphological analysis makes it possible to group the predominant

leukemiccells into myeloblastsand promyeloblasts,monocytes, or atypi-

cal (lympho)blasts. A morphological subclassification of these main

groupswas put forwardin the French–American–British(FAB) classifica-

tion(Table

14).

In practical, treatment-oriented terms, the most relevant factor is

whetherthe acute leukemiais characterized asmyeloid or lymphatic.

Including the very rareforms, there are at least 11 forms of myeloid

leukemia.

Abnormalitiesof theWhite CellSeries

AcuteMyeloblastic Leukemia (Type M

through M

in theFAB Classifica-

tion). Morphologically, the cell populations that dominate the CBC and

bonemarrow analyses (Fig.

31)more or less resemblemyeloblasts in the

course of normal granulopoiesis. Differences maybe foundto varying

degreesin theform of coarserchromatin structure,more prominentlyde-

finednucleoli, and relativelynarrow cytoplasm. Comparedwith lympho-

cytes(micromyeloblasts), the analyzed cellsmay beup tothreefold larger.

In a good smear, the transformed cells can be distinguished from lym-

phaticcells bytheir usually reticularchromatin structureand itsirregular

organization.Occasionally, thecytoplasm containscrystalloid azurophilic

needle-shaped primary granules (Auer bodies). Auerbodies (rods)are

conglomeratesof azurophilic granules. A few cells may begin to display

promyelocyticgranulation. Cytochemistry shows thatfrom stage M

on-

ward,more than 3% ofthe blasts areperoxidase-positive.

Characteristicsof Acute Leukemias

Ageof onset: Anyage.

Clinical findings: Fatigue,fever, and signs of hemorrhage in later

stages.

Lymphnode and mediastinaltumors are typical onlyin ALL.

Generalized involvement of all organs (sometimes including the

meninges)is always present.

CBC and laboratory: Hb ", thrombocytes ", leukocytes usually

stronglyelevated (~ 80%) butsometimes decreased ornormal.

Inthe differential blood analysis, blastspredominate (morphologies

vary).

Beware:Extensive urate accumulation!

Furtherdiagnostics: Bonemarrow, cytochemistry,immunocytochem-

istry,cytogenetics, and moleculargenetics.

Differential diagnosis: Transformed myeloproliferative syndrome

(e.g.,CML) or myelodysplasticsyndrome.

Leukemicnon-Hodgkin lymphomas (incl.CLL).

Aplasticanemias.

Tumors in the bone marrow (carcinomas, but also rhabdomyosar-

coma).

Course,therapy: Usually rapid progressionwith infectious complica-

tionsand bleeding.

Immediate efficient chemotherapy ina hematology facility; bone

marrowtransplant may beconsidered, with curativeintent.

Fundamental characteristic of acute leukemia: variable blasts

driveout other cell series

Fig.31 Acuteleukemia, M

–M

.a Undifferentiated blast withdense, fine chro-

matin,nucleolus (arrow),and narrowbasophilic cytoplasmwithout granules. This

cell type istypical of early myeloid leukemia (M

–M

); the finalclassification is

madeusing cellsurface markeranalysis (see Table14).b The peroxidasereaction,

characteristicof cells in themyeloid series, shows positive(& 3%) only forstage

leukemiaand higher.The imageshows aweakly positiveblast (1),strongly po-

sitiveeosinophil (2), andpositive myelocyte(3). c andd Variants ofM

leukemia.

Someof the cellsalready contain granules(1) and crystal-like Auerbodies (2).

Abnormalitiesof theWhite CellSeries

AcutePromyelocytic Leukemia (FABClassification Type M

andM

). The

characteristicfeature of thecells, which areusually quite largewith vari-

ablystructured nuclei, is extensivepromyelocytic granulation. Auer rods

arecommonly present. Cytochemistryreveals a positiveperoxidase reac-

tionfor almostall cells.All otherreactions arenonspecific. Acuteleukemia

withpredominantly bilobed nuclei is classified as a variantof M

The cytoplasm may appear eitherungranulated (M

) or very strongly

granulated(M

AcuteMyelomonocytic Leukemia (FAB Classification TypeM

).Given the

closerelationship between cellsin the granulopoieticand the monocyto-

poieticseries (seep. 3),it wouldnot be surprisingif thethese twosystems

showeda commonalteration inleukemic transformation.Thus, acutemy-

elomonocyticleukemia shows increased granulocytopoiesis (upto more

than20 % myeloblasts)with altered cell morphologies, togetherwith in-

creased monocytopoiesis yielding morethan 20% monoblasts or pro-

monocytes.Immature myeloid cells(atypical myelocytes to myeloblasts)

arefound in peripheralblood inaddition to monocyte-relatedcells. Cyto-

chemically,the classification calls for more than 3% peroxidase-positive

andmore than20% esterase-positiveblasts inthe bonemarrow. M

issim-

ilar to M

; the difference is that in the M

type the monocyte series is

stronglyaffected. In addition to theabove characteristics, the

Eovari-

antshows abnormal eosinophilswith dark purplestaining granules.

Thediagnosis of acuteleukemia is relevanteven without further

subclassification

Fig.32 Acute leukemiaM

andM

.a Blood analysis in promyelocyticleukemia

):copious cytoplasmic granules.b In type M

,multiple Auer bodiesare often

stackedlike firewood (so-calledfaggot cells). cBlood analysis invariant M

with

dumbbell-shapednuclei. Auerbodies dBone marrowcytology inacute myelomo-

nocyticleukemia M

:in addition to myeloblasts(1) and promyelocytes(2) there

arealso monocytoid cells (3). e Invariant M

Eoabnormal precursors of eosino-

philswith darkgranules are present.f Esteraseas amarker enzyme forthe mono-

cyteseries in M

leukemia.

100

AcuteMonocytic Leukemia(FAB ClassificationTypes M

5a+b

).Two morpho-

logicallydistinct forms of acute monocytic leukemias exist,monoblastic

andmonocytic. In the monoblasticvariant M

,blasts predominate inthe

bloodand bonemarrow. Theblast nucleishow adelicate chromatinstruc-

turewith several nucleoli.Often, only thefaintly grayish-bluestained cy-

toplasmhints at theirderivation.

Inmonocytic leukemia(type M

),the bonemarrow containspromono-

cytes,which aresimilar to theblasts inmonocytic leukemia, buttheir nu-

clei are polymorphic and showridges andlobes. Some promonocytes

showfaintly stained azurophilic granules. Theperipheral blood contains

monocytoidcells indifferent stagesof maturationwhich cannotbe distin-

guishedwith certaintyfrom normal monocytes.Both typesare character-

ized by strong positive esterase reactions in over 80 % of the blasts,

whereasthe peroxidasereactivity is usuallynegative, orpositive inonly a

fewcells.

AcuteEr ythroleukemia(FAB Classification TypeM

)

Erythroleukemiais a malignantdisorder of bothcell series. Itis suspected

when maturegranulocytes are virtually absent, but blasts (myeloblasts)

are present in addition to nucleated erythrocyte precursors, usually

erythroblasts(for morphology,see p. 33).The bonemarrow is completely

overwhelmedby myeloblastsand erythroblasts(more than50 %of cellsin

theprocess of erythropoiesis). Bonemarrow cytology and cytochemistry

confirm the diagnosis. Sporadically, some cases show granulopenia,

erythroblasts, andseverely dedifferentiated blasts, which correspond to

immaturered cell precursors(proerythroblasts and macroblasts).

Thedifferential diagnosis in casesof cytopenia withred blood cell pre-

cursorsfound in the CBCmust include bone marrowcarcinosis, in which

thebone marrow–blood barrieris destroyedand immature redcells (and

sometimeswhite cells)appear inthe bloodstream. Bonemarrow cytology

and/orbone marrow histologyclarifies thediagnosis. Hemolysis withhy-

persplenismcan also showthis constellation of signs.

!Fig.33 Acuteleukemia M

andM

.a Inmonoblastic leukemia M

,blasts witha

finenuclear structureand wide cytoplasmdominate theCBC. b Seeminglymatu-

re monocytes in monocytic leukemia M

. c Homogeneous infiltration of the

bonemarrow by monoblasts(M

).Only residual granulopoiesis(arrow). d Same

asc butafter esterasestaining. Thestage M

blastsshow aclear positivereaction

(redstain). There isa nonspecific-esterase (NSE)-negativepromyelocyte.

Abnormalitiesof theWhite CellSeries

101

Acute leukemias may also derive from monoblasts or erythro-

blasts

Fig.33 eSame as cOnly the myelocytein the centerstains peroxidase-positive

(browntint); themonoblasts are peroxidase-negative.f Inacute erythrocytic leu-

kemia (M

) erythroblasts and myeloblasts are usually found in the blood. This

imageof bone marrowcytology inM

showsincreased, dysplastic erythropoiesis

(e.g.,1) in additionto myeloblasts (2).

102

AcuteMegakaryoblastic Leukemia (FAB Classification TypeM

)

This form of leukemia is very rarein adults andoccurs more oftenin

children. It can also occur as “acute myelofibrosis,”with rapid onset of

tricytopenia and usually small-scale immigration into the blood of de-

differentiated medium-size d blasts without granules. Bone marrow

harvestingis difficultb ecausethe bonemarrow is veryfibrous. Onlybone

marrow histology and marker analysis (fluorescence-activated cell

sorting,FACS) can confirmthe suspected diagnosis.

The differential diagnosis, especially if the spleen is very enlarged,

shouldinclude the megakaryoblastic transformationof CML or osteomy-

elosclerosis(see pp. 112ff.), in which blastmorphology is verysimilar.

AMLwith Dysplasia

TheWHO classification (p.94) givesa specialplace to AMLwith dysplasia

intwo to threecell series, eitheras primary syndromeor following amy-

elodysplasticsyndrome (see pp.106) or amyeloproliferative disease (see

pp.114ff.).

Criteria for dysgranulopoiesis: & 50% of all segmented neutrophils

haveno granulesor very fewgranules, orshow the Pelgeranomaly, orare

peroxidase-negative.

Criteria for dyserythropoiesis: &50 % of the red cell precursor cells

display one of the following anomalies: karyorrhexis, megaloblastoid

traits,more than onenucleus, nuclear fragmentation.

Criteria for dysmegakaryopoiesis: &50% of atleast six megakaryo-

cytesshow one of the followinganomalies: micromegakaryocytes, more

thanone separate nucleus,large mononuclear cells.

HypoplasticAML

Sometimes (mostly in the mild or “aleukemic” leukemias of the FABor

WHOclassifications), the bonemarrow islargely emptyand shows onlya

fewblasts, which usuallyoccur in clusters. Insuch a case, avery detailed

analysisis essentialfor adifferential diagnosisversus aplasticanemia (see

pp.148f.).

Abnormalitiesof theWhite CellSeries

103

New WHO classification: AML with dysplasia and hypoplastic

AML

Fig.34 AMLwith dysplasia andhypoplastic AML. aAML withdysplasia: megalo-

blastoid(dysplastic) erythropoiesis (1)and dysplastic granulopoiesiswith Pelger-

Huët forms (2) and absence of granulation in a myelocyte (3). Myeloblast (4).

b Multipleseparated nuclei in a megakaryocyte (1) in AMLwith dysplasia. Dys-

erythropoiesiswith karyorrhexis (2).c andd HypoplasticAML. c Cellnumbers be-

lownormal forage inthe bonemarrow. dMagnification of thearea indicatedin c,

showingpredominance of undifferentiatedblasts (e.g., 1).

104

Table16 Immunologicalclassification ofacute bilineageleukemias (adaptedfrom BeneMC

etal. (1995)European Group forthe ImmunologicalCharacterization of Leukemias(EGIL) 9:

1783–1786)

Score B-lymphoid T-lymphoid Myeloid

2 CytCD79a* CD3(m/cyt) MP0

CytIgM anti-TCR

1 CD19 CD2 CD117

CD20 CD5 CD13

CD10 CD8 CD33

CD10 CD65

0.5 TdT TdT CD14

CD24 CD7 CD15

CD1a CD64

* CD79amay also be expressedin some casesof precursor T-lymphoblasticleukemia/lym-

phoma.

Abnormalitiesof theWhite CellSeries

AcuteLymphoblastic Leukemia (ALL)

ALLare theleukemias in whichthe cellsdo not morphologicallyresemble

myeloblasts,promyelocytes, or monocytes, nor do they show the corre-

spondingcytochemical pattern. Commonattributes are a usuallyslightly

smaller cell nucleus and denser chromatin structure, the grainy con-

sistencyof whichcan bemade outonly withoptimal smeartechnique (i.e.,

verylight). The classification as ALL isbased on the (often remote)simi-

laritiesof thecells tolymphocytes orlymphoblastsfrom lymphnodes, and

ontheir immunologicalcell markerbehavior. Insufficientlyclose morpho-

logicalanalysis canalso resultin possible confusionwith chroniclympho-

cyticleukemia (CLL),but cell surfacemarker analysis(see below)will cor-

rectthis mistake.Advanced diagnosticsstart withperoxidase andesterase

tests on fresh smears, performed in a hematologylaboratory, together

with(as a minimum) immunologicalmarker studies carried outon fresh

heparinizedblood samples in a specialist laboratory.The detailed differ-

entiationprovided by thiscell surfacemarker analysis hasprognostic im-

plicationsand sometherapeutic relevanceespecially for thedistinction to

bilineageleukemia and AML(Table

16).

105

The cellsin acute lymphocytic leukemia vary, and the subtypes

canbe reliably identified only by immunologicalmethods

Fig.35 Acutelymphocytic leukemias. a Screeningview: blasts (1)and lympho-

cytes(2) in ALL. Further classification of the blastsrequires immunological me-

thods(common ALL).b Samecase as a. Theblasts showa dense,irregular nuclear

structureand narrowcytoplasm (cf.mononucleosis, p.69). Lymphocyte(2). cALL

blasts with indentations must be distinguished from small-cell non-Hodgkin

lymphoma (e.g., mantle cell lymphoma, p.77) by cell surface marker analysis.

dBone marrow: large, vacuolated blasts,typical of B-cell ALL. Theimage shows

residualdysplastic erythropoietic cells (arrow).

106

Table17 Forms ofmyelodysplasia

Formof myelodysplasia Blood analysis Bonemarrow

RA= refractoryanemia Anemia(normo-

chromicor hyper-

chromic);possibly

pseudo-Pelgergranulo-

cytes;blasts '1 %

Dyserythropoiesis

(marginaldysgranulo-

poiesisand dysmega-

karyopoiesis" 10%)

$5% blasts

RAS= refractoryane-

miawith ringsidero-

blasts(( aquired

idiopathicsideroblastic

anemia,p. 137)

Hypochromicand

hyperchromicerythro-

cytesside byside,

sometimesdiscrete

thrombopeniaand

leukopenia;pseudo-

Pelgercells

Morethan 15%of the

redcell precursorsare

ringsideroblasts;

blasts$ 5%

RAEB= refractoryane-

miawith excessof

blasts

Oftenthrombocyto-

peniain additionto

anemia;blasts $5 %,

monocytes$ 1000/

µl,

pseudo-Pelgersyn-

drome

Erythropoietichyper-

plasia(with orwithout

ringsideroblasts);

5–20%blasts

Cont.p. 108

Abnormalitiesof theWhite CellSeries

Myelodysplasia(MDS)

Clinicalpractice has long been familiar withthe scenario in which, after

yearsof bonemarrow insufficiency witha more orless pronounced deficit

in all threecell series (tricytopenia), patients pass into a phase of insid-

iously increasing blast counts and from there into frank leukosis

—although the evolutionmay come to a halt at any of these stages.The

transitions between the forms of myelodysplasticsyndromes are very

fluid,and they havethe following featuresin common:

➤

Anemia,bicytopenia, or tricytopeniawithout known cause.

➤

Dyserythropoiesiswith sometimes pronounced erythrocyteanisocyto-

sis;in the bone marrowoften megaloblastoid cells and/orring sidero-

blasts.

➤

Dysgranulopoiesis with pseudo-Pelger-Huët nuclear anomaly (hypo-

segmentation)and hypogranulation(often no peroxidasereactivity) of

segmentedand band granulocytesin blood and bonemarrow.

➤

Dysmegakaryopoiesiswith micromegakaryocytes.

TheFAB classification isthe best-known schemeso far for organizingthe

differentforms of myelodysplasia(Table

17).

107

In unexplainedanemia and/or leukocytopenia and/or thrombo-

cytopenia,blood cell abnormalitiesmay indicate myelodysplasia

Fig.36 Myelodysplasia and CMML. a–d Different degrees of abnormal matu-

ration(pseudo-Pelger type); the nuclear density canreach that of erythroblasts

(d).The cytoplasmic hypogranulationis also observedin normal segmentedgra-

nulocytes.These abnormalitiesare seenin myelodysplasiaor afterchemotherapy,

amongother conditions.e Bloodanalysis in CMML:monocytes (1),promyelocyte

(2),and pseudo-Pelger cell(3). Thrombocytopenia.

108

Table17 Continued

Formof myelodysplasia Blood analysis Bonemarrow

CMML= chronicmyelo-

monocyticleukemia

Blasts$ 5%, mono-

cytes" 1000/

µl,

pseudo-Pelgersyn-

drome

Hypercellular,blasts

$20%, elevatedpro-

monocytes

RAEBin transformation

(RAEB

Similarto RAEBbut

"5% blasts

Blasts20–30% (some

cellscontain Auer

bodies)

* IntheWHO classification,thecategory RAEB

wouldbelongto thecategoryof acutemyeloid

leukemia.

Table18 WHO classificationof myelodysplasticsyndromes

Disease* Dysplasia** Blasts in

peripheral

blood

Blastsinthe

bonemarrow

Ringsidero-

blastsinthe

bonemarrow

Cytogenetics

5q-syndrome Usually onlyE $ 5% $5% $15 % 5qonly

RA Usually onlyDysE $1 % $ 5% $ 15% Variable

RARS Usuallyonly DysE None $ 5% & 15% Variable

RCMD 2–3 lines Rarely $5% $15% Variable

RCMD-RS 2–3 lines Rarely $5% &15% Variable

RAEB-1 1–3 lines $ 5% 5–9 % $15% Variable

RAEB-2 1–3 lines 5–19% 10–19% $15% Variable

CMML-1 1–3 lines $5 % $ 10% $15% Variable

CMML-2 1–3 lines 5–19% 10–19% $15% Variable

MDS-U 1celllineage None $5% $ 15% Variable

*RA =refractory anemia;RARS =refractory anemiawith ringsideroblasts; RCMD=refractor y

cytopeniawith morethan onedysplastic cellline; RCMD-RC=refractory cytopeniawith more

thanonedysplastic lineageandring sideroblasts;RAEB= refractoryanemia withelevatedblast

count; CMML= chronic myelomonocytic leukemia, persistent monocytosis (more than

1#10

/l) in peripheral blood; MDS-U= MDS, unclassifiable. ** Dysplasia in granulopoie-

sis=Dys G, in erythropoiesis =DysE, in megakaryopoiesis=DysM, multilineage dys-

plasia=two celllines affected;trilineage dysplasia(TLD) =all threelineage showdysplasia.

Abnormalitiesof theWhite CellSeries

Thenew WHO classif icationof myelodysplasticsyndromes definesthe

differencesin cell morphology evenmore precisely thanthe FAB classifi-

cation(Table

18).

Forthe criteria ofdysplasia, see page 106.

The“5q- syndrome” ishighlighted as aspecific type ofmyelodysplasia

inthe WHOclassification; inthe FAB classificationit wouldbe asubtype of

RAand RAS. Amacrocytic anemia, the5q- syndrome manifests withnor-

mal or increased thrombocyte counts while the bone marrow contains

megakaryocyteswith hyposegmented roundnuclei (Fig.

37b).

Naturally,bone marrow analysisis of particularimportance in the my-

elodysplasias.

109

Theclassification of myelodysplasiasrequires bone marrowana-

lysis

Fig.37 Bone marrow analysis in myelodysplasia. a Dysmegakaryopoiesis in

myelodysplasticsyndrome (MDS). Relativelysmall disk-forming megakaryocytes

(1)and multiple singular nuclei (2) areoften seen. b Mononuclearmegakaryo-

cytes(frequent in 5q-syndrome). c Dyserythropoiesis. Particularlystriking is the

coarsenuclear structurewith verylight gapsin thechromatin (arrow1). Someare

megaloblast-likebut coarser(arrow 2).dIro nstaining ofthebone marrow(Prussi-

anblue) inmyelodysplasia ofthe RARStype: dense irongranules forminga partial

ringaround the nuclei(ring sideroblasts).

110

Table19 Diagnostic work-up for anomalies in the white cell series with poly-

nucleated(segmented) cellspredominating

Clinicalfindings Hb MCH Leuko-

cytes

Segmented

cells

(%)

Lympho-

cytes

(%)

Othercells

Acutetempera-

ture,possibly

focalsigns

n n ! ! " Left shift

Patientsmokes

heavily(no

splenomegaly)

n/! n ! ! " !

Slowlydevelop-

ingfatigue,

spleen!

"/n "/n ! ! " Leftshif t

Slowlydevelop-

ingfatigue,

spleen!

" " n/!/" n/!/" " Normoblasts,

leftshift

" !! n ! ! " Somenormo-

blasts

Pruritusor

exanthema

n n n/! n n !

Eosinophils

Diagnosticstepsproceed fromleftto right.The nextstep isusuallyunnecessary; #thenext step

isobligatory.

Prevalence of Polynuclear

(Segmented) Cells

(Table19)

Neutrophilia without Left Shift

Forclarity, conditions in which mononuclear cells (lymphocytes, mono-

cytes) predominate werein the previous section kept distinct from he-

matologicalconditions inwhich cells with segmented nucleiand, insome

cases, their precursorspredominate. Leukocytosis with a predominance

ofsegmented neutrophilic granulocyteswithout the lessmature forms is

calledgranulocytosis or neutrophilia.

Abnormalitiesof theWhite CellSeries

111

Causesof Neutrophilia

➤

Allkinds of stress

➤

Pregnancy

➤

Connectivetissue diseases

➤

Tissuenecrosis, e.g., aftermyocardial or pulmonaryinfarction

➤

Acidosisof various etiologies,e.g., nephrogenic diabetesinsipidus

➤

Medications,drugs, or noxiouschemicals, such as

– Nicotine

– Corticosteroids

– Adrenaline

– Digitalis

– Allopurinol

– Barbiturates

– Lithium

– Streptomycin

– Sulfonamide

Prevalenceof Polynuclear(Segmented) Cells

Throm-

bocytes

Electro-

phore-

sis

Tentative

diagnosis

Evidence/advanced

diagnostics

Bone

marrow

Ref.

page

n n Acute bacterial

infection

(possiblywith

leukemoid

reaction)

Searchfor disease

focus

p.112

n/! n Smoker’s

leukocytosis

Anamnesis Inprogressive

disease:bone

marrowanaly-

sis(DD myelo-

proliferative

disease)

p.112

n/!/" n Chronic

myeloid

leukemia

(CML)

Cytogenetics,

BCR-ABL

Complete

bonemarrow

analysis,all

fractions

p.116

n/!/" n Osteomyelo-

sclerosis

Tear-drop-shaped

erythrocytes

Oftendry tap

#bone mar-

rowhistology

p.122

n/! n Polycythemia

withconcom-

itantleuko-

cytosis

p.162

n n Reactive

eosinophilia

Searchfor disease

focus/allergen

p.124

112

ReactiveLef t Shif t

A relativeleft shift in the granulocyte series means less mature formsin

excessof 5% band neutrophils;the preceding,less differentiated cellforms

are included and all transitional forms are taken into account. This left

shiftalmost alwaysindicates anincrease innew cellproduction inthis cell

series.In most cases, it is associated witha raised total leukocyte count.

However,since total leukocyte counts are subject to various interfering

factorsthat canalso alter thecell distribution, leftshift without leukocyto-

siscan occur, and hasno further diagnostic value.At best, if no otherex-

planationsoffer, aleft shiftwithout leukemiacan promptinvestigation for

splenomegaly, which would have prevented elevation of the leukocyte

countby increased sequestrationof leukocytes asin hypersplenism.

Inevaluating themagnitude ofa left shift,the basicprinciple isthat the

moreimmature thecell forms,the morerarely theyappear; andthat there

is a continuum starting from segmented granulocytes and sometimes

reaching asfar as myeloblasts. Accordingly, a moderate left shift ofme-

diummagnitude mayinclude myelocytes and a severeleft shift maygo as

faras afew promyelocytesand (veryrarely) myeloblasts,all dependingon

how fulminant the triggering process is and how responsive the in-

dividual. The term “pathological left shift” (for a left shift that includes

promyelocytesand myeloblasts) is inappropriate,because such observa-

tionscan reflect a very active physiologicalreaction, perhaps following a

“leukemoidreaction” with pronouncedleft shift and leukocytosis.

Causesof Reactive LeftShif t

Leftshift occurs regularlyin the following situations:

➤

Bacterialinfections (including miliary tuberculosis),

➤

Nonbacterialinflammation (e.g.,colitis, pancreatitis,phlebitis, and

connectivetissue diseases)

➤

Cellbreakdown (e.g., burns,liver failure, hemolysis)

Leftshift sometimes occurs inthe following situations:

➤

Infectionwith fungi, mycoplasm,viruses, or parasites

➤

Myocardialor pulmonary infarction

➤

Metabolicchanges (e.g., pregnancy,acidosis, hyperthyroidism)

➤

Phasesof compensation andrecuperation (hemorrhages, hemoly-

sis,or after medical orradiological immunosuppression).

Abnormalitiesof theWhite CellSeries

113

Predominanceof the granulocytic lineagewith copious granula-

tionand sporadic immature cells: usually areactive left shift

Fig.38 Leftshif t. a andb Typicalblood smearafter bacterialinfection: toxicgra-

nulation in a segmented granulocyte (1), monocyte with gray–blue cytoplasm

(2),metamyelocyte (3), and myelocyte(4). c Bloodanalysis in sepsis: promyelo-

cyte(1) andorthochromatic erythroblast (2).Thrombocytopenia. d ande Reacti-

veleft shiftas faras promyelocytes(1). Particularly strikingare thereddish granu-

lesin a bandneutrophilic granulocyte (2).

114

Chronic MyeloidLeukemia and Myeloproliferative

Syndrome (Chronic MyeloproliferativeDisorders,

CMPD)

The chronicmyeloproliferative disorders (previouslyalso called the my-

eloproliferative syndromes) include chronic myeloid leukemia (CML),

osteomyelosclerosis (OMS), polycythemia vera (PV) and essentialthrom-

bocythemia (ET). Clearly,noxious agents of unknown etiology affect the

progenitorcells at different stages of differentiation and trigger chronic

malignant proliferation in the white cell series (CML),the red cell series

(PV),and the thrombocyte series(ET). Sometimes, they leadto concomi-

tantsynthesis of fibers (OMS). Transitional formsand mixed forms exist

particularlybetween PV,ET, and OMS.

The chronic myeloproliferativedisorders encompass chronic autono-

mousdisorders ofthe bone marrowand theembryonic blood-generating

organs(spleen and liver),which may involveone or severalcell lines.

Thecommon attributesof thesediseases areonset inmiddle age,develop-

mentof splenomegaly, andslow disease progression(Table

20).

In 95% of cases, CML shows a specific chromosome aberration

(Philadelphia chromosome with a specific BCR-ABL translocation) and

maymake the transitioninto a blastcrisis.

PV and ETof tenshow similar traits (high thrombocyte count or high

Hb)and have a tendencyto secondary bone marrow fibrosis.OMS is pri-

marily characterizedby fibrosis in bone marrow and splenomegaly (see

p.122).

Abnormalitiesof theWhite CellSeries

115

Table20 Clinical characteristicsand differential diagnosticcriteria in chronic

myeloproliferativedisease

CML

(seep.116)

(seep.162)

OMS

(seep.122)

(seep.170)

Spleno-

megaly

+ No + No

Changesin

theCBC for

granulopoie-

sisand/or

erythropoie-

sis

Leukocytosis

withleft shift,

eosinophilia,

basophilia!!

Leukocytosis,

hematocrit

!!!

Tear-drop-

shapederyth-

rocytes,left

shiftin the

granulopoiesis,

normoblasts

Thrombo-

cytes

(!) (!)

Giantforms

"to (!) #450 000/

µl!

giantforms

Bonemarrow

cytology

Veryhyper-

cellular,

basophilia,

megakaryo-

cytes,and

eosinophilia

Markedly

hypercellular,

erythropoie-

sis!

Inmost cases

drytap

Mega-

karyo-

cytes

clearly

elevated

and

arranged

innests

Bonemarrow

histology

Granulopoie-

sis!, mega-

karyocytes!

Numberof

cells!

Advanced

fibrosis!

Mega-

karyo-

cytes

clearly

elevated,

arranged

innests

Philadelphia

chromosome

Yes! No No No

Otherchro-

mosomal

alterations

Inthe accel-

erationphase

andblast cri-

sis

del(20q), +8,

+9,+1q, and

others

-7,+8,+9,

+1q,and others

Veryrare

Alkaline

Leukocyte-

phosphatase

(ALP)

""! ! ! Normal to! Normalto

VitaminB

#900 pg/ml

! ! Normal Normal

Prevalenceof Polynuclear(Segmented) Cells

116

Characteristicsof CML

Ageof onset: Anyage. Peak inicidence about50 years.

Clinical findings: Slowly developing fatigue, anemia; in some cases

palpablesplenomegaly; no fever.

CBC:Leukocytosis and a left shift inthe granulocyte series; possibly

Hb", thrombocytes "or !.

Advanced diagnostics: Bone marrow, cytogenetics, and molecular

genetics(Philadelphia chromosome andBCR-ABL rearrangement).

Differential diagnosis: Reactive leukocytoses (alkaline phosphatase,

trigger?); other myeloproliferative disorders (bone marrow, cyto-

genetics,alkaline phosphatase).

Course, therapy: Chronic progression. Acute transformation after

years.New,curative drugsare currentlyunder development.Evaluate

the possibility of a bone marrow transplant (up toage approx. 60

years).

Stepsin the Diagnosis of ChronicMyeloid Leukemia

Left-shift leukocytosis in conjunction with usually low-grade anemia,

thrombocytopenia or thrombocytosis (which often correlates withthe

migrationof smallmegakaryocyte nucleiinto theblood stream),and clini-

calsplenomegaly is typical of CML.LDH and uric acidconcentrations are

elevatedas a resultof the increased cellturnover.

Theaverage“typical” cell compositionis asfollows(in a seriesanalyzed

bySpiers): about 2% myeloblasts,3 %promyelocytes, 24% myelocytes,8 %

metamyelocytes,57% band andsegmented neutrophilic granulocytes,3%

basophils,2 %eosinophils, 3% lymphocytes, and1 %monocytes.

In almost all cases of CML the hematopoietic cells display amarker

chromosome, an anomalously configured chromosome 22 (Philadelphia

chromosome).The translocationresponsible for thePhiladelphia chromo-

some corresponds toa special fusion gene (BCR-ABL) that can be deter-

mined by polymerase chain reaction (PCR) and fluorescence in situ hy-

bridization(FISH).

Abnormalitiesof theWhite CellSeries

117

b c

Left shift as far as myeloblasts, proliferation of eosinophils and

basophilssuggest chronic myeloid leukemia (CML)

Fig.39 CML.a Blood analysisin chronic myeloidleukemia (chronic phase):seg-

mented neutrophilicgranulocytes (1), band granulocyte (2) (looks like a meta-

myelocyteafter turningand foldingof thenucleus), myelocytewith defectivegra-

nulation (3),and promyelocyte (4). b and c Alsochronic phase: myeloblast (1),

promyelocyte(2), myelocytewith defectivegranulation (3), immatureeosinophil

(4),and basophil (5) (the granules are largerand darker, the nuclear chromatin

denserthan in apromyelocyte).

118

Bone Marrow Analysis in CML. In many clinical situations, the f indings

from the CBC, the BCR-ABL transformation and the enlargedspleen un-

equivocally point to a diagnosis of CML.Analysis ofthe bonemarrow

shouldbe performed because itprovides a series ofinsights into the dis-

easeprocesses.

Normally,the cell density isconsiderably elevated and granulopoietic

cells predominatein the CBC. Cells in this series mature properly, apart

froma slight leftshift in thechronic phase ofCML. CML differsfrom reac-

tiveleukocytosesb ecausethereare nosigns ofstress, suchas toxicgranula-

tionor dissociation in thenuclear maturation process.

Matureneutrophils mayoccasionallyshow pseudo-Pelgerforms (p.43)

and the eosinophilic and, especially, basophilic granulocyte counts are

often elevated. The proportionof cells from the red blood cell series

decreases. Histiocytes may store glucocerebrosides, as in Gaucher syn-

drome (pseudo-Gauchercells), or lipids in the form of sea-blue precipi-

tates(sea-blue histiocytes afterRomanowsky staining).

Megakaryocytesare usually increased and areoften present as micro-

megakaryocytes,with oneor twonuclei whichare onlyslightly largerthan

those of promyelocytes. Their cytoplasm typically shows clouds of

granules,as in thematuration of thrombocytes.

Abnormalitiesof theWhite CellSeries

119

b c

Bonemarrow analysis isnot obligatory in chronicmyeloid leuke-

mia,but helpsto distinguish betweenthe variouschronic myelo-

proliferativedisorders

Fig.40 Bone marrowcytology in CML. a Bone marrowcytology in the chronic

phase: increasedcell density due to increased, left-shifted granulopoiesis, e.g.,

promyelocytenest (1) and megakaryopoiesis (2). Eosinophils are increased (ar-

rows), erythropoiesis reduced. b Often micromegakaryocytes are found in the

bonemarrow cytology. cPseudo-Gaucher cells inthe bone marrowin CML.

120

BlastCrisis in Chronic Myeloid Leukemia

Duringthe course ofCML with or withouttherapy, regularmonitoring of

thedifferential smearis particularlyimportant, sinceover periodsof vary-

ing duration the relativeproportions of blasts and promyelocytes in-

creases noticeably.When the blast and promyelocyte fractions together

makeup 30%, and at thesame time Hb hasdecreased to lessthan 10g/dl

andthe thrombocytecount isless than1000 00/

µl,an incipientacute blast

crisismust be assumed.this blastcrisis is oftenaccompanied or preceded

bya markedly increased basophil count. Furtherblast expansion—usually

largely recalcitrant to treatment—leads to a clinical picture notalways

clearly distinguishable fromacute leukemia. If in the chronic phase the

diseasewas “latent” andmedical treatment wasnot sought, enlargement

ofthe spleen, slighteosinophilia and basophilia, andthe occasional pres-

ence of normoblasts, together with the overwhelming myeloblast frac-

tion,are all signsindicating CML as thecause of theblast crisis.

Asin AML, in two-thirds of casescytological and immunological tests

are ableto identify the blasts as myeloid. In the remaining one-thirdof

cases,the cells carrythe same markersas cells inALL. This isa sign of de-

differentiation.A final megakaryoblasticor a finalerythremic crisis is ex-

tremelyrare.

Bonemarrow cytologyis particularlyindicated whenclinical symptoms

such asfatigue, fever, and painful bones suggest an acceleration of CML

whichis notyet manifest inthe CBC.In such acase, bonemarrow analysis

willfrequently show a much moremarked shift to blasts andpromyelo-

cytesthan theCBC. Aproportion ofmore than20 %immature cellfractions

issufficient to diagnose ablast crisis.

Theprominence ofother cell series(erythropoiesis, thrombopoiesis)is

reduced.The basophil count maybe elevated.

Abone marrow aspirationmay turnout to beempty (sicca) orscarcely

yield any material. This suggests fibrosis of the bone marrow, which is

frequentlya complicating symptomof long-standing disease. Stainingof

thefibers will demonstratethis condition in thebone marrow histology.

Abnormalitiesof theWhite CellSeries

121

In the course of chronic myeloid leukemia, an acute crisis may

developin which blasts predominate

Fig.41 Acuteblast crisis in CML.a Myeloblasts (1) with somewhatatypical nu-

clearlobes. Basophilic granulocyte (2) and bandgranulocyte (3). Thrombocyto-

penia.The proliferationof basophilic granulocytesoften precedesthe blast crisis.

bMyeloblasts in an acute CMLblast crisis. Typical sand-likechromatin structure

with nucleoli.A lymphocyte. c Bone marrow cytology inacute CML blast crisis:

blastsof variable sizesaround a hyperlobulatedmegakaryocyte (in thiscase dur-

inga lymphatic blastcrisis).

122

Osteomyelosclerosis

Whenanemia accompaniedby moderatelyelevated (althoughsometimes

reduced) leukocyte counts, thrombocytopenia or thrombocytosis, clini-

callyevident splenic tumor,left shift upto and includingsporadic myelo-

blasts,and eosinophilia, the presenceof a large proportionof red cell pre-

cursors(normoblasts) inthe differentialblood analysis,osteomyelosclero-

sisshould be suspected. BCR-ABLgene analysis isnegative.

Pathologically, osteomyelosclerosis usually originates from mega-

karyocytic neoplasia in the bone marrowand theembryonic hemato-

poietic organs, particularly spleen and liver, accompanied by fibrosis

(=sclerosis) that will eventually predominate in the surrounding tissue.

The centralrole of cells of the megakaryocyte seriesis seen in the giant

thrombocytes, or even small coarsely structured megakaryocyte nuclei

withoutcytoplasm, that migrateinto the blood streamand appear in the

CBC.OMS can be a primaryor secondary disease. Itmay arise during the

course of other myeloproliferativediseases (often polycythemia vera or

idiopathicthrombocythemia).

Tough,fibrous material hampers the samplingof bone marrow mate-

rial, which rarelyyields individual cells. This in itself contributes to the

bone marrowanalysis, allowing differential diagnosis versus reactivefi-

broses(parainfectious, paraneoplastic).

Characteristicsof OMS

Ageof onset: Usuallyolder than 50 years.

Clinical findings: Signs of anemia, sometimes skin irritation, drasti-

callyenlarged spleen.

CBC:Usually tricytopenia, normoblasts,and left shift.

Further diagnostic procedures: Fibrous bone marrow (bone marrow

histology),when appropriate andBCR-ABL (always negative).

Differential diagnosis: Splenomegaly in cases of lymphadenomaor

othermyeloproliferative diseases:bone marrow analysis.

Myelofibrosis in patients with metastatic tumors or inflammation:

absenceof splenomegaly.

Course,therapy: Chronic disease progression;transformation is rare.

Ifthere issplenic pressure:possibly chemotherapy,substitutionther-

apy.

Furthermyeloproliferative diseases aredescribed together withthe rele-

vant cell systems: polycythemia vera (see p. 162)and essentialthrom-

bocythemia(see p. 170).

Abnormalitiesof theWhite CellSeries

123

Enlargedspleen and presenceof immature white cellprecursors

inperipheral blood suggest osteomyelosclerosis

Fig.42 Osteomyelosclerosis(OMS). aand bScreening ofblood cellsin OMS: red

cellprecursors (orthochromatic erythroblast= 1 andbasophilic erythroblast= 2),

basophilic granulocyte (3), and teardropcells (4).c Sometimessmall, dense

megakaryocyte nuclei arealso found in the blood stream in myeloproliferative

diseases. dBlast crisis in OMS: myeloblasts and segmented basophilic granulo-

cytes(1).

124

Elevated Eosinophiland Basophil Counts

In accordance with their physiological role, an increase in eosinophils

(#400/

µl,i.e. fora leukocyte countof 6000, morethan 8% inthe differen-

tialblood analysis) isusually due to parasiticattack (p.5). In the Western

hemisphere, parasitic infestations are investigated on the basis of stool

samplesand serology.

Strongyloidesstercoralis in particular causes strong, sometimesextreme,

elevation of eosinophils (may be up to 50%). However, eosinophilia of

variabledegree is also seenin ameba infection, inlambliasis (giardiasis),

schistosomiasis,filariasis, and evenmalaria.

Bacterialand viralinfections are bothunlikely everto lead toeosinophilia

exceptin a few patients with scarlet fever,mononucleosis, or infectious

lymphocytosis.The secondmost common groupof causes ofeosinophilia

areallergic conditions: theseinclude asthma, hay fever,and various der-

matoses (urticaria, psoriasis). This second group also includes drug-

induced hypersensitivitywith its almost infinitely multifarious triggers,

among which various antibiotics, gold preparations,hydantoin deriva-

tives, phenothiazines, and dextrans appear to be the most prevalent.

Eosinophilia is also seen in autoimmune diseases, especially in

sclerodermaand panarteritis. All neoplasias can lead to“paraneoplastic”

eosinophilia,and in Hodgkin’s disease itappears to play a specialrole in

thepathology, although itis nevertheless notalways present.

A specific hypereosinophilia syndrome withextreme values(usually

#40%) is seen clinically in association with various combinations of

splenomegaly, heart defects, and pulmonary infiltration (Loeffler syn-

drome),and is classified somewhere betweenautoimmune diseases and

myeloproliferativesyndromes. Of the leukemias, CML usually manifests

moderateeosinophilia in addition toits other typical criteria(see p.114).

When moderate eosinophilia dominates the hematological picture, the

termchronic eosinophilic leukemiais used. Acute,absolute predominance

of eosinophil blasts with concomitant decrease in neutrophils,er ythro-

cytes, and thrombocytes suggeststhe possibility of the very rare acute

eosinophilicleukemia.

ElevatedBasophil Counts. Elevationof segmentedbasophils to morethan

2–3% or 150/

µlis rare and, in accordancewith their physiological rolein

theimmune system regulation,is seen inconsistentlyin allergic reactions

tofood, drugs,or parasites (especiallyfilariae andschistosomes), i.e., usu-

ally in conditions in which eosinophilia is also seen. Infectious diseases

thatmay showbasophilia are tuberculosisand chickenpox;metabolic dis-

easeswhere basophiliamay occurare myxedemaand hyperlipidemia.Au-

tonomic proliferations of basophils are part of the myeloproliferative

Abnormalitiesof theWhite CellSeries

125

c d

Eosinophilia and basophilia are usually accompanying pheno-

mena in reactive and myeloproliferative disorders, especially

CML

Fig.43 Eosinophilia and basophilia. a Screening view of blood cellsin reactive

eosinophilia:eosinophilic granulocytes (1), segmented neutrophilicgranulocyte

(2),and monocyte(3) (reactionto bronchialcarcinoma).b andc Theimage shows

an eosinophilicgranulocyte (1) and a basophilic granulocyte (2) (clinicalosteo-

myelosclerosis). d Bone marrow in systemicmastocytosis: tissue mast cell (3),

which,in contrasttoa basophilicgranulocyte, hasan unlobednucleus, andthe cy-

toplasmis widewith atail-like extension.Tissue mastcells containintensely baso-

philicgranules.

126

Table21 Different forms of benign and malignant proliferationof tissue mast

cells

Clinicalpicture Clinical diagnosis Evidence

Inchildhood, solitaryor

multipleskin nodules,

somebrownish

Localizedmastocytosis Histology

"Sometimes transition

Diffusebrownish papules,

withurticaria onirritation

Urticariapigmentosa Typical clinical

picture

Hyperpigmentedspots,

papulesand/or dermo-

graphism;histamine

symptoms:flushing, head-

ache,pruritus, abdominal

spasms,shock

Systemicmastocytosis Histology

Malignanttransformation,

possiblywith osteolysis,

enlargedlymph nodes,

splenomegaly,hepa-

tomegaly

Malignantmastocyto-

sis*

Histology

Migrationof leukemiccells

toperipheral blood

Acutemast cell

leukemia

CBC(bone marrow)

* Mayoccur denovo withoutprecedingst agesandwithout skininvolvement.

pathologies andcan develop to the extent of being termed “chronic ba-

sophilicleukemia.” In the veryrare acute basophilic leukemia,the cells in

thebasophilic granulocytelineage maturein the bonemarrow onlyto the

stageof promyelocytes,giving a picture similarto type M

AML(p. 98).

Beingan expressionof idiopathicdisturbanceof bonemar rowfunction,

elevatedbasophil counts area relatively constantphenomenon in myelo-

proliferativesyndromes (inaddition tothe specificsigns of thesediseases),

especially in CML. Acute basophilic leukemia is extremely rare; in this

condition, some of the dedifferentiated blasts contain more or less ba-

sophilicgranules.

Thetissue-bound analogs of thesegmented basophils, the tissuemast

cells,can show benign or malignant cellproliferation, including the (ex-

tremelyrare) acute mastcell leukemia (Table

21).

Abnormalitiesof theWhite CellSeries

Erythrocyte and Thrombocyte

Abnormalities

128

Clinically RelevantClassification Principle for Anemias:

Mean Erythrocyte Hemoglobin Content (MCH)

Incurrent diagnosticpractice, erythrocyte countand hemoglobincontent

(gramsper 100ml) inwhole bloodare determinedsynchronously. Thisal-

lows calculation of the hemoglobin content per individualerythrocyte

(meancorpuscular hemoglobin,MCH) usingthe followingsimple formula

(p.10):

Hb(g/dl) · 10

Ery(10

/µl)

Themean cellvolume, hematocrit,MCH, and erythrocytesize canbe used

for variouscalculations (Table

22; methodsp. 10, normalvalues Table 2,

p.12). Despitethis multiplicity of possiblemeasures, however, inroutine

diagnosticpractice thedifferential diagnosisin casesof lowHb concentra-

tion or low erythrocyte counts relies above allon the MCH,and most

formsof anemia can safely be classified byreference to the normal data

rangeof 26–32 pgHb/cell(1.61–1.99fmol/cell) as normochromic (within

thenormal range), hypochromic (below the), orhyperchromic (above the

norm). The reticulocyte count (p.11) provides important additional

pathophysiologicalinformation. Anemiaswith increasederythrocyte pro-

duction (hyper-regenerativeanemias) suggesta high reticulocyte count,

whileanemias withdiminished erythrocyteproduction (hyporegenerative

anemias)have low reticulocytecounts (Table

22).

It should be noted that hyporegenerative anemias due to iron or vi-

tamin deficiency can rapidly display hyper-regeneration activity after

onlya short courseof treatment withiron or vitaminsupplements (up to

thedesirable “reticulocyte crisis”).

Thepractical classification ofanemia starts with theMCH:

— 26–32pg =normochromic

— Less than26 pg= hypochromic

— More than32 pg= hyperchromic

Hypochromic Anemias

Iron Deficiency Anemia

Mostanemias are hypochromic.Their usual cause isiron deficiency from

various causes (Fig.

44). To distinguish quickly between real irondefi-

ciencyand an irondistribution disorder, ironand ferritin levelsshould be

determined.

Erythrocyteand Thrombocyte Abnormalities

129

Insufficient iron absorption:

●

Lower than normal acidity, no

acidity, stomach resection,

accelerated passage from

stomach to intestines

●

Substances that inhibit ab-

sorption of iron, such as citric

acids and lactic acids, mucus and

similar materials

●

Substances that facilitate iron

absorption are missing

(e.g. vitamin C)

Endogenous redistribution:

●

Tumors, infections, lung

hemosiderosis

Insufficient iron

supplementation:

●

Iron-deficient diet

●

In newborns: insufficient

iron transfer from the

mother during gravidity

Chronic bleeding and pathological

loss of iron in the following diseases:

●

Disorders of the stomach and

intestines (positive benzidine

test)

●

Diseases of the urethra

(hematuria!)

●

Diseases of the female repro-

ductive cycle (menorrhagia,

metrorrhagia)

Increased iron requirement:

●

Growth/maturation

●

Increased production of the

new blood cells

Physiological iron loss

in females:

●

Menses

●

Lactation

●

Pregnancy

Iron deficiency:

●

Fatigue, koilonychia, infectious angle

(angulus infectiosus oris), Plummer-

Vinson syndrome, possibly “iron

deficiency fever”

●

Anemia

●

Hypochromy, ring-shaped erythrocytes

●

Light serum, deficient in color

components

●

Serum iron very reduced

Fig.44 The most important reasons for iron deficiency (according to

Begemann)

HypochromicAnemias