Laboratory Diagnosis in Neurology E-Book

Brigitte Wildemann

• Herausgeber: Thieme
• Kategorie: Fachliteratur
• Sprache: Englisch

Seitenzahl: 702

Veröffentlichungsjahr: 2010

Beschreibung

A practice-oriented reference for the laboratory diagnosis of neurologic diseases

Up-to-date, comprehensive, and beautifully illustrated, Laboratory Diagnosis in Neurology presents all the measuring parameters and methods relevant to the analysis of cerebrospinal fluid, serum, and tissues affected by neurologic disease and syndromes. Following an introduction to basic concepts, the book guides clinicians through the methods of CSF analysis, neurochemical examinations, clinical applications of neuroimmunology, microbiology and virology, neurogenetic tests, and evaluation of biopsies. Readers will learn about the equipment and various procedures, and how to effectively differentiate similar methods. In the final section of the book, the authors provide a systematic introduction to the pathophysiology and laboratory findings for specific clinical disorders, indications for particular test methods, and criteria for diagnostic interpretation.

Key features:

• Clear presentation of pearls, pitfalls, and practical tips in blue boxes for at-a-glance review
• Contributions by neurologists, psychiatrists with experience in laboratory analysis, clinical chemists, and neurochemists
• More than 140 high-quality illustrations, mostly in full color, demonstrating common findings
• Appendix with basic rules for interpreting disease-specific patterns, recommendations for quality control, and a list of the most important reference values

An indispensable tool for neurologists, laboratory physicians, and pathologists, this book is also a valuable reference for neurosurgeons, internists, and psychiatrists.

Leseprobe

Library of Congress Cataloging-in-Publication Data is available from the publisher.

This book is an authorized translation of the German edition published and copyrighted 2006 by Georg Thieme Verlag, Stuttgart, Germany. Title of the German edition: Neurologische Labordiagnostik.

Translator: Ursula Vielkind, PhD, CTran, Dundas, Canada

Illustrator: Grafik Design Roland Geyer, Weilerswist, Germany

© 2010 Georg Thieme Verlag,Rüdigerstrasse 14, 70469 Stuttgart, Germanyhttp://www.thieme.deThieme New York, 333 Seventh Avenue,New York, NY 10001, USAhttp://www.thieme.com

Cover design: Thieme Publishing GroupTypesetting by primustype R. Hurler GmbH, Notzingen, GermanyPrinted in India by Gopsons Paper Ltd, Delhi

ISBN 978-3-13-144101-0                 123456

Important note: Medicine is an ever-changing science undergoing continual development. Research and clinical experience are continually expanding our knowledge, in particular our knowledge of proper treatment and drug therapy. Insofar as this book mentions any dosage or application, readers may rest assured that the authors, editors, and publishers have made every effort to ensure that such references are in accordance with the state of knowledge at the time of production of the book.

Nevertheless, this does not involve, imply, or express any guarantee or responsibility on the part of the publishers in respect to any dosage instructions and forms of applications stated in the book. Every user is requested to examine carefully the manufacturers’ leaflets accompanying each drug and to check, if necessary in consultation with a physician or specialist, whether the dosage schedules mentioned therein or the contraindications stated by the manufacturers differ from the statements made in the present book. Such examination is particularly important with drugs that are either rarely used or have been newly released on the market. Every dosage schedule or every form of application used is entirely at the user's own risk and responsibility. The authors and publishers request every user to report to the publishers any discrepancies or inaccuracies noticed. If errors in this work are found after publication, errata will be posted at www.thieme.com on the product description page.

Some of the product names, patents, and registered designs referred to in this book are in fact registered trademarks or proprietary names even though specific reference to this fact is not always made in the text. Therefore, the appearance of a name without designation as proprietary is not to be construed as a representation by the publisher that it is in the public domain.

This book, including all parts thereof, is legally protected by copyright. Any use, exploitation, or commercialization outside the narrow limits set by copyright legislation, without the publisher's consent, is illegal and liable to prosecution. This applies in particular to photostat reproduction, copying, mimeographing, preparation of microfilms, and electronic data processing and storage.

Preface by the Editors of this Volume

Laboratory Diagnosis in Neurology gives a practice-oriented overview of the diagnostic relevance of current laboratory tests for neurological diseases and syndromes. The book is intended to provide clinicians and laboratory medical staff with information about the basics of analytical laboratory procedures, the range of indications, and the interpretation of laboratory findings. Also covered are important factors that may interfere with tests.

The book has been designed to supplement other books on laboratory diagnosis (see references in this book) as well as textbooks of neurology. In addition to cerebrospinal fluid analysis, which is of central importance to neurology as a medical specialty, the book also describes laboratory parameters that have relevance for neurological diagnosis in general. The book was written by clinical chemists and neurochemists together with neurologists and psychiatrists with experience in laboratory analysis.

The first part of the book explains the basic methods of cerebrospinal fluid analysis and of laboratory analysis in general. This overview of relevant methodological principles is unique and gives the physician important information about the classification of equipment and procedures, and about how to recognize differences in the qualities of similar methods that are of relevance for the interpretation of their results.

The second part describes the relative importance and diagnostic validity of laboratory findings for specific clinical disorders. The practice-oriented structure of the laboratory integrated cerebrospinal fluid diagnostic report, which collects the individual data of the medical report and presents them in the form of disease-related patterns, appears at the end, together with a tabulation of the most important reference values.

Our thanks are due to the late Prof. Klaus Felgenhauer for suggesting and supporting the writing of this book, and to all the authors and coauthors for their enthusiastic work on individual chapters. We thank Ms. Ursula Vielkind for the professional translation of the book and Mr. Martin Kortenhaus for substantial editing. We also thank the team at Thieme, particularly Ms. Gabriele Kuhn-Giovannini and Mr. Andreas Schabert for excellent collaboration and their helpful and constructive efforts regarding the design and printing of this book.

We would greatly appreciate critical comments from our readers.

Brigitte WildemannPatrick OschmannHansotto Reiber

Contributors

Johannes Brettschneider, MDDepartment of NeurologyUniversity Hospital Ulm, Germany

Friedrich Ebinger, MDDepartment of Child NeurologyUniversity Pediatric HospitalHeidelberg, Germany

Thomas Gasser, MDProfessorDepartment of Neurodegenerative DiseasesHertie Institute for Clinical Brain ResearchUniversity Hospital Tübingen, Germany

Heinrich Konrad Geiss, MDProfessorRhoen ClinicBad Neustadt/Saale, Germany

Patrick Oschmann, MDProfessorDepartment of NeurologyBayreuth Hospital, Germany

Markus Otto, MDProfessorDepartment of NeurologyUniversity Hospital Ulm, Germany

Hansotto Reiber, PhDProfessorCSF and Complexity StudiesUniversity Hospital Göttingen, GermanyInvited ProfessorInstituto Superior de Ciencias Medicas de La Habana, Cuba

Angela Rosenbohm, MDDepartment of PsychiatryUniversity Hospital Ulm, Germany

Erich Schmutzhard, MDProfessorDepartment of NeurologyUniversity of Innsbruck, Austria

Paul Schnitzler, PhDDepartment of VirologyHygiene InstituteUniversity Hospital Heidelberg, Germany

Anne-Dorte Sperfeld, MDHelios ClinicBad Saarow, Germany

Erwin Stolz, MDProfessorDepartment of NeurologyUniversity Hospital Giessen, Germany

Brigitte Storch-Hagenlocher, MDDepartment of NeurologyUniversity Hospital Heidelberg, Germany

Hayrettin Tumani, MDProfessorDepartment of NeurologyUniversity Hospital Ulm, Germany

Manfred Uhr, MD, PhDMax Planck Institute for PsychiatryMunich, Germany

Marlies Vogt-Schaden, MDDepartment of NeurooncologyUniversity Hospital Heidelberg, Germany

Brigitte Wildemann, MDProfessorDivision of Molecular NeuroimmunologyDepartment of NeurologyUniversity Hospital Heidelberg, Germany

Ulrich Wurster, PhDCSF Laboratory, Department of NeurologyMedical School of Hanover, Germany

Markus Zorn, MScCentral LaboratoryUniversity Hospital Heidelberg, Germany

Abbreviations

Ab

antibody

ABGA

anti-basalganglia antibody

ACE

angiotensin-convertingenzyme

AchR

acetylcholinereceptor

ACLA

anti-cardiolipin antibody

ACTH

adrenocorticotropic hormone

ADEM

acutedemyelinatingencephalomyelitis

ADC

AIDSdementia complex

AECA

anti-endothelial cell antibody

AFP

alpha fetoprotein

Ag

antigen

AHLAI

acutehemorrhagicleukoencephalitis antibodyindex

AIDP

acute inflammatory demyelinatingpoly-neuropathy

AIDS

acquired immunodeficiency syndrome

AIP

acuteintermittent porphyria

ALD

adrenoleukodystrophy

ALL

acutelymphoblastic leukemia

ALS

amyotrophic lateral sclerosis

ALS

5-aminolevulinic acid

ALT

alanine aminotransferase

AMA

anti-mitochondria antibody

AMAN

acutemotor axonal neuropathy

AMSAN

acute motorand sensoryaxonal neuropathy

ANA

antinuclear antibody

ANab

antineural antibody

ANCA

anti-neutrophilcytoplasmic antibody

ANNA

anti-neuronal nuclear antibody

APAAP

alkalinephosphatase anti-alkaline phosphatase

APase

alkaline phosphatase

ApoE

apolipoproteinE

APP

amyloid precursor protein

APS

antiphospholipid syndrome

aPTT

activated partialthromboplastin time

ASA

acetylsalicylic acid

ASL

antistreptolysin (titer)

ASAT

aspartateaminotransferase

ATG

anti-thymocyte globulin

ATPase

adenosinetriphosphatase

bAk

Bundesarztekammer (GermanMedical Association)

bDNA

branched DNA

BGA

blood gasanalysis

BHI

brain-heart infusion

BJP

BenceJones protein

BMI

bodymassindex

bp

base pair

CADASIL

cerebralautosomaldominantarteriopathy with subcortical infarcts andleukoencephal-opathy

CANOMAD

chronic ataxic neuropathy, ophthalmoplegia, IgMparaprotein, cold agglutinins, and disialosyl antibodies

CC

cell count

CCT

cranial computed tomography

CD

clusterofdifferentiation

CDC

Centersfor Disease Control andPrevention

CT

computedtomography

CDG

carbohydrate-deficient glycoprotein

cDNA

complementaryDNA

CDR

complementarydeterminingregion

CDT

carbohydrate-deficienttransferrin

CEA

carcinoembryonic antigen

CEE

Central European encephalitis

CFT

complementfixation test

CHD

coronary heart disease

CIDP

chronic inflammatorydemyelinatingpoly-neuropathy

CJD

Creutzfeldt-Jacob disease

CK

creatine kinase

CMT

Charcot-Marie-Toothdisease

CMV

cytomegalovirus

CNPase

cyclic nucleotidephosphohydrolase

CNS

central nervoussystem

COHb

carboxyhemoglobin

COX

cytochrome coxidase

CPE

cytopathic effect

CPEO

chronic progressiveexternalophthalmoplegia

CREST

calcinosis, Raynaud'sphenomenon, esophageal dysfunction, sclerodactyly, and telangiectasia

CRH

corticotropin-releasing hormone

CRMP

collapsin-responsive mediator protein

CRP

C-reactive protein

CSF

cerebrospinalfluid

CV

coefficient of variation

CZF

cerebellar zincfinger

DAD

diabetes mellitusand deafness

DGUOK

deoxyguanosine kinase

DNA

deoxyribonucleic acid

dsDNA

double-strandedDNA

DST

dexamethasone suppression test

DTR

deep tendon reflexes

EBV

Epstein-Barr virus

EEG

electroencephalogram

EDTA

ethylenediaminetetraacetic acid

EIA

enzymeimmunoassay

EITB

enzyme-linkedimmunoelectrotransfer blot

ELISA

enzyme-linked immunosorbent assay

EM

electron microscopy

EMA

epithelialmembraneantigen

ENA

extractable nuclear antigen

ERG

electroretinogram

ESR

erythrocyte sedimentationrate

ET

essentialthrombocythemia

FABP

fatty-acid-binding protein

FFI

fatal familial insomnia

FDG-PET

positron emissiontomographyusing fluoro-deoxyglucose

FIA

fluorescence immunoassay

FITC

fluorescein isothiocyanate

FPIA

fluorescence-polarization immunoassay

FRDA

Friedreich'sataxia

FSH

follicle-stimulatinghormone

FSHD

facioscapulohumeral muscular dystrophy

FSME

Fruhsommer-Meningo-Encephalitis (r CEE, TBE)

FT3

free triiodothyronine

FT4

free thyroxine

FTA-ABS

fluorescenttreponemal antibodyabsorption

FTD

frontotemporal dementia

FTDP-17

frontotemporal dementia with parkinsonism linkedtochromosome 17

FTLD

frontotemporal lobar degeneration

GAD

glutamate decarboxylase

GalNAc

N-acetylgalactosamine

GBS

Guillian-Barre syndrome

GC

gaschromatography

GFAP

glial fibrillaryacidic protein

GGT

gamma-glutamyl transferase

GH

growthhormone

GLG

gasliquid chromatography

GOT

glutamate oxaloacetate transaminase

GPL

IgG-phospholipid antibody unit

GPT

glutamate pyruvate transaminase

GROD

granular osmophilic deposits

GSS

Gerstmann-Strausslersyndrome

HAI

hemagglutination inhibition assay

Hb

hemoglobin

HbA1c

glycosylated hemoglobin

HBV

hepatitis Bvirus

HbS

sickle cell hemoglobin

HC

homocysteine

hCG

human chorionic gonadotropin

HCP

hereditarycoproporphyria

Hct

hematocrit

HCV

hepatitis Cvirus

HD

Huntington'sdisease

HDL

high-density lipoprotein

HE

hematoxylin-eosin

HIV

human immunodeficiency virus

HLA

human leukocyteantigen

HMSN

hereditarymotor sensoryneuropathy

HNA

hereditary neuralgicamyotrophy

HNPP

hereditaryneuropathywith liabilityto pressurepalsy

HSAN

hereditary sensory andautonomic neuropathy

HPLC

high-pressure liquid chromatography

HRP

horseradishperoxidase

HSP

heatshock protein

HSS

Hallervorden-Spatzsyndrome

HSV

herpes simplexvirus

IBM

inclusionbodymyositis

ICAM

intracellular adhesion molecule

ICP

intracranial pressure

IDL

intermediate densitylipoprotein

IEF

isoelectric focusing

IF

intrathecal fraction

IFT

indirect immunofluorescence test

Ig

immunoglobulin

IHA

indirect hemagglutination

IL

interleukin

INCAT

Inflammatory NeuropathyCause and Treatment(group)

INR

international normalizedratio

INSTAND

Institutefor Standardizationand Documentation in theMedical Laboratory

IRMA

immunoradiometric assay

ITPA

intrathecal Treponema pallidum antibody

JCV

John Cunningham virus

kDa

kilodalton

KSS

Kearns-Sayre syndrome

LAL

Limulus amebocyte lysate

LAMP

lysosome-associatedmembrane protein

LBD

Lewybody dementia

LC

liquidchromatography

LDH

lactate dehydrogenase

LDL

low density lipoprotein

LEMS

Lambert-Eatonmyasthenic syndrome

LH

luteinizinghormone

LHON

Leber'shereditaryoptic atrophy

LIA

luminescenceimmunoassay

LP

lumbarpuncture

LPL

lipoprotein lipase

LS

Leigh'ssyndrome

mAb

monoclonal antibody

MAD

myoadenylate deaminase

MADD

myoadenylate deaminase deficiency

MAG

myelin-associatedglycoprotein

Mb

megabase

MBP

myelin basic protein

MCH

meancorpuscularhemoglobin

MCTD

mixedconnective tissuedisease

MCV

meancorpuscular volume

MELAS

mitochondrial encephalomyelopathy, lactic acidosis, and strokelikeepisodes

MERRF

myoclonus epilepsy with ragged-red fibers

MetHb

methemoglobin

MFS

Miller-Fischer syndrome

MG

myastheniagravis

MGUS

monoclonal gammopathy of undetermined significance

MHC

majorhistocompatibilitycomplex

MID

multi-infarctdementia

MLD

metachromaticleukodystrophy

MMA

methylmalonic acid

MMN

multifocal motor neuropathy

MND

muscle-specific tyrosine kinase

MNGIE

myoneurogenicgastrointestinal encephalopathy

MOG

myelin oligodendrocyte glycoprotein

MPL

IgM-phospholipid antibody unit

MPO

myeloperoxidase

MPS

mucopolysaccharidosis

MRI

magnetic resonanceimaging

mRNA

messengerRNA

MRZ

measles, rubella, (varicella) zoster

MS

multiple sclerosis

MS

mass spectrometry

MSA

multiple system atrophy

MuSK

muscle-specific(receptortyrosine)kinase

MW

molecular weight

NAB

neutralizingantibody

NAD

nicotinamideadeninedinucleotide

NADH

reducedform of nicotinamide adenine dinucleotide

NADH-TR

ADHtetrazoliumreductase

NARP

neuropathywith ataxia andretinitis pigmentosa

NASBA

nucleic-acid-sequence-basedamplification

NAT

nucleic acid amplification technique

NC

nitrocellulose

NCL

neuronal ceroid lipofuscinosis

NF

neurofilament

NFT

neurofibrillarytangle

NHL

non-Hodgkinlymphoma

NMO

neuromyelitis optica

Nova

neuro-oncological ventral antigen

NSE

neuron-specific enolase

OCB

oligoclonalIgG band

OD

optical density

oGTT

oralglucose tolerancetest

OKT3

anti-CD3 monoclonalantibody

OMS

Opsoclonus-myoclonussyndrome

OSP

oligodendrocyte-specific protein

Osp

outersurface protein

OTC

ornithinetranscarbamylase

PA

progressiveaphasia

PAGE

polyacrylamide gelelectrophoresis

PAI

plasminogen activator inhibitor

PACNS

primaryangiitis of thecentral nervoussystem

PAN

polyarteritis nodosa

PANDAS

pediatric autoimmune neuropsychiatric dis-ordersassociated withstreptococcal Infections

PAP

peroxidase-anti-peroxidase

PAS

periodic-acid - Schiff

PBG

porphobilinogen

PBG-D

porphobilinogendeaminase

PCA

Purkinjecellantibody

PCD

paraneoplastic cerebellardegeneration

PCNSL

primaryCNS lymphoma

PCR

polymerase chain reaction

PCT

procalcitonin

PDH

pyruvatedehydrogenase

PEEP

positive end-expiratory pressure

PEM

paraneoplastic encephalomyelitis

PERM

progressive encephalitis with rigidity and myoclonus

PET

positron emissiontomography

PHF

pairedhelical filaments

Pi

inorganic phosphate

pI

isoelectric point

PLAP

placentalalkaline phosphatase

PLP

proteolipid protein

PML

progressivemultifocal leukoencephalopathy

PMP

peripheral myelinprotein

PM-Scl

polymyositis scleroderma

PNP

polyneuropathy

PNS

peripheral nervoussystem

POEMS

polyneuropathy, organomegaly, endocrinopathy, Mprotein, and skin changes

POLG

polymerase gamma

PPSB

prothrombincomplex

PrP

prion protein

PT

prothrombintime

PTT

partial thromboplastin time

PVDF

polyvinylidene difluoride

RBC

redblood cell(count)

RBP

retinol-bindingprotein

REAL

Revised European American Lymphoma (classification)

RIA

radioimmunoassay

rilibAk

Richtlinien derBundesdrztekammer (Guidelines of theGermanFederal MedicalSociety)

RNA

ribonucleic acid

RNP

ribonucleoprotein

RRA

radioreceptor assay

rRNA

ribosomalRNA

RT-PCR

reverse transcriptase polymerasechain reaction

RyR

ryanodine receptor

SAE

subcortical arteriosclerotic encephalopathy

SAH

subarachnoid hemorrhage

SAP

sphingolipid activator protein

SCA

spinocerebellar ataxia

sCD25

interleukin-2 receptor

sCJD

sporadicCreutzfeldt-Jacobdisease

Scl-70

scleroderma 70-kDa antigen

SCLC

small-cell lung carcinoma

SD

standard deviation

SD

semanticdementia

SDH

succinatedehydrogenase

SDS

sodiumdodecyl sulfate

SEP

somatosensory evoked potential

SGLPG

sulfate-3-glucuronyl-neolactose paragloboside

SGPG

sulfate-3-glucuronyl paragloboside

SGTC

secondary generalizedtonic-clonic (seizures)

SIADH

syndrome of inappropriate antidiuretic hormone

SIRS

systemic inflammatoryresponse syndrome

SLE

systemic lupus erythematosus

TPHA

Treponemapallidum hemagglutination

SMA

spinal muscularatrophy

TPLA

Treponemapallidum latex agglutination

SMN

survival motorneuron

TPO

thyroidperoxidase

SNE

subacute necrotizing encephalopathy

TPOAb

thyroid peroxidase antibody

SNV

Sin Nombre virus

TP-PA

Treponemapallidum particle agglutination

SPECT

single photon emission computed tomography

TRAb

thyrotropin receptor antibody

SPP

spastic paraplegia(paraparesis)

TRH

thyrotropin-releasinghormone

SPS

stiffpersonsyndrome

TSD

Tay-Sachs disease

SS

Sjogren'ssyndrome

TSH

thyroid-stimulatinghormone

SSN

subacute sensoryneuronopathy

TT

thrombin time

SSPE

subacute sclerosingpanencephalitis

TTR

transthyretin (prealbumin)

TBE

tick-borne encephalitis

vCJD

variant Creutzfeldt-Jacob disease

TBG

thyroxine-binding globulin

VDRL

Venereal Disease Research Laboratory

TCA

trichloroacetic acid

VEP

visual evoked potential

TDM

therapeuticdrugmonitoring

VGCC

voltage-gated calciumchannels

TFPI

tissue factorpathway inhibitor

VGKC

voltage-gated potassiumchannels

TGA, TgAb

hyroglobulin antibody

VLCFA

verylongchain fattyacids

TIA

transientischemic attack(s)

VLDL

very lowdensity lipoprotein

TLC

thin-layer chromatography

VlsE

variable lipoprotein surfaceantigen of Borrelia

TLE

temporal lobe epilepsy

VP

variegate porphyria

TNF

tumor necrosis factor

VZV

varicella-zoster virus

TP

thymidinephosphorylase

WHO

World HealthOrganization

Tp

Treponemapallidum

WNV

West Nile virus

Contents

Basics

1      Cerebrospinal Fluid: Spaces, Production, and Flow

H. Reiber

2      Blood–Brain Barrier and Blood–CSF Barrier Function

H. Reiber

Blood–Brain Barrier

The Blood–CSF Barrier Function

3      Dynamics of Serum and Brain Proteins in CSF and Blood

H. Reiber

Serum Proteins in CSF

Brain Proteins in CSF

Brain Proteins in Blood

Analysis

4      Principles of Analytical Methods

H. Reiber

Immunochemistry

Antigen–Antibody Binding

Methods of Immune Complex Analysis

Qualitative Electrophoretic Methods with or without Immunodetection

Relevance and Principle of Electrophoresis

Protein Electrophoresis

SDS Gel Electrophoresis (One-Dimensional)

Isoelectric Focusing

Electrophoresis Gels

Electrophoretic Methods with Immunodetection

Detection of Oligoclonal IgG Bands

Immunohistochemistry

5      Cerebrospinal Fluid Analysis

B. Storch-Hagenlocher, H Reiber, B. Wildemann, M. Otto

Cerebrospinal Puncture

B. Storch-Hagenlocher

Measurement of Cerebrospinal Pressure

B. Storch-Hagenlocher

Cell count, Cytology

B. Storch-Hagenlocher

Indications

Preanalytical Requirements

Procedures

Findings

Proteins

H. Reiber

Total Protein

CSF/Serum Albumin Quotient

Immunoglobulins

Quotient Diagrams (Reibergrams)

Statistics for Groups in Quotient Diagrams

Oligoclonal IgG

Antibody Index

Relative Sensitivities of Immunodetection Methods

Lactate and Glucose

H. Reiber

Lactate

Glucose

Diagnosis of Pathogens

B. Wildemann, H. K. Geiss, P. Schnitzler

General Remarks

Microscopic Detection of Pathogens

Rapid Antigen Tests

Propagation of Pathogens in Culture

Detection of Pathogen-Specific Genome Sequences Using Nucleic Acid Amplification Techniques

Antibody Detection

Markers of Dementia and Neuronal Destruction

M. Otto

6      Serum Analysis

M. Zorn, M. Uhr

General Serum Analysis

M. Zorn

Inflammation Markers

Angiotensin-Converting Enzyme (ACE)

Ceruloplasmin (Cp)

Folic Acid and Vitamin B12 (Cobalamins)

Diabetes Markers

Alcohol Markers

Endocrine Markers

Atherosclerosis Markers

Creatine Kinase (CK)

Neuron-Specific Enolase (NSE) and S-100 Protein

Special Serum Analysis

M. Uhr

Determination of Drug Levels

Intoxication

Determination of Vitamins

7      Autoantibodies and Antineural Antibodies

B. Wildemann, U. Wurster

Autoantibodies

B. Wildemann

Antineural Antibodies

U. Wurster

8      Neurogenetics

T. Gasser

Basics

Monogenic Hereditary Neurological Disorders

Polygenic or Multifactorial Hereditary Neurological Disorders

9      Biopsy

A. Rosenbohm, A. Sperfeld, H. Tumani

Brain Biopsy

Nerve Biopsy

Muscle Biopsy

Basics

Typical Muscle Biopsy Findings

Clinical Pictures

10     Inflammatory and Autoimmune Diseases

B. Storch-Hagenlocher, P. Oschmann, B. Wildemann

Infections

Bacterial Infections

B. Storch-Hagenlocher

Abscesses

B. Storch-Hagenlocher

Ventriculitis

B. Storch-Hagenlocher

Viral Infections of the Nervous System

P. Oschmann

Infection of the Nervous System by Fungi and Other Opportunistic Pathogens

P. Oschmann

Neuroborreliosis

P. Oschmann

Neurosyphilis

P. Oschmann

Chronic Infections of the Nervous System

P. Oschmann

Non-Pathogen-Related Inflammatory Disorders: Autoimmune Diseases

B. Wildemann

Multiple Sclerosis

Acute Demyelinating Encephalomyelitis and MS Variants

Neurosarcoidosis

Stiff Person Syndrome and Other Neurological Diseases Associated with GAD Antibodies

Myasthenia Gravis and Other Disorders of Neuromuscular Transmission

Lambert-Eaton Myasthenic Syndrome

Neuromyotonia

Polymyositis, Dermatomyositis, and Inclusion Body Myositis

Guillain-Barré Syndrome and Other Immune-Mediated Neuropathies

Systemic Vasculitis and Connective Tissue Diseases

Paraneoplastic Neurological Syndromes

11     Dementia and Other Psychiatric Disorders

M. Otto, M. Uhr

Alzheimer's Disease

M. Otto

Lewy Body Dementia

M. Otto

Frontotemporal Lobar Degeneration

M. Otto

Multiple System Atrophy

M. Otto

Subcortical Arteriosclerotic Encephalopathy

M. Otto

Creutzfeldt-Jakob Disease

M. Otto

Psychiatric Disorders

M. Uhr

12     Cerebral Ischemia and Hemorrhage

E. Stolz, P. Oschmann

Ischemic Cerebral Infarction

Intracerebral Hemorrhage

Subarachnoid Hemorrhage

Cerebral Hypoxia

13     Polyneuropathies

B. Wildemann

14     Pseudotumor Cerebri, Ventricular Drainage, and Neurosurgery

E. Stolz, P. Oschmann

Pseudotumor Cerebri

External Ventricular Drainage, Ventriculoatrial and Ventriculoperitoneal Shunts

Diagnosis of Cerebrospinal Rhinorrhea and Otorrhea

15     Epileptic Seizures

J. Brettschneider, H. Tumani

16     Brain Tumors

B. Storch-Hagenlocher, M. Vogt-Schaden

Primary Brain Tumors

B. Storch-Hagenlocher

Secondary Brain Tumors

M. Vogt-Schaden

Neoplastic Meningitis in Malignant Non-Hodgkin Lymphoma and Leukemia

M. Vogt-Schaden

Malignant Non-Hodgkin Lymphoma

Leukemia

Lymphomatous Meningitis

17     Metabolic Diseases

F. Ebinger

Mitochondrial Diseases

Lysosomal Diseases

Peroxisomal Diseases

Cerebrotendinous Xanthomatosis

Leukodystrophy/Leukoencephalopathy in Organic Aciduria

Disorders of Copper Metabolism

18     Parasitoses and Tropical Diseases

H. Reiber, E. Schmutzhard

Appendix

19     CSF Analysis Report

H. Reiber

Integrated CSF Report

CSF Analysis Procedure

Evaluation

Interpretation

20     Quality Assessment in the CSF Laboratory

H. Reiber

Quality Assessment in Laboratory Medicine

Internal Quality Control for CSF proteins (Total Protein, Albumin, IgG, IgA, IgM)

The INSTAND Interlaboratory CSF Survey

EQA for CSF proteins (Total Protein, Albumin, IgG, IgA, IgM)

CSF Survey: Specific Antibodies in CSF and Serum

CSF Survey: Oligoclonal IgG

Manufacturer-Related Versus Patient-Related Evaluation in Interlaboratory Surveys

CSF Survey: Lactate, Glucose and Surrogate Markers

CSF Cytology: Quality Control and Interpretation Training

21     Reference Ranges of Analytes in CSF and Serum

H. Reiber

Electrolytes and Substrates

Proteins

Amino Acids, Lipids, and Vitamins

Cells

Basics

1 Cerebrospinal Fluid: Spaces, Production, and Flow

H. Reiber

Cerebrospinal fluid. The cerebrospinal fluid (CSF) [Latin: liquor cerebrospinalis; German: Liquor (CSF), French: liquide cephalo-rachidien (LCR), Spanish: liquido cefalorracideo (LCR)] is aclear fluid containing few or no cells. Its protein concentration is very low, and its salt concentration is similar to that of blood.

Its two main physiological functions are:

• To cushion the brain against sudden movement or physical shock.

• To drain brain- and blood-derived CSF molecules into venous blood.

For the practical purposes of clinical medicine, it functions as:

• A contributing element in the diagnosis of neurological disease.

• A source of information in brain research.

• An aid to treatment monitoring.

CSF analysis plays an important role in the laboratory-supported diagnosis of neurological diseases, but its relevance depends on knowledge-based interpretation of the results. This chapter details the relevant physiological background.

CSF physiology. CSF is produced in the choroid plexus of the ventricular system, flows from the ventricles through the two lateral apertures (foramina of Luschka) and the median aperture (foramen of Magendie) into the cisterns (Fig. 1.1), and divides into the cerebral and the spinal subarachnoid space. The subarachnoid space is the space between the arachnoidea mater (the middle cerebral membrane) and the pia mater (the innermost membrane lining the cerebral tissue).The CSF drains without filtration (“bulk flow“) into the venous blood viathe arachnoid villi in the pacchionian bodies (Davson and Segal, 1996; Zettl et al., 2005). Arachnoid villi are found both in the cranium (sagittal sinus) and in the spinal subarachnoid space (spinal nerve roots).

The driving force of CSF flow is the pressure difference between arterial and venous blood.

CSF volume. In adults, the total volume of CSF is about 140mL, with the ventricles containing 12 to 23 mL on aver age and the spinal subarachnoid space containing about 30 mL. The CSF volume is influenced by heart function, respiration, coughing, and body posture, all of which cause random movements to and fro and the mixing of CSF from regions with differing flow rates.

CSF flow. In humans, CSF flow begins around the time of birth, when the arachnoid villi are maturing, and peaks about 4 months after birth, when the villi have matured. This peak in the flow rate is accompanied by a trough in protein concentration (Fig. 1.2). In adulthood, mean CSF production is 500 mL/day, and the flow rate is 0.4 mL/min. With increasing age, CSF production in the ventricles decreases and the flow rate declines to only 0.1 mL/min (May et al., 1990). CSF flow is made visible by imaging procedures (for references see Reiber, 2003).

Fig. 1.1 Subarachnoid space, CSF flow, and molecular diffusion. Produced by the choroid plexus of the ventricular system (1, first and second lateral ventricles; 2, third ventricle; 3, fourth ventricle with the two lateral apertures), the CSF flows through various apertures (4, interventricular foramen, also called foramen of Monro; 5, cerebral aqueduct, also called aqueduct of Sylvius; followed by the lateral foramina of Luschka and the median foramen of Magendie) into the cisterns (6, cerebellomedullary cistern; 7, inter-peduncular cistern; 8, chiasmatic cistern; 9, ambient cistern), after which the subarachnoid space branches into a cerebral and a spinal space, and branch. Passing through these, the CSF then drains through the arachnoid villi into the venous blood.

Fig. 1.2a,b Albumin CSF/serum quotient (QAlb; see also Table 3.1). Threshold reference values for the age-related QAlb are calculated as follows: QAlb =(4+ age in years/15) × 10−3 (for ages of >5 years).

a Around the time of birth (point 1), the albumin quotient is very high, since the arachnoid villi are still immature and the CSF flow is restricted. However, as the villi mature, the rate at which the CSF flows into the venous blood increases and the albumin quotient drops. It reaches its lowest value about 4 months after birth (point 3) when the villi are fully mature, and then slowly rises again during adult life as CSF production slows.

b The IgG quotient as a function of the albumin quotient shows that the selectivity of the barrier function is fully developed at birth (point 1). With the changing flow rate, the IgG/albumin ratio in the CSF changes hyperbolically (points 1–3 correspond to those in a).

Brain area of relevance in CSF analysis. Various observations have led to the recognition of a defined cerebral region that is of relevance in CSF analysis (Felgenhauer, 1998):

• The albumin concentration in lumbar CSF does not change if there is local dysfunction of the blood–brain barrier in the frontal, temporal, or parietal region (e. g., stroke, MS plaque).

• Local intrathecal synthesis of proteins far away from the subarachnoid space has no effect on the concentration of these proteins in the CSF; for example, brain metastases of a CEA-synthesizing tumor located in the frontal brain far away from the subarachnoid space do not change CEA levels in the cerebrospinal fluid.

Thus, the “catchment area” for CSF in the brain parenchyma is limited. This is partly because of the long diffusion distances and partly because the intercellular fluid moves very slowly (CSF flows 10 times faster). Furthermore, in the cerebellar subarachnoid space the CSF flows toward the convexity (Fig. 1.1) and drains into the superior longitudinal sinus: local changes in protein levels in this brain region are there fore not detectable in the lumbar CSF.

This physiological spatial limitation to the study of changes in the brain by CSF analysis is another example of why we have to distinguish between the blood–brain barrier and the blood–CSF barrier function.

References

Davson H, Segal MB (eds). Physiology of the CSF and blood–brain barriers. Boca Raton: CRC Press; 1996

May C, Kaye JA, Atack JR, Schapiro MD, et al. Cerebrospinal fluid production is reduced in healthy aging. Neurology 1990; 40: 500–503

Reiber H. Proteins in cerebrospinal fluid and blood: barriers, CSF flowr ate and source-related dynamics. Restor Neurol Neurosci 2003; 21: 79–96

Zettl UK, Lehmitz R, Mix E (eds). Klinische Liquordiagnostik. Berlin: Walter de Gruyter; 2005

2 Blood–Brain Barrier and Blood–CSF Barrier Function

H. Reiber

The restriction of free exchange of molecules and cells between the blood and the perivascular extracellular spaces is referred to as a “barrier.” Passage rates may vary greatly because of the different morphological structures, but all molecules, including the largest proteins, and entire cells can pass these structures, even the blood–brain barrier with its particularly tight intercellular junctions.

Blood–Brain Barrier

Structure. The blood–brain barrier is a morphologically defined structure. Its special components include capillaries, the basal membrane, and a perivascular layer of astroglial cells. Unlike other capillaries, some brain capillaries have an endothelium with cells connected by tight junctions. The tight junctions around brain capillary cells form a three-dimensional maze, which is not as impenetrable as their morphology in two-dimensional tissue sections suggests. In addition, the capillary structures in brain are not uniform but also contain fenestrated capillaries. All these morphological features are sufficient to explain the passage of large particles by passive diffusion from blood to brain and CSF. However, it is neither morphologically nor functionally justified to refer to any of these passageways as “pores.”

Permeability. The permeability and selectivity of the blood–brain barrier for proteins is determined by the diffusion of the macromolecules, which is dependent on molecular size. Properly speaking, what we are speaking of is a particular barrier function for proteins, as distinct from other facilitated or active transfer processes for other molecules—for in fact a variety of different barrier functions exist based on the different conditions of passage for different classes of substances, e. g., amino acids (Kruse et al., 1985), sugars, and vitamins (Reiber et al., 1993).

The Blood–CSF Barrier Function

Definition

It is the blood–CSF barrier function which is the really important function in CSF analysis. Empirically, it is described by the ratio of protein concentrations in venous blood and (lumbar) CSF. Unlike the blood–brain barrier, it includes dynamic aspects (e. g., CSF flow) that cannot be described in morphological terms (Reiber, 1994 a).

A blood-derived protein, such as albumin, reaches the CSF via diffusion from local blood vessels directly into the ventricles, the cisterns, and the cerebral and spinal subarachnoid spaces. Thus, its concentration increases steadily along the pathway of CSF flow, and hence also (secondarily —Reiber, 2003) depends on the flow rate (Fig. 2.1).

The multiplicity of morphological structures restricting the diffusion of molecules between blood and CSF, and the additional functional effects of CSF flow rate on CSF protein concentrations, have given rise to the term blood–CSF barrier function.

The blood–brain barrier is defined in morphological terms, whereas the blood–CSF barrier is defined in terms of function: after all, CSF protein concentrations are measured a long way away from the actual “barrier”—after a long CSF pathway with constant opportunities for exchange—and then related to the serum concentration in venous blood.

Biophysical Model of Blood–CSF Barrier Function

Comparison with earlier models. The new paradigm of blood–CSF barrier function and dysfunction was derived from the laws of diffusion (Reiber, 1994 a, 1994 b). It replaces many linear and nonlinear empirical data fits that were not supported by any physiological or biophysical theory (for references see Reiber, 1994 a). The essential difference of the new paradigm from earlier models, which assumed that the overall concentration gradient between blood and CSF was linear (Fig. 2.2 b), is that it assumes a nonlinear concentration gradient between blood and CSF, with a steady state between molecular diffusion and CSF flow rate (Figs. 2.1, 2.2 a). In this molecular flux/CSF flow model, proteins diffusing from the blood through the tissue into the CSF are eliminated by bulk flow with the passing CSF, and these two processes create a dynamic equilibrium—a steady state. Without CSF flow, the protein concentration in CSF would gradually approach the serum concentration (as it does in a corpse shortly after death, when CSF flow ceases).

Protein concentrations in CSF. The sigmoid change in tissue concentration along the diffusion barrier from the blood into the CSF (Fig. 2.2 a) is determined by:

• Diffusion.

• CSF flow rate.

The rate of protein diffusion into the CSF space—the molecular flux (J in Fig. 2.1)—depends on:

• The size of the protein molecules (Table 3.1).

• The local concentration gradient at the border between endothelium and subarachnoid space.

It is important for the mathematical treatment that the local concentration gradient (dc/dx) (Fig. 2.2 a) at the endothelial surface (s) facing the subarachnoid space is used (Reiber, 1994 a), rather than the (linear) overall concentration gradient between blood and CSF (Fig. 2.2 b).

The crucial point of the theory—particularly for the evaluation of pathological processes—is that the slope of the local concentration gradient is affected by the CSF flow rate, and in a nonlinear way.

Fig. 2.3 Hyperbolic function. The figure shows one of four branches of a complete hyperbolic function: ya/b (x2-y2)0.5–c, where a/b is the slope of the asymptote and c the coordinate transformation of the y-axis (the intersection of the asymptotes is not identical with the origin of the diagram).

With this discovery it became possible to derive a hyperbolic function (Fig. 2.3) for the description of the relationship between CSF concentrations of molecules of different sizes—for example, between QIgG and QAlb (Figs. 2.4, 2.5). Figure 2.4 shows an example of hyperbolic functions for a fraction of the empirical data collected from 4300 patients (Reiber, 1994 a) as a basis for the establishing of the reference values in the CSF/serum quotient diagrams (Reiber and Peter, 2001). This diffusion/CSF flow theory requires no assumptions to be made about the morphology of the structures participating in the blood–CSF barrier function, since the same diffusion conditions apply for all molecules whose ratio during passage is being considered. The molecularsize-dependent “selectivity” for the passage of blood proteins into the CSF space (Table 3.1) is fully explained by the molecular-size-dependent diffusion coefficient.

Impact of the CSF Flow Rate

Decreasing CSF flow rate. A pathologically reduced CSF flow rate has the following consequences (Reiber, 1994 a):

• The protein concentration in the CSF increases as a primary consequence, because the volume turnover is reduced but the molecular flux J remains constant (Figs. 2.1, 2.2 a). QC thus becomes QD (Fig. 2.2 a).

• As the result of the higher concentration in the CSF, the mean concentration in the tissue also rises (curve C becomes curve D, Fig. 2.2 a).

• This in turn alters the local concentration gradient (dc/dx) in a nonlinear fashion, thus increasing the molecular flux J in a nonlinear fashion (Fick's Second Law).

• This facilitates the entrance of molecules into the subarachnoid space (for QAlb < 0.5) and causes a further (secondary) increase in protein concentration in the CSF until a new steady state is reached (curve E, Fig. 2.2 a).

Thus, this process contains a positive feedback mechanism—like autocatalysis—which enhances the effect of a process once begun. This nonlinearity is what distinguishes this model from earlier, linear models.

Hyperbolic function. If we now relate the concentration of two molecules of different sizes in CSF (e. g., QIgG/QAlb), their ratio to one another changes as CSF flow diminishes (and the protein concentration in the CSF increases) in accordance with the following function (Reiber, 1994 a):

This function has been recognized as a hyperbolic function (Reiber, 1994 a) in which the ratio of QIgG/QAlb depends entirely on the ratio of the diffusion coefficients (DIgG/DAlb). This equation, which uses complicated trigonometric series (error function complement, erfc) for the diffusion pathway (z), can now also be described by a common hyperbolic function (Fig. 2.4):

This is the function introduced earlier on a purely empirical basis (Reiber and Felgenhauer, 1987). Parameters a/b, b, and c (see Table 5.3) have now been improved by empirical fit of the data measured for IgG, IgA, and IgM in the CSF, using a larger study group (Reiber, 1994 a). This concept is valid for all blood-derived proteins in CSF (Fig. 2.5).

Rostrocaudal Concentration Gradient

The continuous diffusion of serum molecules into the CSF from the blood vessels along the CSF flow paths, e. g., along the spine, creates a rostrocaudal concentration gradient in the lumbar subarachnoid space. This gradient explains the observation that the diffusion of molecules from the blood into lumbar CSF is faster than their diffusion into ventricular CSF. The gradient is again nonlinear (Reiber, 2003). The increasing CSF concentration down the spine also induces an increasing concentration in the tissue (Reiber, 1994 a) and consequently an increase in the local concentration gradient going down the lumbar CSF space, to be understood as locally different steady states—as in a standing wave in acoustics, with locally different amplitudes.

Fig. 2.5 Molecular-size-dependent changes in the mean CSF/serum quotients (Q) of serum proteins in CSF with diminishing CSF flow rate. The description of protein quotients as a function of the albumin quotient by means of a hyperbolic function is valid for IgG, IgA, and IgM, as well as for all other serum proteins in the CSF studied so far. Proteins with molecular sizes larger than that of albumin lie below the 45° line, and the larger the molecule, the less steep the slope of the line (see also Table 3.1). Transthyretin (TT, 54 kDa), which is associated with retinol-binding protein (RBP, 21 kDa), passes the barrier at nearly the same molecular size as albumin (67 kDa). Consequently, their quotient ratio follows a 45° line.

References

Kruse T, Reiber H, Neuhoff V. Amino acid transport across the human blood–CSF barrier. An evaluation graph for amino acid concentrations in cerebrospinal fluid. J Neurol Sci 1985;70:129–138

Reiber H. Flow rate of cerebrospinal fluid (CSF)—a concept common to normal blood–CSF barrier function and to dysfunction in neurological diseases. J Neurol Sci 1994 a;122:189–203.

Reiber H. The hyperbolic function: a mathematical solution of the protein flux/CSF flow model for blood–CSF barrier function J Neurol Sci 1994 b;126:240–242.

Reiber H. Proteins in cerebrospinal fluid and blood. Barriers, CSF flow rate and source-related dynamics. Restor Neurol Neurosci 2003;21:79–96.

Reiber H, Albaum W. Statistical evaluation of intrathecal protein synthesis in CSF/serum quotient diagrams. Acta Neuropsychiatr 2008; 20 (S 1):48–49 (www.comed-com.de)

Reiber H, Peter JB. Cerebrospinal fluid analysis—disease-related data patterns and evaluation programs. J Neurol Sci 2001;184:101–122.

Reiber H, Felgenhauer K. Protein transfer at the blood–CSF barrier and the quantitation of the humoral immune response within the central nervous system. Clin Chim Acta 1987;163:319–328

Reiber H, Ruff M, Uhr M. Ascorbate concentration in human cerebrospinal fluid (CSF) and serum. Intrathecal accumulation and CSF flow rate. Clin Chim Acta 1993;217:163–173

3 Dynamics of Serum and Brain Proteins in CSF and Blood

H. Reiber

Serum Proteins in CSF

Factors Affecting the Normal Blood CSF–Barrier Function

The concentration of a serum protein in CSF depends on its concentration in the serum: the higher the serum concentration, the higher the CSF concentration. The molecular size of a protein determines the relationship between its concentrations in CSF and serum: the larger the protein molecule, the more slowly it passes through the barriers and the steeper the concentration gradient between blood and CSF. This is demonstrated in Table 3.1 for normal conditions with an albumin quotient of 5 × 10−3. When the serum/CSF gradient for albumin is 200:1, the ratio observed for the much larger IgM molecule is 3300:1. The diagram in Fig. 2.5 illustrates how the ratio between the CSF/serum quotients of serum proteins and the albumin quotient (e. g., QIgG: QAlb) follows a theoretically based hyperbolic function. Age-related variations in the CSF flow rate and length of flow path make it necessary to determine an age-related reference range of the normal albumin quotient (normal barrier function).

Blood–CSF Barrier Dysfunction: Leakage vs. Reduced CSF Flow Rate

Many neurological diseases are accompanied by an increased protein concentration in CSF. In the past, this increased protein concentration was mistakenly thought to be caused by a change in morphological structure (leakage model). Today, this increasing concentration of serumderived proteins in CSF is explained quantitatively by a reduced CSF flow rate (Reiber, 1994 a and b; Reiber, 2003).

The leakage model was based on the assumption that the dysfunction of the blood–CSF barrier is due to a change in morphological structure—in other words, to holes (leaks) in the barrier. The model was also supposed to explain a seemingly reduced selectivity dependent on molecular size during protein transfer between blood and CSF. However, careful analysis of the empirical CSF protein data already contradicts this assumption of a morphological change. The following arguments, in particular, are immediately apparent:

• Unchanged selectivity: Even in the most severe blood–CSF barrier dysfunction, selectivity for molecules of the various sizes remains unchanged (Fig. 3.1 a; Table 3.2).

• Dynamics of proteins in blood–CSF barrier dysfunction: The data obtained from CSF of a patient with bacterial meningitis (who underwent lumbar puncture on days 1 and 2 after the first clinical symptoms) contradict the leakage model (Table 3.3). At the time of the first puncture, the albumin concentration was 47% of the value on day 2, as compared to 21% for IgG, 12% for IgA, and only 5% for IgM. According to the leakage model (with concomitant loss of selectivity), the relative increase between normal values and those obtained on day 1 (Table 3.3) should as a matter of course be larger for a larger molecule (IgM), with the steeper blood/CSF gradient, than for a smaller molecule (albumin). Clearly, however, this is not the case: the empirical changes in the quotient diagrams (Fig. 3.1 a) do not agree with the hypothesis of a leakage model, the consequences of which are presented in Fig. 3.1 b. The dynamics of the process in Fig. 3.1 a are explained quantitatively by the biophysical model (Reiber, 1994a and b; Reiber, 2003).

• Blood–brain barrier in newborns: The high total protein concentration in CSF of newborns is caused not by an immature barrier (as animal studies have shown, the barrier is already formed during the early fetal phase), but by the fact that the CSF flow starts only around time of birth with the maturation of the arachnoid villi (see Chap. 1). In CSF of the newborn, the ratio of molecules of different sizes (QIgG: QAlb) already follows the same hyperbolic function as in adults (see Fig. 1.2).

Fig. 3.1a, b Changes in the CSF concentration of immunoglobulins (QIg) as a function of increasing albumin quotients (QAlb) (blood–CSF barrier dysfunction).

a Mean values of the empirical data for the immunoglobulin CSF/serum quotients with increasing albumin quotients, which may be described as hyperbolic functions (Reiber, 1994 a).

b Experimental simulation of a leakage model in which serum proteins pass in bulk flow from the tissue into the CSF. The immunoglobulin CSF/serum quotients follow a linear function of the increasing albumin quotient. In this in-vitro experiment a patient's serum was added stepwise to the patient's CSF sample, and the resulting concentrations of IgG, IgA, IgM, and albumin were measured. Calculation of the CSF/serum quotients for each protein reveals a linear increase, in each case parallel to the 45° line. The empirical data from a large group (a), which show that the molecular-size-dependent discrimination (selectivity) is maintained even in case of the most severe barrier dysfunction, are confirmed by the extreme data from individual patients with different diseases given in Table 3.2. The leakage model (b) cannot explain the reality seen in patients, which is explained quantitatively by the diffusion/flow model.

• Early detection of serum proteins in lumbar CSF: Serum proteins can be detected earlier in lumbar CSF than in ventricular CSF (for references see Reiber, 1994 a). This is explained in the new biophysical model by the steeper local concentration gradient in the spinal subarachnoid space (Reiber, 2003).

• CSF-flow-dependent dynamics of brain proteins: The connection between barrier dysfunction and protein concentration in the CSF is valid for the passage of serum proteins from serum into CSF, but not for the passage of brain proteins into CSF. Whereas the leakage model gives no explanation of the dynamics of brain proteins, the diffusion/flow model provides a sufficient explanation of the dynamics of brain proteins of various origins. Thus, the diffusion/flow model is a more general theory explaining the dynamics of all proteins, both blood- and brain-derived.

The following physiological observations in the context of actual neurological diseases confirm our understanding of the barrier function as changes in the CSF flow rate:

• Leukemia of the CNS: This disease is primarily associated with changes in the trabeculae of the arachnoidea mater. Histopathological studies suggest a reduced CSF flow rate.

• Purulent bacterial meningitis: This disease is associated with increased CSF viscosity and meningeal adhesions. Post-mortem studies reveal protein complexes and cellular depositions in the arachnoid villi. All these features impede CSF drainage.

• Guillain–Barré syndrome: The high protein concentrations associated with this disease, too, are related to reduced CSF turnover caused by diminished outflow into the veins accompanying the spinal nerve roots, due to swellings in the area around the spinal roots.

• Complete spinal block: In the case of spinal stenosis or complete spinal block, high protein levels are measured in the lumbar CSF caudal to the blockade, despite normal cisternal and ventricular CSF levels. In contrast to bloodderived proteins, proteins originating from the brain, such as transthyretin (formerly called prealbumin), decrease relative to albumin caudal to the blockade. Here, too, the molecular-size-dependent discrimination (selectivity) for protein transfer between blood and CSF is undisturbed.

Brain Proteins in CSF

Site of synthesis of brain proteins. About 20% of the proteins in the CSF originate from the CNS (Thompson, 2005). Of these, only a few are brain-specific (i. e., synthesized exclusively in the CNS). Diagnostically relevant brain proteins come from three main sources (Reiber, 2001; Reiber, 2003):

• Proteins synthesized in the plexus epithelium (Aldred et al., 1995):

– Transthyretin (formerly called prealbumin)

– Transferrin (asialo form)

– Cystatin C

• Proteins synthesized in brain cells:

– Neuron-specific enolase (NSE) (γ-homodimer of enolase, derived from neurons)

– S-100B (β-homodimer derived from glial cells)

– Tau protein (axonal microtubules of neurons)

• Leptomeningeal proteins:

– β-Trace protein (prostaglandin D synthetase activity)

– Cystatin C

Concentration of brain proteins. Brain-derived proteins in CSF shown in Table 3.4 are characterized by the following:

• A higher concentration in the CSF than in the serum or, at least, a brain-derived fraction in the CSF that contributes more than 90%.

• A concentration that decreases from the ventricular to the lumbar CSF for proteins that originate from brain cells and choroid plexus and diffuse into the ventricular and cisternal CSF, but increases for leptomeningeal proteins (Reiber, 2001; Reiber, 2003).

• No or linear changes in cases of “barrier dysfunction” due to reduced CSF flow rate; i. e., with increasing QAlb in the lumbar CSF (Fig. 3.2), the CSF concentrations of brain cell proteins remain constant, while those of leptomeningeal proteins show a linear increase (Reiber, 2001; Reiber, 2003).

Dynamics of brain proteins. The biophysical model of the blood–CSF barrier function for blood-derived proteins allows us for the first time to provide a theoretically sound explanation of the dynamics of brain-derived proteins in the CSF (Fig. 3.2). Here, too, the change in CSF flow is sufficient to explain quantitatively the dynamics of proteins derived from glial cells and neurons, and from leptomeninges (Reiber, 2001; Reiber, 2003). The direct influence of CSF flow rate is shown by an example in which the physiologically increasing CSF flow rate induces an increasing albumin concentration: during the first few months of life, the concentration of the leptomeningeal protein cystatin C decreases in parallel with the concentration of albumin (Fig. 1.2). The positive feedback that is typical of serum proteins passing from blood into CSF does not exist for brain proteins, released from brain cells into extracellular fluid and CSF; the dynamics of these proteins therefore remain linear with changing CSF flow rates (leptomeningeal proteins, such as β-trace protein) (Reiber, 2001). Proteins derived from brain cells (e. g., tau protein, NSE) show a rostrocaudal drop in concentration (Table 3.4) and are constant in their lumbar CSF concentrations, i. e., they are independent of the CSF flow rate (Fig. 3.2)—as the theory predicts.

Fig. 3.2 Changes in the mean CSF concentrations of brain proteins (P) at increasing albumin quotients (QAlb) (“blood–CSF barrier dysfunction” due to decreasing CSF flow rate). Proteins getting into the CSF from the choroid plexus or from regions near the ventricles (tau protein, S-100, NSE) remain constant in their lumbar CSF concentrations, irrespective of CSF flow rate (compensation of molecular diffusion in and out of CSF). Proteins with pronounced leptomeningeal release (β-trace protein, cystatin C) show increasing CSF concentrations with decreasing CSF flow rate. In contrast to the hyperbolic function for serum-derived proteins (Fig. 2.5), this increase is linear because the primary increase of protein concentration in CSF in response to reduced CSF turnover does not exercise positive feedback on the rate of intracellular production or release of the proteins from the brain cells (Reiber, 2003).

Brain Proteins in the Blood

Because of their diagnostic relevance as marker proteins for cerebral dysfunctions that are measurable in blood (Schaarschmidt et al., 1994), some brain proteins are also of theoretical interest for understanding the dynamics of brain-derived proteins in blood (Reiber, 2003). Brain proteins enter the blood in two ways: by drainage of CSF into venous blood, and by diffusion through the blood–brain barrier (i. e., in the reverse direction, from brain to blood).

CSF-mediated passage. The β-trace protein is an example of CSF-mediated passage from brain to blood. The β-trace protein concentration (Table 3.4) is about 34 times higher in lumbar CSF than in blood. Based on the volume of CSF produced daily (500 mL) and the total volume of the blood (4–5 L), it has been calculated that the blood concentration is accounted for by CSF drainage (Reiber, 2003). This pathway of CSF-mediated passage into the blood is supported by an earlier report that the mean vitamin C concentration in blood decreases with decreasing CSF flow rate (barrier dysfunction), since the vitamin C concentration in CSF is 12 times higher than in blood (Reiber et al., 1993).

Diffusion from brain to blood. The second pathway by which brain-derived proteins can enter the blood is exemplified by the neuron-specific enolase (NSE). Even in the case of massive hypoxia with high NSE values in the CSF (Jacobi and Reiber, 1988), the NSE concentrations measured in the blood are too great to be explained by CSF-mediated passage. In fact, the increased release of NSE from brain cells leads to an increased concentration in the extracellular fluid and this creates a brain–blood concentration gradient, thus leading to diffusion of NSE from the brain directly into the blood vessels. For the rapid increase in NSE concentration observed in blood in, e. g., hypoxia (Schaarschmidt et al., 1994), no morphological blood–brain barrier dysfunction is required—again, diffusion is a sufficient explanation (Reiber, 2003).

For proteins moving only by diffusion from one compartment into another, the blood–brain barrier has by definition the same permeability in both directions (blood–brain and brain–blood), since—by definition—the direction of net diffusion depends only on the local concentration gradient of the protein.

References

Aldred AR, Brack CM, Schreiber G. The cerebral expression of the plasma protein genes in different species. Comp Biochem Physiol B Biochem Mol Biol 1995;111:1–15

Felgenhauer K. Protein size and cerebrospinal fluid composition. Klin Wochenschrift 1974;52:1158–1164

Jacobi C, Reiber H. Clinical relevance of increased neuron-specific enolase concentration in cerebrospinal fluid. Clin Chim Acta 1988;177:49–54

Reiber H. Flow rate of cerebrospinal fluid (CSF): a concept common to normal blood–CSF barrier function and to dysfunction in neurological diseases. J Neurol Sci 1994 a;122:189–203

Reiber H. The hyperbolic function: a mathematical solution of the protein flux/CSF flow model for blood–CSF barrier function. J Neurol Sci 1994 b;126:240–242

Reiber H. Dynamics of brain-derived proteins in cerebrospinal fluid. Clin Chim Acta 2001;310:173–186

Reiber H. Proteins in cerebrospinal fluid and blood: barriers, CSF flow rate and source-related dynamics. Restor Neurol Neurosci 2003;21:79–96

Reiber H, Felgenhauer K. Protein transfer at the blood–CSF barrier and the quantitation of the humoral immune response within the central nervous system. Clin Chim Acta 1987;163: 319–328

Reiber H, Peter JB. Cerebrospinal fluid analysis: disease-related data patterns and evaluation programs. J Neurol Sci 2001;184:101–122

Reiber H, Ruff M, Uhr M. Ascorbite concentration in human CSF and serum. Intrathecal accumulation and CSF flow rate. Clin Chim Acta 1993;217:163–173

Reiber H, Walther K, Althaus H. Beta-trace protein as sensitive marker for CSF rhinorhea and CSF otorhea. Acta Neurol Scand 2003;108:359–362

Schaarschmidt H, Prange H, Reiber H. Neuron-specific enolase concentrations in blood as a prognostic parameter in cerebrovascular diseases. Stroke 1994;24:558–565

Thomas L (ed). Labor und Diagnose. 5th ed. Frankfurt: TH-Books; 1998

Thompson, EJ. The CSF proteins: a biochemical approach. 2nd ed. Amsterdam: Elsevier; 2005

Analysis

4 Principles of Analytical Methods

H. Reiber

Immunochemistry

Antigen–Antibody Binding

Immunochemical analysis is modeled on the biological process of an immune reaction in which antibodies are produced against foreign antigens and the antigens are then eliminated as immune complexes. The formation of an immune complex, which is very specific evidence of a macromolecule, is now used as the basis for a multitude of different procedures for demonstrating the presence of molecules in body fluids and tissues. The range of techniques for quantitative detection of these immune complexes has become extremely wide; the following selection describes only the most commonly used.

Properties of Antigen–Antibody Binding

Immune complex formation. The binding of an antibody molecule to its specific antigenic determinant (epitope, see below) with the corresponding antigen creates a very stable immune complex due to the often extremely high affinity between the two molecules (Gharavi and Reiber, 1996) (Fig. 4.1).

Reaction equilibrium. Two molecules that can react with each other or that can bind to each other, such as an antigen (Ag) and an antibody (Ab), form an equilibrium between their bound state (Ag-Ab complex) and their dissociated state (free Ag and Ab molecules), as described by the following equation:

The concentration of an immune complex (AB) in equilibrium depends on both the equilibrium constant (affinity) and the concentrations of antigen and antibody.

The practical consequence is that raising the antibody concentration in the solution (e. g., at low antigen concentration) has the effect that more immune complex is formed: the velocity (vk1 × Ag × Ab) rises as the antibody concentration rises, and the equilibrium shifts in the direction of the immune complex (Ag-Ab), increasing the sensitivity of the test.

Avidity. Since both viral epitopes and antibody molecules are multivalent, their association calls for a more general presentation of the equilibrium constant, i. e., one that incorporates the effective antibody valency and the effective antigen valency. The maximum valency of the IgG molecule is 2, that of the IgA molecule is 4, and that of the IgM molecule is 5. The effective valency of viruses (with up to 1000 identical antigenic subunits) depends very much on the steric hindrance between the antibody molecules on adjacent epitopes. This physically somewhat imprecise overall attraction between an antibody and a complex antigen is called avidity (rather than affinity). For methods of determining avidity, see Gharavi and Reiber (1996).

Fig. 4.1a–d Formation of immune complexes.

a Antibody excess.

b Equivalence (optimal for precipitation).

c Antigen excess (impedes cross-linking).

d Heidelberger-Kendall curve for the intensity of scattered light as a function of antigen concentration at constant antibody concentration. Methodologically, the measuring method needs to avoid the zone of antigen excess because of the falsely low concentration measurements that result: the signal measured in this zone (right-hand point) mimics a 10-fold lower antigen concentration (left-hand point).

Specificity. The binding strength (avidity) of two macroglobulins grows with increasing numbers of complementary binding sites. This capacity of the macromolecules to reduce the dissociation constant significantly is a newly emerging property known as the binding specificity between two molecules. This means that an antibody's specificity (a quality) for a given antigen is defined by its higher affinity (a quantity) for it, compared to its affinities for other antigens.

Cross-reactivity is therefore only a question of avidity: i. e., the chances of cross-reactivity increase with decreasing avidity of the antibody.

Epitopes and Antigen–Antibody Complex

Epitopes. Proteins carry several antigenic determinants (epitopes) on their surface. These epitopes are limited structures formed by 6–8 amino acids or by carbohydrate residues. The protein–protein interaction is based on:

• Formation of noncovalent hydrogen bonds between amino acids.

• Electrostatic and hydrophobic properties.

• Van der Waals forces between the reaction components.

Antigen–antibody complex. Antisera used for protein determination usually contain different antibodies directed against many different epitopes. Furthermore, since every antibody molecule (IgG class) has two identical antigen binding sites, a three-dimensional molecular aggregate is created during formation of the immune complex with a protein antigen (Fig. 4.1 b). This is the basic principle of agglutination and precipitation methods. The classic precipitation reaction, corresponding to the equivalence zone of the Heidelberger-Kendall curve (Fig. 4.1 d) (Heidelberger and Kendall, 1935) is also the basis of immunodiffusion methods (see below). Nephelometry and turbidimetry, including particle-enhanced reactions, are used in the zone of antibody excess (Fig. 4.1 a,d).

Methods of Immune Complex Analysis