J Clin Pathol 1997;50:187-192

Origins of...

Cerebrospinal fluid analysis in clinical diagnosis

A 0 Olukoga, J Bolodeoku, D Donaldson

Department of ClinicalBiochemistry, HopeHospital, Salford,ManchesterA 0 Olukoga

Nuffield Departinentof Pathology andBacteriology,University of Oxford,John RadcliffeHospital, Headington,OxfordJ Bolodeoku

Consultant ChemicalPathologist, EastSurrey Hospital,Redhill, Surrey andCrawley Hospital,Crawley, West SussexD Donaldson

Correspondence to:Dr A 0 Olukoga,Department of ClinicalBiochemistry, HopeHospital, Eccles Old Road,Salford, Manchester M68HD.

Accepted for publication5 November 1996

IntroductionThis article reviews important milestones inthe evolution of the biochemical analysis ofcerebrospinal fluid (CSF). Historically, knowl-edge of the biochemistry of CSF lagged farbehind that ofblood and urine, mainly becausethe fluid was much less readily accessible.However, by the time procurement of CSF bylumbar puncture became established as a rou-tine clinical procedure, chemical methods forassaying a large number of analytes in bloodhad already been adequately established. Manyof these methods were later adapted to CSFanalysis and this, no doubt, facilitated morerapid development of the subject. Examinationof CSF was, indeed, the first ancillary investi-gation to be introduced into the practice ofclinical neurology and, for a considerable time,stood alone as a diagnostic aid to the physician.

It is most appropriate to regard the history ofCSF as commencing with descriptive accountsof the meninges and the ventricles; these arethe anatomical constraints of the space inwhich the fluid flows (fig 1).The earliest of these accounts can be traced

back to one of the Hippocratic writers(430-350 BC) who made reference to the falxcerebri. This was followed by descriptions ofthe meninges and ventricles-the lateral, third,and fourth-by the then two best knownphysicians of the city of Alexandria, namelyErasistratus (c 260 BC) and Herophilus (c 300BC), but Rufus of Ephesus (AD c 98-117) alsocontributed some knowledge. Subsequently,Claudius Galen of Pergamum (AD 129-99)gave the first detailed account of the ventricu-lar system; his work, however, was based on oxbrain dissections. Nevertheless, there was to bea long wait until 1543 before a more accuratedescription of the system was provided byAndreas Vesalius (1514-64). Admittedly,Leonardo da Vinci (1452-1519) had produceda wax cast of the human ventricular system inabout 1504, but his work was unknown and ithad therefore been without influence until the19th century. Vesalius's account of the ven-tricular system was extended and improvedupon by Giulio Aranzi (1530-89) in 1587; heprovided a clearer description of the temporalhorns of the lateral ventricles and of thechoroid plexus. Moreover, he was the first torefer to the passage leading from the third tothe fourth ventricle as an "aqueduct"; this pas-sage was later named after Fransois de le BoeSylvius (1614-72) (fig 2).'

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Figure 1 Portrait showing two views of the brains ofdissected heads. Courtesy of the Wellcome Institute for theHistory ofMedicine, London.

It was in the second century that ClaudiusGalen found there to be a clear fluid residue inthe ventricles of the living brain. His work hadbeen based on animal studies and he describedthe fluid as a vaporous humour produced bythe brain-which he surmised provided energyfor the whole body. This historical accountcomprised the first recognition of CSF. More-over, this description of the fluid was an exten-sion of the earlier concept of "vitalism", onwhich hinged the basic beliefs of Greekmedicine of the time as to how the human bodyfunctioned. According to their views the heartproduced a vital spirit which was the vaporouslife determinant (referred to as psychic

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Olukoga, Bolodeoku, Donaldson

Figure 2 Portrait ofFranfois de le Boe Sylvius(1614-72). Courtesy of the Wellcome Institute for theHistory of Medicine, London.

"pneuma") and which was distributed periph-erally to every organ in the body.2The next historical reference to CSF came

from Antonio Valsalva (1666-1723), but notuntil 1692 (fig 3); he drained clear fluid fromthe lumbar sac of the dog and compared it withsynovial fluid. The first really clear andcomplete description of CSF was made byDomenico Cotugno (1736-1822) in 17643; hisrecognition of continuity between the cerebraland spinal fluids can be regarded, in retrospect,as the proper beginning of modern CSF physi-ology, although this particular observationremained in obscurity for many years until itwas rediscovered by Francois Magendie(1783-1855) some 60 years later (fig 4).

Prior to Magendie's descriptions of the nor-mal presence of CSF in the brain andventricles, in both 1825 and 1827,4 5any CSFfound around the brain or within the ventriclesat autopsy was regarded as being a conse-quence of disease.

Evidence that CSF was produced by thechoroid plexus was provided in 1854 by JFaivre.6 In 1855, Hubert von Luschka (1820-75) described the lateral recesses of the fourthventricle through which CSF flowed into thesubarachnoid space. These openings, whichwere subsequently named after him and calledthe "foramina of Luschka", establish commu-nication between the ventricular and subarach-noid fluids in humans.7 The definitive demon-strations of CSF formation, its flow and itsabsorption were made in 1876 by Ernest Key(1832-1901) and Gustav Retzius (1842-1919).8

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Figure 3 Portrait ofAntonio Maria Valsalva(1666-1723). Courtesy of the Wellcome Institute for theHistory ofMedicine, London.

Figure 4 Portrait ofFrancois Magendie (1783-1855).Courtesy of the Wellcome Institute for the History ofMedicine, London.

The formal examination of CSF started withdevelopment and perfection of the technique oflumbar puncture in 1891 by Heinrich Quinke(1842-1922)9; this was achieved while he wassearching for a safe and simple way to removethe excess fluid in children with hydrocephalus.Quinke is credited as being the first to examinein detail the constituents of CSF. He counted

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Origins of ... Cerebrospinalfluid analysis in clinical diagnosis

the cells, measured total protein by theKjeldahl method and identified the presence ofbacteria in the fluid in pathological circum-stances. However, it took another 20 yearsbefore the first comprehensive description ofthe chemical composition of CSF was made in1911 by William Mestrezat (1883-1928).1oThe earliest report of the diagnostic value ofanalysing CSF biochemically was made in1893 by Ludwig Lichtheim (1845-1928) whenhe observed that glucose levels were low inboth bacterial and tuberculous meningitis."lSince that time glucose has been routinelymeasured in CSF.The bacteriological diagnosis of meningitis

was made possible at this time because ofprogress that had been made previously in thefield of microbiology. Such advances includedthe description of a technique for the stainingof bacteria in 1875 by Carl Weigert (1845-1904). This was succeeded by a method forcultivating bacteria in 1877, followed by amodification of it in 1881 in order to obtainpure cultures, both techniques having beendeveloped by Robert Koch (1843-19 10). Sub-sequently, Christian Hans Gram (b. 1853)published his now famous method for thedifferential staining of bacteria, the "Gramstain", in 1884. In the following year, theZiehl-Neelsen stain for identifying the tuberclebacillus was described; it was based on thework of Franz Ziehl (1857-1926) and Freid-rich Neelsen (1854-94).' All these procedureswere also applied to CSF. In 1906, August vonWasserman (1866-1925) applied his serologi-cal test, the "Wasserman reaction" (WR test),to CSF for the diagnosis of syphilis.'3 Thismethod was a modification of the complement-fixation test which had been described earlierby Jules Bordet (1870-1961) in 1901 12; it hadgreatly facilitated the diagnosis of generalparalysis of the insane (GPI, general paresis).Interestingly, in 1903, while working inUganda, East Africa, Aldo Castellani (1877-1971) also found the trypanosome parasite, Tgambiense, in the CSF ofpatients suffering fromsleeping sickness. 14 Diagnostic cytology ofCSF, other than routine total and differentialcell counts, commenced with the recognition ofneoplastic cells in 1904 by H Dufour"5; routineexamination of CSF for tumour cells wasthereby firmly established in confirming dis-ease of the central nervous system, especiallywhen a cerebral tumour was suspected.That the CSF occasionally contained yellow

pigment was discovered soon after lumbarpuncture had been adopted as a routine clinicalprocedure. This yellow discoloration was alsoobserved in the supernatant CSF in cases ofsubarachnoid haemorrhage by Millian andChiray in 1902 and they proposed the term"xanthochromia" to describe the phe-nomenon.'6 In the following seven years, casesof xanthochromia, each being in associationwith a seemingly different aetiopathogenesis,were described. Georges Froin (1874-1903) in1903 reported three clinical cases in which theCSF was not only yellow but also coagulated-and contained an abundance of lymphocytes;he associated this CSF "picture" (later to be

termed "Froin's syndrome") with syphiliticmeningitis.'7 In 1910, Max Nonne (1861-1959) described six cases of spinal cordtumour, each having an increase in CSFglobulin but without lymphocytosis. ThisCSF picture was, thereafter, called "Nonne'scompression syndrome"; another CSF patterncomprising an increase in protein, xanthochro-mia and spontaneous coagulation, was latertermed the "Nonne-Froin syndrome". 18 Aninsight into the mechanisms of xanthochromiawas subsequently provided in 1936 by Robin-son and Miller who produced experimentalcompression of the spinal cord in dogs andlater recovered a xanthochromic high protein-containing CSF.'9 Lawrence J Barrows et alestablished in 1955 that oxyhaemoglobin,bilirubin and methaemoglobin were pigmentsthat could be detected in CSF in certainpathological conditions.20The study of CSF proteins progressed in

1912 when Karl Friedrich Lange (b. 1883)demonstrated that the ability of certain patho-logical fluids to precipitate a gold sol could beused in the clinical diagnosis of generalparesis.2' This was the birth of the famous"colloidal gold test" which detected increasesin the globulin content of CSF. The test wasspecific for CSF and had never been used onany other body fluid-including urine andblood. The original version of the test wasimproved upon in 1944 by reducing the pH ofthe chemical reaction, a modification thatenhanced precipitation of the gold sol.22Although globulin precipitates colloidal gold,albumin has no such property. For the test,serial dilutions of CSF in saline were preparedin 10 test tubes (concentrations ranging from1:10 to 1:5120) and left to stand overnight inthe presence of cherry red colloidal goldsolution. Numerical codes for the colourchanges observed in pathological states wereallocated as follows: no change 0, a bluish tingeto the red 1, a reddish tinge to the blue 2, blue3, light blue supernatant with purple precipita-tion 4, and a colourless supernatant with com-plete precipitation of the gold 5. The colloidalgold test was used to study several neurologicalconditions; number sequences correspondedwith specific diseases for example,0012344310 equated with a meningitic curveand was seen in syphilitic or bacterial meningi-tis. Likewise, 5555432100 equated with aparetic curve and was obtained mainly in gen-eral paresis, but sometimes was seen in tabesdorsalis and multiple sclerosis.There were other qualitative methods for

detecting excess globulin in CSF which werebased on somewhat similar principles to that ofthe colloidal gold test, although they were lesssensitive and less popular. These includedPandy's test in which globulins were precipi-tated by a saturated solution of carbolic acid,and the Nonne-Apelt reaction which usedammonium sulphate for globulin precipitation.The next major landmark in CSF protein

analysis was application of the electrophoreticmethods developed by Hesselvik in 1939, thusenabling more detailed study of individual pro-teins. CSF electrophoresis was developed as a

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clinical tool in 1942 by Elvin Kabat (b. 1914)and associates who, on employing the Tiseliusfree boundary electrophoretic method, foundincreased concentrations of y globulin in thefluid of both neurosyphilis and multiple sclero-sis patients. Moreover, they showed that thesechanges were independent of those in serum,suggesting that immunoglobulins were pro-duced within the CNS in such diseases.23 In1948, Kabat et al, for the first time, used animmunochemical (precipitin) method to quan-tify albumin and globulin in human CSF with-out prior concentration of the fluid. Theyestablished reference ranges for these analytesand confirmed the increase in y globulinfractions in multiple sclerosis.24 However, asevere limitation of the precipitin method actu-ally used was that it required no less than6-7 ml of CSF for duplicate albumin and yglobulin estimations.Measurement of CSF sugar and proteins

had become established as being an importantdiagnostic aid by the first quarter of the 20thcentury. Biochemical developments in this areasubsequently concentrated on further refiningthe analytical techniques and their applicationsto measuring CSF analytes. Examples includedthe practice of interpreting CSF sugar concen-trations in relation to plasma values, which canbe traced back to 1925 when George Goodwinand Harold Shelley established the dependenceof the former on the latter.25 An importantdevelopment in CSF glucose assay during thecourse of this century was the advent of theenzymatic glucose-oxidase and hexokinasemethods. Hitherto, CSF glucose had been esti-mated principally by non-specific reducingmethods, involving reduction of copper salts.Vincent Marks (b. 1930) studied glucose inCSF, blood, and urine in 1959; he comparedlumbar and ventricular CSF glucose using theglucose-oxidase method in 1960.26 In 1973, KA Greenwald and associates used a hexokinasemethod for quantifying CSF glucose and theyre-established the previously described rela-tionship between CSF and serum glucose.27 Anew methodology for protein included the tur-bidimetric estimation of CSF total proteinusing trichloracetic acid, which had beendeveloped by William Mestrezat (1883-1928)in 1921.28 F P Kingsbury et al used sulphosali-cylic acid instead of trichloracetic acid in asimilar turbidimetric procedure in 1926.28These were the forebears of the methods incommon use today. A sensitive colorimetricmethod for CSF protein was also described byHowe Oliver Lowry in 1952 using the Folin-Ciocalteu reagent following treatment with analkaline copper solution.29The early 20th century also marked the

advent of measurement of lactate in CSF as apotential disease marker. Lactate concentra-tions were first noted to be increased in theCSF from patients with tuberculous andbacterial meningitis in 1924 by KikugoroNishimura.30 However, in marked contrast toglucose, lactate has only recently been occa-sionally used as an early indicator of bacterialmeningitis-as a differential aid in distinguish-ing this entity from aseptic meningitis.

Another diagnostic test applied to CSF andwhich was widely used during this period in theassessment of the blood-CSF barrier ("menin-geal permeability"), was the "bromide test"described by Friedrich Walter in 1925 and1929.31 Briefly, the patient takes bromide orallyfor several days; 24 hours after the last intake,blood and CSF are withdrawn and the ratio ofthe concentrations ofbromide in plasma to thatin CSF is determined; this was called the "per-meability quotient". This quotient, which innormal individuals is three, was reported to bereduced in patients with meningitis and multi-ple sclerosis.32The second half of the 20th century has wit-

nessed even more rapid development indiagnostic CSF analysis. Quantitative measure-ments of CSF immunoglobulins were refined,using very precise methods and only requiringminute quantities of fluid; these remained use-ful in the diagnosis of multiple sclerosis untilthe early 1970s. The need to distinguish trueintrathecal synthesis of immunoglobulins fromincreases in CSF protein concentrations due toleakage from plasma, as a consequence ofimpairment in the blood-CSF barrier, led todevelopment of the "immunoglobulin index"in 1972 by B Delpech and E Lichtblau.33 Thisindex relates concentrations of CSF immuno-globulins and albumin to the correspondingserum values. However, qualitative abnormali-ties in CSF immunoglobulins in the form ofoligoclonal bands were also reported in pa-tients with multiple sclerosis in 1959 by EwaldFrick, who used immunoelectophoresis, and in1967 by Hans Link, using agar gel elec-trophoresis.343 The demonstration of oligo-clonal bands in CSF by isoelectric focusing wasfirst reported in 1972 by P Delmotte.36 Sincethen, routine detection of oligoclonal bands, byisoelectric focusing followed by immunofixa-tion has supplanted quantitative CSF immuno-globulin measurements in the diagnosis ofmultiple sclerosis. Nevertheless, not all cases ofCSF oligoclonal bands will turn out to be mul-tiple sclerosis, as demonstrated in 1977 by K GPorter et al who reported oligoclonal bands inthe CSF of patients with cryptococcal menin-gitis; these bands were shown to be antibodiesto Cryptococcus neoformans.37 Inflammatory dis-orders of the CNS other than multiple sclerosishave also been shown to be associated with oli-goclonal IgG bands.38 Extensive work was alsoundertaken with proteins which enabled fur-ther understanding of the blood-CSF barrier.These studies included application of specificprotein measurements as markers of integrityof the blood-CSF barrier in different patho-logical states.39"2Many more CSF analytes were evaluated as

potential biochemical indicators of centralnervous system diseases during this period.These have included biogenic amines (for Par-kinson disease43 44), enzymes (for infectiousdiseases and neoplasia4sAB), C-reactive protein(for infectious diseases49), glutamine (forhepatic encephalopathy"0), cytokines (for infec-tious diseases and multiple sclerosis5' 52), 02microglobulin,53 neopterin,54 and myelin basicprotein55 56 (for acquired immunodeficiency

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Origins of ... Cerebrospinalfluid analysis in clinical diagnosis

syndrome dementia complex), amyloidprecursor,57 58 and acetyl cholinesterase57 (forAlzheimer disease), and I2 microglobulin,59 andneuron specific enolase60 (for neoplasia).Measurement of these analytes has been possi-ble for some time but they have not yetachieved routine application. However, withfurther clinical evaluation in well controlledstudies a few of them, such as P, microglobulin,neuron specific enolase, lactate dehydrogenase,and amyloid precursor, may yet have a role.6'

Further applications of cytological methodsto CSF examination include characterisationof B and T lymphocytes by cell surface antigenand rosetting techniques. These methods havebeen useful in evaluating immunological dis-eases of the CNS, such as multiple sclerosis;they have also enabled distinction betweeninfectious meningitis (in which T cells pre-dominate) and lymphomatous meningitis inpatients with lymphomas or leukaemia (inwhich B cells predominate).6264 However, theyare now seldom required for routine diagnosis.Rapid diagnosis of viral encephalitis by specificimmunofluorescent staining of CSF cells wasalso reported in 1973.65 Advances in themicrobiology ofCSF have included newer testsbased on immunological methods or geneamplication techniques; these hold great prom-ise for the diagnosis of infections caused byorganisms that are difficult to culture or whichare present in only very small numbers.66

ConclusionsThe origins of knowledge relating to the analy-sis of CSF, together with its diagnostic applica-tions, have been chronicled in this brief review.We have learned that the history of CSF inclinical diagnosis has lagged somewhat behindthat of urine and blood. Development of thelumbar puncture as a routine clinical proce-dure for obtaining CSF facilitated rapid growthin the formal examination of this fluid.Furthermore, the analytical techniques andinstrumentation, which had been designed forblood analysis, were then adapted to studyingCSF. This enabled intensive investigation ofthe fluid and, consequently, a fairly large bodyof information on the subject now exists. NewCSF analytes continue to emerge as potentialmarkers of CNS disease although many ofthem have yet to be accepted as routine proce-dures.

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