European Handbook of Neurological Management

Section 1 Investigations

CHAPTER 1

Routine cerebrospinal fluid (CSF) analysis

F. Deisenhammer,¹ A. Bartos,² R. Egg,¹ N. E. Gilhus,³ G. Giovannoni,⁴ S. Rauer,⁵ F. Sellebjerg,⁶ H. Tumani⁷

¹Innsbruck Medical University, Austria;

²Charles University, Prague, Czech Republic;

³University of Bergen, and Haukeland University Hospital, Bergen, Norway;

⁴University College London, Queen Square, London, UK;

⁵Albert-Ludwigs University, Freiburg, Germany;

⁶Copenhagen University Hospital, Denmark;

⁷University of Ulm, Germany

Introduction

The cerebrospinal fluid (CSF) is a dynamic, metabolically active substance that has many important functions. It is invaluable as a diagnostic aid in the evaluation of inflammatory conditions, infectious or non-infectious, involving the brain, spinal cord, and meninges, as well as in CT-negative subarachnoidal haemorrhage and in lepto-meningeal metastases. CSF is obtained with relative ease by lumbar puncture (LP). Alterations in CSF constituents may be similar in different pathologic processes and cause difficulties in interpretation. Combining a set of CSF variables referred to as routine parameters (i.e. determination of protein, albumin, immunoglobulin, glucose, lactate, and cellular changes, as well as specific antigen and antibody testing for infectious agents) will increase the diagnostic sensitivity and specificity.

The aim of this guideline paper was to produce recommendations on how to use this set of CSF parameters in different clinical settings and to show how different constellations of these variables correlate with diseases of the nervous system (table 1.1) [1].

Table 1.1 Typical constellation of CSF parameters in some neurological diseases.

Search strategy

A MEDLINE search using the search terms cerebrospinal fluid (CSF), immunoglobulin G (IgG) immunoglobulin M (IgM), immunoglobulin A (IgA), and albumin was conducted. Also, the key words ‘cerebrospinal fluid’ or ‘CSF’ were cross-referenced with ‘glucose’, ‘lactate’, ‘cytology’, ‘cell* in title’ excluding ‘child*’. Furthermore, a search for ‘cerebrospinal fluid’ and ‘immunoglobulin’ and ‘diagnosis’ and ‘electrophoresis’ or ‘isoelectric focusing’ was performed limited to the time between 1 January 1980 and 1 January 2005, and returned only items with abstracts, and English language (274 references). A search for ‘cerebrospinal fluid’ AND ‘infectious’ limited for time (1 January 1980 until now) returned 560 abstracts. Abstracts that primarily did not deal with diagnostic issues and infectious CSF (e.g. non-infectious inflammatory diseases, vaccination, general CSF parameters, pathophysiology, cytokines and therapy) were excluded, resulting in 60 abstracts. Searching the items ‘cerebrospinal fluid’ AND ‘serology’ limited for time (1 January 1980 until now) and excluding abstracts not directly related to the topic returned 35 abstracts and a search for ‘cerebrospinal fluid’ AND ‘bacterial culture’ limited for time (1 January 1980 until now) resulted in 28 abstracts.

For the current update (deadline October 2009) all the above search terms and selection criteria were applied for the time between 2005 and now.

Because this was not included in the first edition an additional MEDLINE search for the items ‘cerebrospinal fluid analysis’ AND ‘quality assurance’ from 1981 until now returned 87 references. Only 15 of these references dealt primarily with quality assurance aspects of cerebrospinal fluid analysis.

The abstracts were selected by the author in charge of the respective topic.

In addition, textbooks and articles identified in reference lists of individual papers were selected if considered appropriate.

There are no guidelines for CSF analysis published by the American Academy of Neurology (AAN). Individual task force members prepared draft statements for various parts of the manuscript. Evidence was classified as Class I–IV and recommendations as Level A–C according to the scheme agreed for EFNS guidelines [1]. When only Class IV evidence was available but consensus could be reached, the task force has offered advice as Good Practice Points (GPP) [1]. The statements were revised and adapted into a single document that was then revised until consensus was reached.

Quantitative analysis of total protein and albumin

The blood–CSF barrier is a physical barrier, consisting of different anatomical structures, for the diffusion and filtration of macromolecules from blood to CSF. The integrity of these barriers and CSF bulk flow determine the protein content of the CSF [2, 3]. In newborns, CSF protein concentrations are high, but decrease gradually during the first year of life, and are maintained at low levels in childhood. In adults, CSF protein concentrations increase with age [4, 5] (Class I). The CSF to serum albumin concentration quotient (Qalb) can also be used to evaluate blood–CSF barrier integrity [6]. The Qalb is not influenced by intrathecal protein synthesis, is corrected for the plasma concentration of albumin, and is an integral part of intrathecal immunoglobulin synthesis formulae. The Qalb is a method-independent measure, allowing the use of the same reference values in different laboratories [7, 8]. However, there are no conclusive data on how the Qalb performs compared to total protein as a measure of blood–CSF barrier function in large cohorts of unselected patients.

There is a concentration gradient for total protein and the Qalb along the neuraxis, with the lowest concentrations in the ventricular fluid and the highest concentrations in the lumbar sac [2, 9]. A significant decrease of the Qalb was observed from the first 0–4 ml of CSF to the last 21–24 ml of CSF obtained by LP [7] (Class I). The Qalb is also influenced by body weight, sex, degenerative lower back disease, hypothyroidism, alcohol consumption (Class II), and smoking (Class III) [10–13]. Posture and physical activity may influence the CSF protein concentration, resulting in higher CSF protein concentrations in inactive, bed-ridden patients [13] (Class III). Elevated CSF protein concentrations can be found in the majority of patients with bacterial (0.4–4.4 g/l), cryptococcal (0.3–3.1 g/l), tuberculous (0.2–1.5 g/l) meningitis and neuroborreliosis [14–17] (Class II). A concentration of >1.5 g/l is specific (99%), but insensitive (55%) for bacterial meningitis as compared to a variety of other inflammatory diseases [18] (Class I).

In viral neuroinfections, CSF protein concentrations are raised to a lesser degree (usually <0.95 g/l) [16] (Class II). The concentration in herpes simplex virus encephalitis is normal in half of the patients during the first week of illness [19] (Class IV).

Non-infectious causes for an increased CSF protein and sometimes with an increased cell count include sub-arachnoidal haemorrhage, central nervous system (CNS) vasculitis, and CNS neoplasm [20] (Class IV). Elevated total protein concentration with normal CSF cell count (albuminocytologic dissociation) is a hallmark in acute and chronic inflammatory demyelinating polyneuropathies but protein levels may be normal during the first week [21, 22] (Class IV). Total CSF protein is elevated in 80% of patients with leptomeningeal metastases with a range of a median concentrations between 1 and 2.4 g/l and even wider individual ranges [23, 24] (Class III). In addition, normal pressure, hydrocephalus, spinal stenosis, polyneuropathy, and high body weight and body mass index have been associated with increased CSF-serum albumin quotients [25] (Class III).

In conclusion, there is Class I evidence that increased Qalb and total CSF protein concentrations are mainly supportive of bacterial, cryptococcal, and tuberculous meningitis as well as leptomingeal metastases. As Qalb or protein is usually not the only CSF investigation, the combination with other CSF variables will increase the diagnostic specificity, like albuminocytologic dissociation in Gullain–Barré syndrome.

Quantitative intrathecal immunoglobulin synthesis

Intrathecal Ig synthesis is found in various, mainly inflammatory CNS diseases (table 1.2). There is a close correlation between the Qalb and the CSF-serum IgG concentration quotient (QIgG), which led to the development of the IgG index (QIgG/Qalb) [26–28]. Reiber’ s hyperbolic formula and Öhman’ s extended immunoglobulin indices are based on the demonstration of non-linear relationships between the Qalb and CSF-serum concentration quotients for IgG, IgA, and IgM [3, 29, 30]. For the detection of intrathecal IgG synthesis, the detection of IgG oligoclonal bands is superior to the IgG index and the non-linear formulae both in terms of diagnostic sensitivity and specificity. However, the detection of IgG oligoclonal bands is technically more demanding than the quantitative measures, and it has been suggested that in the setting of suspected multiple sclerosis (MS), oligoclonal bands analysis may be omitted in patients with an IgG-index value above 1.1, as almost 100% of such patients turn out to have intrathecally synthesized IgG oligoclonal bands (F. Deisenhammer, unpublished data).

Table 1.2 Percentage of patients in different categories of disease with elevated IgA-index, IgG-index, IgM-index, or non–linear intrathecal synthesis formula values (data from [31–35]). Unexpected increases are more common with the IgA index, IgG index, and IgM index than with corresponding non-linear formulae.

In studies comparing CSF findings in patients with MS and other neurological diseases, non-linear formulae were superior [33, 34]. Intrathecal IgA, IgG, and IgM synthesis formulae may be helpful in discriminating between different infectious diseases of the nervous system [36, 37] (Class III). However, one study suggested that increased values of the Reiber formula do not always reflect intrathecal IgM synthesis as increased values were observed in several patients with non-inflammatory diseases without IgM oligoclonal bands in CSF [38] (Class II). In conclusion, there is no evidence to support the routine use of quantitative assessment of intrathecal immunoglobulin synthesis in the diagnosis of neurological diseases, but in the setting of suspected MS, the IgG index may be used as a screening procedure to determine intrathecal IgG synthesis.

Qualitative (oligoclonal) intrathecal IgG synthesis

The detection of intrathecal oligoclonal IgG in the CSF is useful diagnostically, particularly as it is one of the laboratory criteria supporting the clinical diagnosis of MS [39]. In addition, it can be used to assist in the diagnosis of other putative autoimmune disorders of the CNS, such as paraneoplastic disorders and CNS infections [40–42].

Using electrophoresis techniques it is possible to classify the humoral responses according to the number of antibody clones produced (i.e. monoclonal, oligoclonal, and polyclonal responses; figure 1.1). Earlier methods have now been superseded by the development of the more sensitive technique of isoelectric focusing (IEF) and immunofixation [6].

Figure 1.1. IEF immunoblots of the five consensus patterns of various CSF and serum isoelectric focusing patterns for local/ systemic synthesis. The pattern number is given above the paired samples.

Type 1 (C −S −): No bands in CSF and serum. Normal.

Type 2 (C + S −): Oligoclonal IgG is present in the CSF with no apparent corresponding abnormality in serum, indicating local intrathecal synthesis of IgG. Typical example: MS.

Type 3 (C + > S +): There are IgG bands in both the CSF and serum, with additional bands present in the CSF. The oligoclonal bands that are common to both CSF and serum imply a systemic inflammatory response, whereas the bands that are restricted to the CNS suggest that there is an additional CNS-only response.

Typical examples: MS, systemic lupus erythematosus (SLE), sarcoid, etc.

Type 4 (C + S +): There are oligoclonal bands present in the CSF, which are identical to those in serum. This is not indicative of local synthesis, but rather, the pattern is consistent with passive transfer of oligoclonal IgG from a systemic inflammatory response. Typical examples: Guillain–Barré syndrome, acute disseminated encephalomyelitis (ADEM), and systemic infections.

Type 5 (Para): There is a monoclonal IgG pattern in both CSF and serum, the source of which lies outside the CNS. Typical examples: Myeloma, monoclonal gammopathy of undetermined significance (MGUS).

Isoelectric focusing uses a pH gradient to separate IgG populations on the basis of charge, which are then transferred onto a nitro-cellulose or other membrane before immunostaining using an anti-human immunoglobulin [43]. Some laboratories continue to use silver staining to detect oligoclonal bands (OCBs) with good results [44].

As CSF is an ultrafiltrate of plasma, it contains immunoglobulins that are passively transferred from the plasma, as well as immunoglobulins synthesized locally. Any systemic pattern of immunoglobulin production seen in plasma or serum will therefore be mirrored in the CSF. It is imperative that any CSF analysis for oligoclonal bands is accompanied by a paired blood analysis.

An oligoclonal intrathecal IgG antibody response is not specific. Table 1.3 provides a list with the proportion of cases with oligoclonal bands (for a more detailed list please see [32]). Local synthesis of oligoclonal bands is therefore not diagnostic and has to be interpreted in the clinical context. A recently published recommendation regarding detection of oligoclonal bands concluded as follows [45]:

The single most informative analysis is a qualitative assessment of CSF for IgG, best performed using IEF together with some form of immunodetection (blotting or fixation). This qualitative analysis should be performed using unconcentrated CSF and must be compared directly with serum run simultaneously in the same assay in an adjacent track. Optimal runs utilize similar amounts of IgG from paired serum and CSF. Recognised positive and negative controls should be run with each set of samples.

In putative non-infectious inflammatory disorders of the CNS there is Class I evidence to support the use of CSF IEF for both predictive and diagnostic testing in the diagnosis of MS. In other non-infectious inflammatory disorders of the CNS, Class II and III evidence exists to support the use of CSF IEF to supplement other diagnostic tests (table 1.3).

Table 1.3 Inflammatory diseases of the CNS associated with CSF oligoclonal IgG bands [32].

CNS, central nervous system; CSF, cerebrospinal fluid; IgG, immunoglobulin G; SLE, systemic lupus erythematosus.

aThis is based on studies using the Poser diagnostic criteria [46] that were validated against the original Schumacher criteria [47]. None of these criteria has been validated using population-based studies. Therefore, it could be argued that the diagnostic ‘gold standard’ is a flawed standard.

CSF glucose concentration, CSF/serum glucose ratio and lactate

As glucose is actively transported across the blood–brain barrier the CSF glucose levels are directly proportional to the plasma levels and therefore simultaneous measurement in CSF and blood is required. Normal CSF glucose concentration is 50–60% of serum values [20] (Class IV). A CSF/serum glucose ratio less than 0.4–0.5 is considered to be pathological [48] (Class IV). CSF glucose takes several hours to equilibrate with plasma glucose; therefore, in unusual circumstances, levels of CSF glucose can actually be higher than plasma levels for several hours. During CSF storage glucose is degraded. Therefore, glucose determination must be performed immediately after CSF collection.

A high CSF glucose concentration has no specific diagnostic importance and is related to an elevated blood glucose concentration, for example in diabetics.

The behaviour of the CSF/serum glucose ratio in different neurological diseases is shown in table 1.1.

The relevance of CSF lactate is similar to that of the CSF/serum glucose ratio. CSF lactate is independent of blood concentration [49] (Class IV). The normal value is considered to be <2.8–3.5 mmol/l [50] (Class II). Except for mitochondrial disease, CSF lactate correlates inversely with CSF/serum glucose ratio. An increased level can be detected earlier than the reduced glucose concentration.

Decreased CSF/serum glucose ratio or increased CSF lactate indicates bacterial and fungal infections or lepto-meningeal metastases.

Cytological examination

Cytological evaluation should be performed within 2 h after puncture, preferably within 30 min because of a lysis of both red blood cells and white blood cells [51] (Class IV).

Cerebrospinal fluid leukocytes are usually counted in a Fuchs-Rosenthal chamber (volume 3.2 µl) and therefore counts are reported as ‘/3’ cells to correct for a standard volume of 1 µl. A cytocentrifuge (cytospin), the Sayk sedimentation chamber, or membrane filtration can be used to obtain a sufficient number of cells for cytology [52]. For cellular differentiation May–Gruenwald–Giemsa staining is widely used but specific methods may be performed, especially for the detection of malignant cells [53, 54] (Class II).

Lymphocytes and monocytes at the resting phase and occasionally ependymal cells are found in normal CSF.

An increased number of neutrophilic granulocytes can be found in bacterial and acute viral CNS infections [54, 55] (Class II). In the postacute phase a mononuclear transformation occurs.

Upon activation, lymphocytes can enlarge or become plasma cells indicating an unspecific inflammatory reaction [54, 56] (Class IV). Resting monocytes enlarge and display vacuoles when activated. Macrophages are the most activated monocytes. These cell forms can occur in a great variety of diseases.

Erythrophages occur 12–18 h after haemorrhage. Siderophages containing haemosiderin are seen as early as 1–2 days after haemorrhage and may persist for weeks. Macrophages containing haematoidin (crystallized bilirubin) degraded from haemoglobin may appear about 2 weeks after bleeding and are a sign of a previous subarachnoid bleeding [54] (Class IV). However, spectrophotometry of CSF involving bilirubin quantitation has been recommended as the method of choice to prove CT-negative subarachnoid bleeding up to 2 weeks after onset [57].

Lipophages indicate CNS tissue destruction. The presence of macrophages without detectable intracellular material is a non-specific finding, occurring in disc herniation, malignant meningeal infiltration, spinal tumours, head trauma, stroke, MS, vasculitis, infections, and subarachnoid haemorrhage [54] (Class IV).

Eosinophils are normally not present in CSF. The presence of 10 or more eosinophils/µl in CSF or eosinophilia of at least 10% of the total CSF leukocyte count is associated with a limited number of diseases, including parasitic infections and coccidioiodomycosis. It can occur in malignancies and react to medication and ventriculo-peritoneal shunts [58].

Malignant CSF cells indicate leptomeningeal metastases. False-positive results often occur when inflammatory cells are mistaken for tumour cells or due to contamination with peripheral blood [59]. False-negative detection of malignant cells on cytologic examination of CSF is common. Factors increasing the detection rate of malignant cells include a volume of at least 10.5 ml and repeating this procedure once if the cytology is negative. The detection rate of 50–70% after the first investigation can be increased to 85–92% after a second puncture [60] (Class III). Further LPs will only slightly increase the diagnostic sensitivity [61, 62] (Class III).

In conclusion, cell count is generally useful because most of the indications for CSF analysis include diseases that are associated with elevated numbers of various cells. Cytological staining can be helpful in distinguishing CNS diseases when the cell count is increased.

Investigation of infectious CSF

There are many small to medium-sized studies investigating the diagnostic sensitivity and specificity of tests for various infectious agents but no controlled study evaluating a work-up of infectious CSF in general. Therefore, there are no valid data on the indication, sensitivity, and specificity of microbiological procedures in general (i.e. how to proceed with CSF in obvious CNS infections). Existing proposals for the general work-up of infectious CSF are based on clinical practice and theoretically plausible procedures [63–65].

There are a great number of methods for antigen or specific antibody detection and their use depends mainly on the type of antigen (table 1.4).

Table 1.4 List of infectious agents responsible for the vast majority of infectious CNS diseases.

The following pathogens should be considered in acute myelitis [Recommendation Level B]: HSV type 1 and 2 (PCR), VZV (PCR), enteroviruses (PCR), Borrelia burgdorferi sensu latu (serology, AI), HIV (serology), tick-borne encephalitis virus (only in endemic areas) (serology, AI).

aNested PCR technique has been shown to be substantially more sensitive and specific than conventional single step PCR techniques [66].

* * Culture from CSF and blood;

* * * aerobic and anaerobic culture from abscess aspirate, CSF, and blood.

In neuroinfections specific antigen or antibody detection should be performed depending on the clinical presentation and the results of basic CSF analysis. The formula for the estimation of the relative intrathecal synthesis of specific antibodies in the CSF (Antibody Index [AI] is as follows:

Estimation of intrathecal synthesis of specific antibodies in the CSF (Antibody Index [AI])

AI=Antibody ratio/IgGratio (positive > 1, 5)

Cerebrospinal fluid polymerase chain reaction can be performed rapidly and inexpensively and has become an integral component of diagnostic medical practice. A patient with a positive PCR result is 88 times more likely to have a definite diagnosis of viral infection of the CNS as compared to a patient with a negative PCR result. A negative PCR result can be used with moderate confidence to rule out a diagnosis of viral infection of the CNS (the probability of a definite viral CNS infection was 0.1 in case of a negative PCR result compared to a positive PCR result) [67]. It should be considered that false-negative results are most likely if the CSF sample is taken within the first 3 days after the illness or 10 days and more after the onset of the disease [68, 69].

In general, PCR is indicated in the following situations:

when microscopy, culture or serology is insensitive or inappropriate;

when culture does not yield a result despite clinical suspicion of infectious meningitis/meningoencephalitis; and

in immunodeficient patients.

Quality assurance in CSF diagnostics

Some CSF quality assurance programmes have been published showing that to ensure optimal performance and results, standardized protocols should be in place for the spinal tap and sample processing [8] (Class 1). Furthermore it is important to analyse the CSF in a specialized laboratory which is routinely evaluated for its performance and uses standardized analytical techniques and interpretation of the laboratory findings in the clinical context [8] (Class 1); [70] (Class 4). If proteins are measured that potentially originate from blood or brain compartments, CSF and serum samples should be run in parallel in the same assay to minimize variability [8] (Class I, Level A).

A cytology training programme resulted in an increase of the number of correctly identified CSF cells from as low as 11% to 93% [71]. In a recent study investigating inter -laboratory variation of neurofilament light chain detection, it turned out that the lack of preparation of accurate and consistent protein standards was the main reason for a very poor inter-laboratory accordance [72] (Class I).

Recommendations

CSF should be analysed immediately (i.e. <1 h) after collection. If storage is required for later investigation this can be done at 4–8°C (short term) or at -20°C (long term). Only protein components and RNA (after appropriate preparation) can be analysed from stored CSF (GPP).

The Level B recommendation regarding CSF partitioning and storage states that 12 ml of CSF should be partitioned into three to four sterile tubes. It is important that the CSF is not allowed to sediment before partitioning. Store 3–4 ml at 4°C for general investigations, cultivation and microscopic investigation of bacteria and fungi, antibody testing, polymerase chain reaction (PCR), and antigen detection. Larger volumes (10–15 ml) are necessary for certain pathogens like Mycobacterium tuberculosis, fungi, or parasites.

Normal CSF protein concentration should be related to the patient’s age (higher in the neonate period and after age of 60 years) and the site of LP (Level B). Exact upper normal limits of protein concentration differ according to the technique and the examining laboratory.

The Qalb should be preferred to total protein concentrations, partly because reference levels are more clearly defined and partly because it is not confounded by changes in other CSF proteins (Level B).

The glucose concentration in CSF should be related to the blood concentration. Therefore CSF glucose/serum ratio is preferable. Pathological changes in this ratio or in lactate concentration are supportive for bacterial or fungal meningitis or leptomeningeal metastases (Level B).

Intrathecal IgG synthesis can be measured by various quantitative methods, but at least for the diagnosis of MS, the detection of oligoclonal bands by appropriate methods is superior to any existing formula (Level A). Patients with other diseases associated with intrathecal inflammation, for example patients with CNS infections, may also have intrathecal IgA and IgM synthesis as assessed by non-linear formulae (Reiber hyperbolic formulae or extended indices), which should be preferred to the linear IgA and IgM indices (Level B).

Cellular morphology (cytological staining) should be evaluated whenever pleocytosis is found or leptomeningeal metastases or pathological bleeding is suspected (Level B). If cytology is inconclusive in case of query CSF bleeding, measurement of bilirubin is recommended up to 2 weeks after the clinical event.

For standard microbiological examination sedimentation at 3000×g for 10 min is recommended (Level B). Microscopy should be performed using Gram or methylene blue, Auramin O or Ziehl-Nielsen (M. tuberculosis), or Indian ink stain (Cryptococcus). Depending on the clinical presentation, incubation with bacterial and fungal culture media can be useful. Anaerobic culture media are recommended only if there is suspicion of brain abscess. A viral culture is generally not recommended. A list of infectious agents and their association with different diseases as well as the recommended method of detection is provided in table 1.4. The results of bacterial antigen detection have to be interpreted with respect to the microscopical CSF investigation and culture results. It is not routinely recommended in cases of negative microscopy. A diagnosis of bacterial nervous system infection based on antigen detection alone is not recommended (risk of contamination).

CSF laboratories need to participate in regular internal and external quality assessment (Level A). In addition, to avoid possible erroneous differential diagnostic interpretations due to inadequate CSF findings, clinicians should make sure that the co-operating laboratory adheres to the essential quality standards (proof of education and training, certification of the CSF laboratory, continuous participation in internal and external controls) [70] (GPP).

Conflicts of interest

The authors have reported no conflicts of interest.

Acknowledgement

We are grateful to Professor Christian Bogdan (Director of the Department for Microbiology and Hygiene, Albert Ludwigs-Universität Freiburg, Germany) and to Professor Rüdiger Dörries (Head of the Department of Virology, Institute of Medical Microbiology und Hygiene Ruprecht-Karls-Universität Heidelberg, Germany) for critical review of the microbiological part of the manuscript (infectious CSF).

References

1. Brainin M, Barnes M, Baron JC, et al. Guidance for the preparation of neurological management guidelines by EFNS scientific task forces–revised recommendations 2004. Eur J Neurol 2004;11:577–81.

2. Thompson EJ. The CSF Proteins: A Biochemical Approach. Amsterdam, Netherlands: Elsevier, 1988.

3. 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;122:189–203.

4. Eeg-Olofson O, Link H, Wigertz A. Concentrations of CSF proteins as a measure of blood brain barrier function and synthesis of IgG within the CNS in ’normal’ subjects from the age of 6 months to 30 years. Acta Paediatr Scand 1981;70:167–70.

5. Statz A, Felgenhauer K. Development of the blood– CSF barrier. Develop Med Child Neurol 1983;25:152–61.

6. Andersson M, Alvarez- Cermeño J, Bernadi G, et al. Cerebrospinal fluid in the diagnosis of multiple sclerosis: a consensus report. J Neurol Neurosurg Psychiatry 1994;57:897–902.

7. Blennow K, Fredman P, Wallin A, Gottfries C-G, Långström G, Svennerholm L. Protein analyses in cerebrospinal fluid. I. Influence of concentration gradients for proteins on cerebrospinal fluid/serum albumin ratio. Eur Neurol 1993;33:126–8.

8. Reiber H. External quality assessment in clinical neurochemistry: survey of analysis for cerebrospinal fluid (CSF) proteins based on CSF/serum quotients. Clin Chem 1995;41:256–63.

9. Kornhuber J, Kaiserauer CH, Kornhuber AW, Kornhuber ME. Alcohol consumption and blood–cerebrospinal fluid barrier dysfunction in man. Neurosci Lett 1987;79:218–22.

10. Skouen JS, Larsen JL, Vollset SE. Cerebrospinal fluid protein concentrations related to clinical findings in patients with sciatica caused by disk herniation. J Spinal Disord 1994;7:12–8.

11. Nyström E, Hamberger A, Lindstedt G, Lundquist C, Wikkelsö C. Cerebrospinal fluid proteins in subclinical and overt hypothyroidism. Acta Neurol Scand 1997;95:311–4.

12. Seyfert S, Kunzmann V, Schwertfeger N, Koch HC, Faulstich A. Determinants of lumbar CSF protein concentration. J Neurol 2002;249:1021–6.

13. Stockstill MT, Kauffman CA. Comparison of cryptococcal and tuberculous meningitis. Arch Neurol 1983;40:81–5.

14. Kaiser R. Neuroborreliosis. J Neurol 1998;245:247–55.

15. Negrini B, Kelleher KJ, Wald ER. Cerebrospinal fluid findings in aseptic versus bacterial meningitis. Pediatrics 2000;105:316–9.

16. Lindquist L, Linne T, Hansson LO, Kalin M, Axelsson G. Value of cerebrospinal fluid analysis in the differential diagnosis of meningitis: a study in 710 patients with suspected central nervous system infection. Eur J Clin Microbiol Infect Dis 1988;7:374–80.

17. Koskiniemi M, Vaheri A, Taskinen E. Cerebrospinal fluid alterations in herpes simplex virus encephalitis. Rev Infect Dis 1984;6:608–18.

18. Jerrard DA, Hanna JR, Schindelheim GL. Cerebrospinal fluid. J Emerg Med 2001;21:171–8.

19. Segurado OG, Kruger H, Mertens HG. Clinical significance of serum and CSF findings in the Guillain - Barre syndrome and related disorders. J Neurol 1986;233:202–8.

20. Twijnstra A, Ongerboer de Visser BW, van Zanten AP, Hart AA, Nooyen WJ. Serial lumbar and ventricular cerebrospinal fluid biochemical marker measurements in patients with leptomeningeal metastases from solid and hematological tumors. J Neurooncol 1989;7:57–63.

21. Bruna J, González L, Miró J, Velasco R, Gil M, Tortosa A. Neuro-Oncology Unit of the Institute of Biomedical Investigation of Bellvitge. Leptomeningeal carcinomatosis: prognostic implications of clinical and cerebrospinal fluid features. Cancer 2009;115:381–9.

22. Brettschneider J, Claus A, Kassubek J, Tumani H. Isolated blood– cerebrospinal fluid barrier dysfunction: prevalence and associated diseases. J Neurol 2005;252:1067–73.

23. Delpech B, Lichtblau E. Étude quantitative des immuno-globulines G et de l ’ albumine du liquide cephalo rachidien. Clin Chim Acta 1972;37:15–23.

24. Ganrot K, Laurell C- B. Measurement of IgG and albumin content of cerebrospinal fluid, and its interpretation. Clin Chem 1974;20:571–3.

25. Link H, Tibbling G. Principles of albumin and IgG analyses in neurological disorders. III. Evaluation of IgG synthesis within the central nervous system in multiple sclerosis. Scand J Clin Lab Invest 1977;37:397–401.

26. Schipper HI, Bardosi A, Jacobi C, Felgenhauer K. Meningeal carcinomatosis: origin of local IgG production in the CSF. Neurology 1988;38:413–6.

27. McLean BN, Luxton RW, Thompson EJ. A study of immunoglobulin G in the cerebrospinal fluid of 1007 patients with suspected neurological disease using isoelectric focusing and the Log IgG-Index. A comparison and diagnostic applications. Brain 1990;113:1269–89.

28. Öhman S, Ernerudh J, Forsberg P, Henriksson A, von Schenck H, Vrethem M. Comparison of seven formulae and isoelectrofocusing for determination of intrathecally produced IgG in neurological diseases. Ann Clin Biochem 1992;29:405–10.

29. Sellebjerg F, Christiansen M, Rasmussen LS, Jaliachvili I, Nielsen PM, Frederiksen JL. The cerebrospinal fluid in multiple sclerosis. Quantitative assessment of intrathecal immunoglobulin synthesis by empirical formulae. Eur J Neurol 1996;3:548–59.

30. Korenke GC, Reiber H, Hunneman DH, Hanefeld F. Intrathecal IgA synthesis in X-linked cerebral adrenoleukocystrophy. J Child Neurol 1997;12:314–20.

31. Felgenhauer K. Differentiation of the humoral immune response in inflammatory diseases of the central nervous system. J Neurol 1982;228:223–37.

32. Felgenhauer K, Schädlich H-J. The compartmental IgM and IgA response within the central nervous system. J Neurol Sci 1987;77:125–35.

33. Sharief MK, Keir G, Thompson EJ. Intrathecal synthesis of IgM in neurological diseases: a comparison between detection of oligoclonal bands and quantitative estimation. J Neurol Sci 1990;96:131–43.

34. McDonald WI, Compston A, Edan G, et al. Recommended diagnostic criteria for multiple sclerosis: guidelines from the international panel on the diagnosis of multiple sclerosis. Ann Neurol 2001;50:121–7.

35. Rauer S, Kaiser R. Demonstration of anti-HuD specific oligoclonal bands in the cerebrospinal fluid from patients with paraneoplastic neurological syndromes. Qualitative evidence of anti-HuD specific IgG-synthesis in the central nervous system. J Neuroimmunol 2000;111:241–4.

36. Stich O, Graus F, Rasiah C, Rauer S. Qualitative evidence of anti-Yo-specific intrathecal antibody synthesis in patients with paraneoplastic cerebellar degeneration. J Neuroimmunol 2003;141:165–9.

37. Storstein A, Monstad SE, Honnorat J, Vedeler CA. Paraneoplastic antibodies detected by isoelectric focusing of cerebrospinal fluid and serum. J Neuroimmunol 2004;155:150–4.

38. Keir G, Luxton RW, Thompson EJ. Isoelectric focusing of cerebrospinal fluid immunoglobulin G: an annotated update. Ann Clin Biochem 1990;27:436–43.

39. Blennow K, Fredman P. Detection of cerebrospinal fluid leakage by isoelectric focusing on polyacrylamide gels with silver staining using the PhastSystem. Acta Neurochir 1995;136:135–9.

40. Freedman MS, Thompson EJ, Deisenhammer F, et al. Recommended standard of cerebrospinal fluid analysis in the diagnosis of multiple sclerosis. Arch Neurol 2005;62:865–70.

41. Poser CM, Paty W, Scheinberg LC, et al. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann Neurol 1983;13:227–31.

42. Schumacher FA, Beebe GW, Kibler RF, et al. Problems of experimental trials of therapy in multiple sclerosis. Ann N Y Acad Sci 1965;122:552–68.

43. Feigin RD, McCracken GH Jr, Klein JO. Diagnosis and management of meningitis. Pediatr Infect Dis J 1992;11:785–814.

44. Watson MA, Scott MG. Clinical utility of biochemical analysis of cerebrospinal fluid. Clin Chem 1995;41:343–60.

45. Steele RW, Marmer DJ, O’Brien MD, Tyson ST, Steele CR. Leukocyte survival in cerebrospinal fluid. J Clin Microbiol 1986;23:965–6.

46. Lamers KJB, Wevers RA. Cerebrospinal fluid diagnostics: biochemical and clinical aspects. Klin Biochem Metab 1995;3:63–75.

47. Roma AA, Garcia A, Avagnina A, Rescia C, Elsner B. Lymphoid and myeloid neoplasms involving cerebrospinal fluid: comparison of morphologic examination and immunophenotyping by flow cytometry. Diagn Cytopathol 2002;27:271–5.

48. Adam P, Taborsky L, Sobek O, et al. Cerebrospinal fluid. Adv Clin Chem 2001;36:1–62.

49. Spanos A, Harrell FE Jr, Durack DT. Differential diagnosis of acute meningitis. An analysis of the predictive value of initial observations. JAMA 1989;262:2700–7.

50. Zeman D, Adam P, Kalistova H, Sobek O, Andel J, Andel M. Cerebrospinal fluid cytologic findings in multiple sclerosis. A comparison between patient subgroups. Acta Cytol 2001;45:51–9.

51. UK National External Quality Assessment Scheme for Immunochemistry Working Group. National guidelines for analysis of cerebrospinal fluid for bilirubin in suspected subarachnoid haemorrhage. Ann Clin Biochem 2003;40:481–8.

52. Twijnstra A, Ongerboer de Visser BW, van Zanten AP. Diagnosis of leptomeningeal metastasis. Clin Neurol Neurosurg 1987;89:79–85.

53. Glantz MJ, Cole BF, Glantz LK, et al. Cerebrospinal fluid cytology in patients with cancer: minimizing false–negative results. Cancer 1998;82:733–9.

54. Wasserstrom WR, Glass JP, Posner JB. Diagnosis and treatment of leptomeningeal metastases from solid tumors: experience with 90 patients. Cancer 1982;49:759–72.

55. Kaplan JG, DeSouza TG, Farkash A, et al. Leptomeningeal metastases: comparison of clinical features and laboratory data of solid tumors, lymphomas and leukemias. J Neurooncol 1990;9:225–9.

56. Schlossberg D. Infections of the Nervous System. Berlin: Springer-Verlag, 1990.

57. Kaiser R. Entzündliche und infektiöse Erkrankungen. In: Hufschmidt A, Lücking CH (eds) Neurologie Compact, Leitlinie Für Klinik Und Praxis. Stuttgart: Georg Thieme Verlag, 2002;pp. 121–172.

58. Takahashi T, Nakayama T, Tamura M, et al. Nested polymerase chain reaction for assessing the clinical course of tuberculous meningitis. Neurology 2005;64:1789–93.

59. Jeffery KJ, Read SJ, Peto TE, Mayon- White RT, Bangham CR. Diagnosis of viral infections of the central nervous system: clinical interpretation of PCR results. Lancet 1997;349:313–7.

60. Davies NW, Brown LJ, Gonde J, et al. Factors influencing PCR detection of viruses in cerebrospinal fluid of patients with suspected CNS infections. J Neurol Neurosurg Psychiatry 2005;76:82–7.

61. Kennedy PG. Viral encephalitis. J Neurol 2005;252:268–72.

62. Reiber H, Thompson EJ, Grimsley G, et al. Quality assurance for cerebrospinal fluid protein analysis: international consensus by an internet - based group discussion. Clin Chem Lab Med 2003;41:331–7. Available at: www.teamspace.net/CSF.

63. Linke E, Wieczorek V, Zimmermann K. Qualitätskontrolle in der Liquorzytodiagnostik. In: Zettl UK, Lehmitz R, Mix E (eds) Klinische Liquordiagnostik, 2nd edn. Berlin/New York: Walter de Gruyter, 2005;pp. 380–91.

64. Petzold A, Altintas A, Andreonie L, et al. Neurofilament ELISA validation. J Immunol Methods 2009;Oct 24. 352:23–31;[Epub ahead of print] doi:10.1016/j.jim.2009.09.014.

65. Fishman RA. Cerebrospinal Fluid in Diseases of the Nervous System. Philadelphia, PA: W.B. Saunders, 1992.

66. Jordan GW, Statland B, Halsted C. CSF lactate in diseases of the CNS. Arch Intern Med 1983;143:85–7.

67. Kniehl ER, Dörries HK, Geiss B, et al. In: Mauch H, Lütticken R (eds) MiQ 17: Qualitätsstandards in Der Mikrobiologisch-Infektiologischen Diagnostik. München, Jena: Urban & Fischer, 2001;pp. 1–77.

68. Lo Re V 3rd, Gluckman SJ. Eosinophilic meningitis. A m J Med 2003;114:217–23.

69. Öhman S, Forsberg P, Nelson N, Vrethem M. An improved formula for the judgement of intrathecally produced IgG in the presence of blood brain barrier damage. Clin Chim Acta 1989;181:265–72.

70. Öhman S, Ernerudh J, Forsberg P, von Schenck H, Vrethem M. Improved formulae for the judgement of intrathecally produced IgA and IgM in the presence of blood CSF barrier damage. Ann Clin Biochem 1993;30:454–62.

71. Sabetta JR, Andriole VT. Cryptococcal infection of the central nervous system. Med Clin North Am 1985;69:333–44.

72. Seneviratne U. Guillain-Barré syndrome. Postgrad Med J 2000;76:774–82.

CHAPTER 2

Use of imaging in cerebrovascular disease

P. Irimia,¹ S. Asenbaum,² M. Brainin ³ H. Chabriat ⁴ E. Martínez-Vila ¹ K. Niederkorn,⁵ P. D. Schellinger,⁶ R. J. Seitz,⁷ J. C. Masdeu¹

¹University of Navarra, Pamplona, Spain;

²Medical University of Vienna, Austria;

³Donauklinikum and Donau-Universität, Maria Gugging, Austria;

⁴Lariboisiere Hospital, University of Paris, France;

⁵Karl Franzens University, Graz, Austria;

⁶University Clinic at Erlangen, Germany;

⁷University Hospital Düsseldorf, Germany

Objectives

The objective of the task force is to actualize the EFNS Guideline on the use of neuroimaging for the management of acute stroke published in 2006. The Guideline is based on published scientific evidence as well as the consensus of experts. The resulting report is intended to provide updated and evidence-based recommendations regarding the use of diagnostic neuroimaging techniques, including cerebrovascular ultrasonography (US), in patients with stroke and thus guide neurologists, other healthcare professionals, and healthcare providers in clinical decision making and in the elaboration of clinical protocols. It is not intended to have legally binding implications in individual situations.

Background

Stroke is the second most common cause of death worldwide, and one of the major determining factors of hospital admission and permanent disability in the developed countries [1]. The proportion of the population over the age of 65 years is growing and this trend is likely to increase stroke incidence in the next decades [2]. Major advances in the understanding of the mechanisms of stroke and its management have been made thanks to the substantial progress in neuroimaging techniques. However, the multiplicity and continuous advances of neuroimaging techniques available for the evaluation of stroke patients has increased the complexity of decision making for physicians. Neurologists, who have been educated to manage acute stroke patients, should be trained in the use of neuroimaging, which allows for the development of a pathophysiologically oriented treatment.

Successful care of acute stroke patients requires a rapid and accurate diagnosis because the time window for treatment is narrow. In the case of intravenous thrombolysis for ischaemic stroke, the treatment is safer and more effective the earlier it is given [3]. Current recommendations call for a 4.5-h time limit for intravenous thrombolysis [4] that can be extended to 6 h for intra-arterial thrombolysis [5]. Thus, the neuroimaging protocol designed to determine the cause of stroke should delay treatment as little as possible. Neuroimaging can provide information about the presence of ischaemic but still viable and thus salvageable tissue (penumbra tissue) and vessel occlusion in the hyperacute phase of ischemic stroke. This information is critical for an improved selection of patients who could be treated with intravenous thrombolysis up to the 4.5-h limit and beyond [5]. Thus, neuroimaging criteria have been used for patient selection and outcome in different trials, using thrombolysis beyond 3 h after stroke onset [6, 7]. Determining stroke type using neuroimaging goes well beyond separating ischaemic from haemorrhagic stroke. For instance, the depiction of multiple cortical infarcts may lead to a fuller work-up for cardiogenic emboli [8, 9]. In arterial dissection, the characteristic semilunar high-intensity signal in the vessel wall on high-resolution T1-weighted magnetic resonance imaging (MRI) alerts to the presence of this cause of stroke [10].

Search strategy

The Cochrane Library was consulted and no studies were found regarding the use of neuroimaging techniques in stroke. A comprehensive literature review using the MEDLINE database has been conducted by searching for the period 1965–2009. Relevant literature in English, including existing guidelines, meta-analyses, systematic reviews, randomized controlled trials, and observational studies have been critically assessed. Selected articles have been rated based on the quality of study design, and clinical practice recommendations have been developed and stratified to reflect the quality and the content of the evidence according to EFNS criteria [11].

Method for reaching consensus

The author panel critically assessed the topic through analysis of the medical literature. A draft guideline with specific recommendations was circulated to all panel members. Each panellist studied and commented in writing on this draft, which was revised to progressively accommodate the panel consensus. After the approval of the panellists, two independent experts gave their opinion on the final version.

Results

Imaging of the brain

The primary objectives of brain imaging in acute stroke are to exclude a non-vascular lesion as the cause of the symptoms and to determine whether the stroke is caused by an ischaemic infarction or a haemorrhage. It is not possible to exclude stroke mimics, such as a neoplasm, and distinguish between ischaemic and haemorrhagic stroke based exclusively on the history and physical examination [12]. Determining the nature of the lesion by brain imaging is necessary before starting any treatment, particularly thrombolysis and antithrombotic drugs (Class I, Level A).

Secondary objectives of brain imaging are to facilitate the identification of stroke mechanisms, to detect salvageable tissue, and to improve the selection of patients who could be candidates for reperfusion therapies.

Computed tomography (CT)

Conventional CT of the head is the examination most frequently used for the emergent evaluation of patients with acute stroke because of its wide availability and usefulness (Class II, Level B). It has been utilized as a screening tool in most of the major therapeutic trials conducted to date [3]. It is useful to distinguish between ischaemic stroke and intracerebral or subarachnoid haemorrhage (SAH), and can also rule out other conditions that could mimic stroke, such as brain tumours. Signs of early ischemia may be identified as early as 2 hrs from stroke onset, although they may appear much later [13]. Early infarct signs include the hyperdense middle cerebral artery (MCA) sign [14, 15] (indicative of a thrombus or embolus in the M1 segment of the vessel), the MCA dot sign [16, 17] (indicating thrombosis of M2 or M3 MCA branches), the loss of grey-white differentiation in the cortical ribbon [18] or the lentiform nucleus [19], and sulcal effacement [20]. The presence of some of these signs has been associated with poor outcome [20–22]. In the European Cooperative Acute Stroke Study (ECASS) I trial those patients with signs of early infarction involving more than one-third of the territory of the MCA had an increased risk of haemorrhagic transformation following treatment with thrombolysis [23]. A secondary analysis of other thrombolytic trials with a 6-h time window (ECASS II and Multicentre Acute Stroke Trial–Europe (MAST-E)) demonstrated that the presence of early CT changes was a risk factor for intracerebral haemorrhage (ICH) [24, 25], and similar results have been observed in larger series of patients [26]. However, in the National Institute of Neurological Disease and Stroke (NINDS) trial and the Australian Streptokinase Trial there was no relation between intracranial haemorrhage and early CT changes [27, 28], and it has been argued that the poorer outcome in patients with CT changes may have more to do with delayed treatment than with the changes themselves, with additional damage of the potentially salvageable tissue in the larger, CT-visible infarcts [29]. Because ischaemic changes are difficult to detect for clinicians without an adequate training in reading CT [30, 31], scoring systems have been developed to quantify early CT changes, such as the Alberta Stroke Programme Early CT Score (ASPECTS). More extensive early changes using ASPECTS correlate with high rates of intracranial haemorrhage and poor outcome at long term. Therefore, its use could improve the identification of ischaemic stroke patients who would particularly benefit from thrombolysis and those at risk of symptomatic haemorrhage [32, 33]. However, given the conflicting evidence, the presence of decreased attenuation on early CT, even affecting more than one-third of the MCA territory, cannot be construed as an absolute contraindication for the use of thrombolytic therapy in the first 3 h after stroke (Class IV, Good Clinical Practice Point (GCPP)).

Conventional CT contrast enhancement is not indicated for the acute diagnosis of stroke, and seldom may be helpful to show the infarcted area in the subacute stage (2–3 weeks after stroke onset) when there may be obscuration of the infarction by the ‘fogging effect’ [34, 35] (Class IV, Level C).

Computed tomography shows acute ICHs larger than 5 mm in diameter as areas of increased attenuation. Not depicted by CT are petechial haemorrhages and bleedings in patients with very low haemoglobin levels [36], because the high density of blood on CT is a function of haemoglobin concentration. CT demonstrates the size and topography of the haemorrhage and gives information about the presence of mass effect, hydrocephalus, and intraventricular extension of the bleeding. In addition, it may identify (although not as well as MRI) possible structural abnormalities (aneurysms, arteriovenous malformations, or tumours) that caused the haemorrhage. The characteristic hyperdensity of ICH on CT disappears with time, becoming hypodense after approximately 8–10 days [37, 38]. For this reason, CT is not a useful technique to distinguish between old haemorrhage and infarction. With newer CT helical units, SAH can be detected in 98–100% of patients in the first 12 h from the onset of symptoms [39, 40] and in 93% of patients studied within the first 24 h [41, 42].

CT is the imaging procedure of choice to diagnose SAH (Class I, Level A). Some experts recommend performing the study with thin cuts (3 mm in thickness) through the base of the brain, because small collections of blood may be missed with thicker cuts [39, 43] (Class IV, GCPP). CT cannot identify SAH in patients with low haemoglobin levels, because blood may appear isodense, and in those scanned after 3 weeks of the bleeding, when blood has usually been metabolized [44]. Lumbar puncture with CSF analysis should be performed when CT scan is negative or doubtful [45].

Cerebral venous thrombosis (CVT) is an uncommon cause of stroke [46, 47]. CT can show direct signs of venous thrombosis and other indirect non–specific signs, but in about one-third of cases CT is normal [48, 49]. Direct signs on unenhanced CT are the cord sign, corresponding to thrombosed cortical veins, and the dense triangle sign, corresponding to a thrombus in the superior sagittal sinus, and, on enhanced CT of the sagittal sinus, the delta sign [50]. Indirect signs such as local hypodensities caused by oedema or infarction, hyperdensities secondary to haemorrhagic infarction, or brain swelling and small ventricles suggest the diagnosis of CVT. CT venography has emerged as a good procedure to detect CVT [51–53] (Class III, Level C).

Perfusion-CT (PCT) techniques, despite being less sensitive than diffusion-weighted (DWI) MRI can show the area of ischaemia [54, 55]. Also, may it help distinguish between reversible (ischaemic penumbra) and irreversible (infarction) areas of ischaemia with standardized methodology [56–58]. Different preliminary studies comparing PCT with MRI have shown comparable results to depict penumbra tissue [59–62]. There is only one study to date (in which the primary end point was negative) demonstrating that perfusion CT may be used for the selection of candidates to thrombolytic therapy beyond the 3-h window [63]. Pregnancy, diabetes, renal failure, and allergy to contrast material are relative contraindications to performing a perfusion brain CT. Perfusion CT may be useful to characterize the presence of marginally perfused tissue (Class II, Level B).

Magnetic resonance imaging (MRI)

Magnetic resonance imaging has a higher sensitivity than conventional CT and results in lower inter-rater variability in the diagnosis of ischaemic stroke within the first hours of stroke onset [64–69] (Class I, Level A). MRI is particularly useful to show lesions in the brain stem or cerebellum, identify lacunar infarcts, and document vessel occlusion and brain oedema [65, 66, 68] (Class I, Level A).

In addition, MRI techniques can provide information about tissue viability. DWI and perfusion (PI) MRI studies may inform about the presence of reversibly and irreversibly damaged ischaemic tissues in the hyperacute phase of stroke [70–78] (Class II, Level B). DWI may demonstrate deeply ischaemic or infarcted brain tissue within minutes of symptom onset [77]. However, areas of abnormal DWI signal are not always infarcted and the finding may disappear spontaneously or after thrombolysis [79–81]. PI requires the intravenous administration of gadolinium and provides information about brain tissue perfusion at a given time. Different perfusion parameters give different perfusion lesion volumes in the same patient [82, 83]. The absolute volume difference or ratio of the PI area and the DWI area (diffusion–perfusion mismatch) is a useful method to estimate the presence of ischaemic penumbra tissue [84]. Not only the volume of abnormally perfused tissue but also the degree of perfusion drop predict the extent of ischaemic brain damage [85]. PI/DWI mismatch has been evaluated in several studies as a selection tool for thrombolytic therapy beyond 3 h [86–89] and in a phase II trial it was used as a selection tool and surrogate parameter for thrombolysis within 3–9 h [63]. A proposal for the standardization of perfusion and penumbral imaging techniques has been published [56]

Some neuroimaging findings on MRI, such as the presence of leukoaraiosis [90] and large DWI lesions [91], are associated with an increased risk for symptomatic intracerebral haemorrhage associated with thrombolytic treatment.

MRI may be useful to predict which patients will develop massive swelling with an MCA infarct. The measurement of infarct volume on DWI allows the prediction of malignant infarction [92, 93], and may be helpful for early management of these patients [94, 95].

MRI can help identify occluded intracranial arteries by the loss of the normal intravascular flow voids [65]. Some sequences, such as T2 *-weighted MRI or fluid-attenuated inversion recovery (FLAIR; hyperintense artery sign), may demonstrate acute MCA thromboembolism with a higher sensitivity than CT, but the type of arterial change on MRI does not predict recanalization, clinical outcome, or ICH after intravenous thrombolysis [96, 97].

Intracranial haemorrhage is easily detectable on MRI using T2*-weighted images [98–100]. MRI can identify intraparenchymal haemorrhage within the first 6 h after symptom onset as accurately as CT [100, 101] (Class I, Level A). Susceptibility-weighted T2 * sequences (gradient echo) can also detect clinically silent parenchymal microbleeds, not visible on CT, which may leave enough local haemosiderin to remain detectable for months or years after the bleeding. Microbleeds are associated with a history of ICH and prospectively have been shown to pose a 3% risk of ICH [102]. Although some retrospective studies reported an increased risk of symptomatic haemorrhage after thrombolysis [103, 104], this risk was not found in more recent studies [82, 105, 106]. The risk of bleeding after thrombolysis in patients with microbleeds is small [106] and their presence is not a contraindication to the use of thrombolytic therapy in the first 3 h after stroke (Class III, Level C).

MRI is also useful to date the haemorrhagic event accurately and to detect lesions (as tumours, vascular malformations, or aneurysms) that may underlie the ICH [107]. To detect these lesions, repeated studies may be needed after some of the swelling and vasospasm have subsided.

Subarachnoid haemorrhage (SAH) can be detected using T2* [108] and FLAIR [109, 110] MR sequences, but at present CT remains the imaging method of choice for this diagnosis (Class I, Level A).

Arterial dissection is a leading cause of stroke in young persons [47]. MRI is the initial procedure of choice [66, 68, 111, 112], replacing conventional angiography as the gold standard (Class II, Level B), because MRI can show the mural haematoma of the dissected vessel on the axial images [112] (high signal in the wall). Visualization of these changes in the vertebral artery is more difficult than for the larger carotid artery, making diagnosis of vertebral dissection less reliable. The study can be completed with magnetic resonance angiography (MRA) to visualize occlusion of the artery, pseudoaneurysms, or a long stenotic segment with tapered ends [113, 114]. Other techniques, including US [115–117] or CT angiography [113, 114, 118, 119], may be useful for the non-invasive diagnosis of arterial dissection.

MRI combined with MRA is the method of choice for the diagnosis and follow-up of CVT [49, 120–123]. MRI is more sensitive than CT to show parechymal abnormalities and the presence of thrombosed veins.

In summary, MRI is very helpful in the clinical setting for the management of acute stroke and to guide decisions regarding thrombolysis (Class I, Level A). It is particularly helpful for the study of stroke patients for whom perfusion CT may be dangerous, such as those with renal failure or diabetes. However, MRI in the acute phase of stroke is not widely available at European hospitals [124]. Other limitations and contraindications for the use of MRI are: claustrophobia, agitation, morbid obesity, the presence of intracranial ferromagnetic elements, an aneurysm recently clipped or coiled, otic or cochlear implants, some old prosthetic heart valves, pacemakers, and some, not all, neurostimulators.

SPECT and PET

Single photon emission computed tomography (SPECT) and positron emission tomography (PET) are functional neuroimaging techniques based on the principles of tracer technology using radiolabelled substances as systemically administered tracers. In the setting of stroke, SPECT has been used for the evaluation of cerebral perfusion. Earlier perfusion SPECT studies failed to show any advantage of SPECT over the structured clinical evaluation (NIH, Canadian, Scandinavian stroke scales) in the prediction of the evolution of acute stroke [125]. However, using ethyl cysteinate dimer (ECD) SPECT in the first 6 h after stroke, Barthel et al. [126] were able to determine which patients would develop massive MCA-territory necrosis, with hemispheric herniation. These patients have a high risk of haemorrhage following thrombolysis and could potentially be helped by early decompressive hemicraniectomy [127]. Complete MCA infarctions were predicted with significantly higher accuracy with early SPECT compared with early CT and clinical parameters. The predictive value increased when the findings on CT, clinical examination, and SPECT were considered [126]. Other studies have found SPECT to add predictive value to the clinical score on admission [128–130]. Those studies suggest that a patient with a normal SPECT study performed within 3 h of stroke onset will most likely recover spontaneously and therefore may not benefit from thrombolysis. A patient with a dense deficit in the entire MCA distribution has a high risk of haemorrhage with thrombolysis, and, depending on age and other factors, should be considered for decompressive hemicraniectomy. The patients most likely to benefit from thrombolysis are the ones with less massive lesions [126, 128]. Thus, SPECT is helpful in the evaluation of acute stroke (Class III, Level C). Unfortunately, the need to perform either CT or MRI in acute stroke renders the performance of SPECT difficult within the time frame allotted for the evaluation of these patients. SPECT is also helpful in the evaluation of cerebral perfusion in non-acute cerebrovascular disease, for instance in the days after a SAH [131] (Class III, Level C).

PET can be used to evaluate a large variety of physiological variables including cerebral blood flow, cerebral blood volume, and cerebral glucose metabolism, as well as the density of neurotransmitters and neuroreceptors, such as benzodiazepine receptors with flumazenil, an accurate marker of neuronal loss [132]. As PET has been considered the gold standard for these kinds of measurements in humans, it is also extremely well suited to help identify the degree of ischaemic damage in the brain. Heiss et al. compared ¹⁵ O-water PET and MRI in patients with acute ischaemic stroke. They observed that DW/ PW–MRI mismatch overestimates the penumbra defined by PET [133]. Although PET is the reference method for quantitative perfusion imaging, it does not allow for the reliable identification of lesions in the vessels or non-vascular lesions giving rise to the stroke syndrome. This, coupled with the cost and current lack of availability of this technique, renders it less useful than MRI and CT for most practising neurologists.

Imaging of the extracranial vessels

Imaging of the extracranial and intracranial vessels will help identify the underlying mechanism of the stroke (atherothrombotic, embolic, dissection, or other). Non-invasive imaging methods are increasingly accepted as replacements of digital substraction angiography (DSA) in the evaluation of carotid stenosis prior to endarterectomy [134] (Class IV, GCPP). US, comprising Doppler sonography and colour-coded duplex sonography, is probably the most common non-invasive imaging examination performed to aid in the diagnosis of carotid disease. The peak systolic velocity and the presence of plaque on greyscale and/or colour Doppler/Duplex US images are the main parameters that should be used when diagnosing and grading internal carotid artery (ICA) stenosis [135]. The examination may be limited by the presence of extensive plaque calcifications, vessel tortuosity and in patients with tandem lesions. In addition, Doppler US is both technician-and equipment-dependent and all sonographers should be able to demonstrate that they have validated their testing procedures. Meta-analyses of published criteria for US have demonstrated sensitivities of 98% and specificities of 88% for detecting > 50% ICA stenosis; and 94% and 90% respectively for detecting > 70% ICA stenosis [136].

Magnetic resonance angiography using time-of-flight angiography (TOF) and contrast-enhanced MRA (CEMRA) are powerful means to assess vascular pathology. Either technique provides specific information: while TOF visualizes changes of flow in the arteries or veins depending on imaging parameters, CEMRA visualizes the vascular lumen. MRA and US have yielded comparable findings. Two meta-analyses [137, 138] and several reviews [139, 140] have compared the diagnostic value of Doppler US, MRA, and conventional DSA for the diagnosis of carotid artery stenosis. The meta-analysis published by Blakeley et al. [137] in 1995 concluded that Doppler US and MRA had similar diagnostic performance in