Laboratory Diagnosis in Neurology, 1 Ed.

Proteins

H. Reiber

Total Protein

Importance

Quantification of the total protein in CSF has two main purposes:

• Rapid emergency information about the barrier function.

• Guidance for subsequent analysis.

In Germany and most other European countries, the total protein value is not used for differential diagnosis (except for emergency analysis) because the CSF/serum albumin quotient is a much better indicator of blood–CSF barrier dysfunction; it is more sensitive and more specific.

Detection Methods

Possible procedures. While there is a reference method for total protein analysis in serum (biuret reaction), the same is not true for CSF: most of the procedures used for protein detection in serum are not sensitive enough for CSF analysis. Turbidimetric and nephelometric methods are currently used in addition to dye-binding reactions. Pandy's test is a semiquantitative method that has been used in the past in the context of emergency analysis. In Europe, it has been increasingly replaced by quantitative total protein analysis. In developing countries, however, this simple, fast, and inexpensive phenol reaction is still in use.

Analytical tests. The best method of total protein analysis in CSF and other fluids is kinetic detection of the maximum intensity of scattered light in 40% trichloroacetic acid (TCA) (Reiber, 1980 a; Reiber, 1983; Chap. 4, this volume, “Immunochemistry”). This method may be recommended as a reference method in CSF analysis because it is mass-related, i. e., independent of the molecular form or amino acid composition of proteins, and is not influenced by any contaminations in the CSF.

Nephelometric two-point measurement in 20% TCA is far more prone to inaccuracy than kinetic measurement (Reiber, 1983). Turbidimetric assays with low TCA concentration or sulfosalicylic acid together with an endpoint detection method are biased by instability of the agglomerated proteins, which have a tendency to flocculate. The method using benzethonium chloride in alkaline solution yields a more stable and homogenous turbidity (Luxton et al., 1989). Dye-binding reactions with Coomassie blue and pyrogallol red–molybdate show different accuracies for proteins with different numbers of aromatic amino acids.

Thus, the dye-binding reactions depend on the fraction of aromatic amino acids, whereas precipitation reactions (e. g., turbidimetry) depend on the molecular form (globulin vs. albumin). Unbiased accuracy is achieved with kinetic nephelometry in 40% TCA.

Reference Range

The CSF reference values for proteins in Table 21.2 are provided only for orientation in laboratory analysis, not as a basis for clinical evaluation of CSF data. The relationship between total protein and QAlb in CSF is shown in Table 21.3.

Pitfalls of Total Protein in CSF

The upper limit of the normal protein concentration in CSF (about 400–500 mg/L) depends very much on the serum concentration and the intrathecal synthesis (Chap. 21, “Proteins”). The limit value for the reference range of total protein in CSF thus very much depends on the blood values. This is the primary reason why it is necessary to use the CSF/serum albumin quotient for sensitive and specific characterization of a blood–CSF barrier dysfunction (see next section, “CSF/Serum Albumin Quotient”).

CSF/Serum Albumin Quotient

Importance

Albumin is synthesized exclusively outside the brain (in the liver) and is therefore an ideal marker to characterize all the influences and limitations on the passage of a protein from the blood into the lumbar CSF, including the individual CSF flow rate.

Albumin quotient as a measure of blood–CSF barrier function. The generally accepted quantitative measure of blood–CSF barrier function is the CSF/serum albumin quotient (QAlb), i. e., the ratio of albumin in CSF, Alb (CSF), to albumin in serum, Alb (Ser) (Andersson et al., 1994; Reiber et al., 2003):

image

Relevance of the CSF/serum quotient. Calculating a CSF/serum concentration quotient eliminates the modulating effect of the individual blood concentration of a protein on its concentration in the CSF. In mathematical terms, this means that a normalized CSF concentration is obtained, a dimensionless quotient with values between 0 and 1.

Albumin quotient as a reference for serum proteins in CSF. Relating the CSF/serum concentration quotients of other serum proteins (QIgGQIgAQIgM) to the albumin quotient as a marker of barrier function (Fig. 1.2) (Reiber, 1994) gives us an excellent way of interpreting the intrathecal synthesis of a serum protein in CSF (IF in Fig. 2.3) irrespective of individual barrier function (CSF flow rate). With the CSF/serum quotient, the interpretation of a protein in CSF becomes independent of its variation in the blood, and by relating this quotient to the albumin CSF/serum quotient the interpretation of the intrathecal fraction of this protein additionally becomes independent of the individual blood–CSF barrier function.

Methods for the Detection of Albumin

The most common procedure for analysis of CSF albumin (combined with serum analysis) is immunochemical nephelometry with two-point or kinetic analysis (Chap. 4, “Methods of Immune Complex Analysis”). Turbidimetric immunochemical methods are also sufficiently sensitive. The precipitation methods of radial immunodiffusion and electroimmunodiffusion (Fig. 4.2) will do as well.

Reference Range

The age-dependent reference ranges for the CSF/serum albumin quotient are presented in Chap. 21, “Proteins,” which gives a formula for adults and a table of values (Table 21.4) for children. The dynamics of albumin quotients in infants are also illustrated in Fig. 1.2Chapter 21, in the section on “Proteins,” gives the conversion factors for the reference ranges of the albumin quotient in ventricular and cisternal CSF.

Since the albumin concentration in CSF also depends (because of the rostrocaudal concentration gradient) on the volume of fluid collected at lumbar puncture—which is not usually recorded—the age-related limit of the reference range should be interpreted generously: ± 10% for volumes of 6 ± 5 mL. For statistics two levels of significance (+10% and + 20%) are proposed (see p. 254).

Immunoglobulins

For over 100 years, the real challenge for developments in CSF analysis has been to demonstrate intrathecal synthesis of immunoglobulins and specific antibodies.

Nomenclature of Immunoglobulins, Antibodies, and Isotypes

image The term immunoglobulin (Ig) refers to all antigen-binding molecules; they are either bound to the cell surface (receptors on B lymphocytes) or present as soluble antibodies.

image The term antibody usually refers to soluble immunoglobulins that are secreted by plasma cells into body fluids.

image The term isotype refers to various classes of immunoglobulins (IgG, IgA, IgM, IgD, IgE), which differ in their heavy chains.

Immunoglobulin classes (isotypes). A monomeric immunoglobulin consists of two heavy chains (H) and two light chains (L). There are two types of light chains (κ and λ; they differ in the amino acid sequence of their constant domains, but this has no effect on their function). The heavy chains of different isotypes differ in size, charge, amino acid composition, and carbohydrate content. There are five types of heavy chains (γ, α, μ, δ, ε) and, accordingly, five classes of immunoglobulins (IgG, IgA, IgM, IgD, IgE) and several subclasses. IgA may occur as a monomer (blood) or as a dimer (in secretions and intrathecal synthesis), whereas IgG, IgD, and IgE are always monomers, and IgM is a pentamer. The different isotypes are identified by laboratory methods based on differences in their heavy chains.

Isotype switch. In the course of an infection, a change takes place in the serum from the primary IgM class response to the more prolonged and more specific IgG class response. This “isotype switch” is notseen in this form in the brain (or in CSF, Fig. 5.22), and this fact is of fundamental importance to the identification of disease-specific diagnostic patterns in CSF analysis (see Chap. 19).

Immunoglobulin Analysis

Detection of an intrathecal humoral immune response is based on analysis of:

• Immunoglobulin classes: total IgG, IgA, and IgM.

• Oligoclonal IgG (qualitative isoelectric focusing, IEF).

• Specific antibodies (antibody index, AI). These tests, with their various sensitivities and specificities, are of varying clinical relevance in both acute and chronic diseases (Felgenhauer and Reiber, 1992).

Quantitative Analytical Methods for Immunoglobulins

Immunoglobulins IgG, IgA, and IgM are routinely determined in parallel in CSF and (appropriately diluted) serum; i. e., paired samples are analyzed in the same run. Reference to a common standard curve rules out the worst of the errors that can arise from the use of two different analytical runs or methods with, unavoidably, different standardizations. Subsequent calculation of the CSF/serum quotient furthermore neutralizes any inaccuracy in the absolute values measured in CSF and serum (Reiber et al., 2003).

Possible Methods. In Europe, the most commonly used method for determining immunoglobulins in CSF and serum is automated immunochemical nephelometry (Chap. 4, “Methods of Immune Complex Analysis”). Less widely used are turbidimetric immunochemical methods. The once widely used precipitation methods of immunodiffusion and electroimmunodiffusion (Fig. 4.2), although still widely used in developing countries as cost-effective, easy-to-perform methods of IgG analysis, have lost ground in Europe because they are not sensitive enough for determination of IgA and IgM in CSF.

The only methods that measure IgA and IgM in CSF with sufficient sensitivity are ELISA and particle-enhanced immunochemical nephelometry (Chap. 4, “Methods of Immune Complex Analysis,” and Fig. 4.6). IgA and IgM ELISAs are the most sensitive and cost-effective analytical methods available, but they are less precise than nephelometry for routine analysis.

Analysis using an automated nephelometer. Among the currently used analytical methods for albumin and immunoglobulins, two automated nephelometer systems are available on the market:

• Instruments from the Dade Behring company (now owned by Siemens) use forward scatter and two-point evaluation of the nephelometric signal (Fig. 4.5). Online evaluation procedures allow the analysis to be worked through on the basis of the total protein content, resulting in a cost-effective analytical system that requires only a few test repeats in various concentration ranges. The recent generation of software (PROTIS 2) automatically performs a simple knowledge-based interpretation and plausibility check and provides a printout of the diagnostic findings.

• Beckman Coulter automated nephelometers measure scattered light at a 70° angle relative to the reflected light (Fig. 4.4), analyze the scattered light signal by rate nephelometry (Chap. 4, “Methods of Immune Complex Analysis” and Fig. 4.5), and perform an antigen excess check. The readings are evaluated and interpreted by knowledge-based interpretation software and checked for implausibility (Information Science and Technology in Medicine, www.wormek.de).

image

Fig. 5.20 Comparison of various evaluation methods for the reference range of blood-derived IgG in CSF (Reiber and Peter, 2001). R, Reiber's hyperbolic discrimination line, QLim (where IgGIF = 0); I, Link's IgG index: graphical representation of the usual discrimination line for I= 0.7; T, Tourtellotte's IgG synthesis rate: the formula is mathematically simplified by using the daily CSF production rate (500 mL) (Reiber and Peter, 2001). The data points represent the restricted range of QAlb = 20 to 30 × 10−3 as a representative fraction taken from a clinical study involving 4300 patients without intrathecal IgG synthesis (Reiber, 1994 a). For example, a representative patient (large dot) with spinal canal stenosis and no signs of inflammation (normal cell count, no oligoclonal IgG) would yield a false-positive result if the intrathecal IgG synthesis were evaluated by Tourtellotte's IgG synthesis rate (T) or Link's IgG index (I). By contrast, the correct interpretation is obtained when evaluation is based on Reiber's hyperbolic discrimination line (R) and the “gold standard”, oligoclonal IgG. (When no oligoclonal IgG is detected, the intrathecal fraction is < 0.5% of total IgG in the CSF.) Statistical evaluation of the data (Reiber and Peter, 2001) for albumin quotients of QAlb = 60 × 10−3 and 120 × 10−3 showed that 11/14 and 16/17, respectively, would be false-positive for intrathecal IgG synthesis when evaluated by the IgG synthesis rate, while 6/14 and 8/17, respectively, would be false-positive when evaluated by the IgG index.

Reference values for immunoglobulin concentrations in CSF are provided in Chap. 21, “Proteins,” for orientation. Evaluation for clinical purposes, however, uses hyperbolic reference ranges of the CSF/serum quotients, either numerically or in a quotient diagram.

Detection of Intrathecal Immunoglobulin Synthesis: Overview

From linear to nonlinear evaluations. In the past 40 years various procedures have been established for quantitative interpretation of intrathecal IgG synthesis (Fig. 5.20):

• In the United States, Tourtellotte (1970) established the formula named after him.

• Delpech and Lichtblau (1972) originally evaluated CSF IgG relative to CSF albumin—which is physiologically irrelevant. (If this quotient is divided by the corresponding serum quotient, the result is numerically the same as the IgG index [see below]—but not much use either graphically or physiologically.)

• The CSF/serum quotient diagram for IgG and albumin introduced by Ganrot and Laurell (1974) has proven useful as a basis for discrimination of the intrathecally synthesized fraction from a purely blood-derived fraction in CSF.

• Link and Tibbling (1977) defined the linear IgG index; it has been in use for the last 30 years, mainly because it is easy to evaluate numerically: IgG index = QIgG/QAlb. The bias of the IgG index (see below) comes from the linear reference value, which in fact depends nonlinearly on the barrier function, QAlb. This is even more of a problem with the corresponding IgM index introduced later (Stauch et al., 2010).

• After a variety of attempts (e. g., Reiber, 1980 b) to account for the changing relation of IgG and albumin in CSF with increasing blood–CSF barrier dysfunction (Ganrot-Norlin, 1978), the first approaches to describing the entire range of barrier function and dysfunction by a uniform mathematical function were published several years later (the sigmoid form by Reiber, 1986, and an imaginary curve by Laurell, 1987).

• Empirically based (Reiber and Felgenhauer, 1987) and theoretically based (Reiber, 1994 a,b) hyperbolic functions (Fig. 5.20Chap. 2, this volume) have put an end to speculation about the correct function that discriminates between CSF protein fractions originating from the blood and those originating from the brain.

The main reason why discrepancies between linear evaluation and hyperbolic function (Fig. 5.20) were not evident in the past is that most comparisons between evaluation procedures (Öhmann et al., 1992) used data derived from patients with multiple sclerosis. However, these patients usually have normal or only slightly increased albumin quotients, i. e. they do not really have serious barrier dysfunction. Yet it is precisely in the region between mild and severe barrier dysfunction that a huge difference exists between the various evaluation methods, with up to 90% false-positive interpretations (see legend to Fig. 5.20).

Quotient Diagrams (Reibergrams)

Function of the Diagrams

Hyperbolic Discrimination Functions. The CSF/serum quotient diagrams for IgG, IgA, and IgM in a double-logarithmic presentation with hyperbolic discrimination lines (Fig. 5.21)—also called reibergrams (Dorta-Contreras et al., 2002)—allow discrimination between blood-derived and brain-derived immunoglobulin fractions in CSF.

The diagrams can be used for the analysis of lumbar, cisternal, and ventricular CSF for the detection of intrathecal synthesis of IgG, IgA, and IgM in patients of all ages. The only difference is in the age-related reference ranges for the albumin quotient (Chap. 21, “Proteins”).

The hyperbolic function is particularly important for interpreting intrathecal IgG synthesis with very low albumin quotients (QAlb < 0.6 × 10−3), when QIgG values are normally (ventricular CSF, or lumbar CSF in children) higher than QAlb. The linear IgG index is unable to explain the changes in the QIgGQAlb ratios in this range; the theoretically based hyperbolic function, by contrast, explains the empirical data in a quantitative manner.

image

Fig. 5.21 CSF/serum quotient diagrams for IgG, IgA, and IgM with hyperbolic discrimination functions (Reiber, 1994 a). The reference ranges of blood-derived IgG, IgA, and IgM fractions in CSF (ranges 1 and 2) between the upper (QLim) and lower hyperbolic discrimination lines include 99% (± 3 SDs) of the data obtained from 4300 patients. The upper hyperbolic curve (thick line, QLim; Lim stands for limit) of the reference range represents the discrimination line between brain-derived and blood-derived immunoglobulin fractions in the CSF. Values above QLim are presented as intrathecal fractions (IF) as percentages of the total CSF concentration: IgGIF, IgAIF, or IgMIF. These intrathecal fractions can be read directly from the diagram by interpolation of the percentage lines (20%, 40%, 60%, 80% of intrathecal synthesis) with reference to QLim as 0% synthesis. The limit of the reference range for the albumin quotient, QAlb, between normal and increased CSF protein concentration (blood–CSF barrier dysfunction) is age-dependent (vertical line, QAlb = 8 × 10−3 for a 60-year-old patient; for calculation, see Chap. 21, “Proteins”). The diagrams show five ranges: (1) normal range; (2) plain blood–CSF barrier dysfunction (i. e., reduced CSF flow rate); (3) intrathecal IgG synthesis with barrier dysfunction; (4) intrathecal IgG synthesis without change in CSF flow rate; (5) values below the lower hyperbolic line, which indicate methodical error. Characterization of the hyperbolic functions takes into account the analytical inaccuracy for quotients of albumin, IgG, IgA, and IgM with a variation coefficient between 3% and 8%. If there are extensive variations between laboratories, intrathecal immunoglobulin synthesis is only confirmed when IgIF is higher than 10%. Arranging the individual diagrams vertically one above the other maintains the reference to the albumin quotient common to all the diagrams, thus facilitating pattern recognition. The data in the diagrams (•) are from a patient with facial paresis with intrathecal IgM synthesis (IgMIF = 40%) and oligoclonal IgG (IgGIF = 0). This basic information led to further analysis of the Borrelia-specific antibody index (IgGAI = 4.3; IgM-AI = 3.2), allowing Borrelia to be identified as the cause of the disease.

Importance. Although qualitative methods such as the detection of oligoclonal IgG are more sensitive than these statistically based quantitative methods, the quantitative methods are nevertheless necessary. In particular, sensitive detection of intrathecal synthesis of specific IgG antibodies requires a physiologically correct limit of the reference range (QLim). Demonstration of diagnostic immunoglobulin class response patterns, too, which uses the relative amounts of intrathecally formed immunoglobulins of the various classes, is impossible without quantitative methods, (Reiber and Peter, 2001).

Construction of the quotient diagrams. The hyperbolic curves of the upper limit (QLim), the mean value (QMean), and the lower limit (QLow) (Fig. 2.3) have been characterized using the values shown in Table 5.3 for a/bb2, and c(Reiber, 1994 a).

Numerical Evaluation of CSF Protein Data

Calculation of QLim. The general hyperbolic function (Fig. 2.4) uses the following equations to describe the upper discrimination line QLim (Ig) for the reference range in the CSF/serum quotient diagram (Table 5.3):

image

Values for QIgGQIgA, and QIgM above these hyperbolic discrimination lines indicate intrathecal synthesis (99% limit; ± 3 SDs range).

image

Quantification of Intrathecal Synthesis

IgLoc – Increased Immunoglobulin Concentration in CSF

The extent of intrathecal synthesis of an immunoglobulin of the IgG, IgA, or IgM class can be characterized by quantitation of the brain-derived contribution (IgLoc) to CSF concentration, which depends on the serum concentration (IgSerum):

image

Intrathecally synthesized immunoglobulins in CSF, which primarily originate from cerebral tissue, can be treated like glial or neuronal proteins with respect to their dynamics (Fig. 3.2). This means that the IgLoc value does not change with CSF flow rate (QAlb). The example of a patient with neuroborreliosis (Table 5.4) shows how intrathecal IgM synthesis IgMLoc decreases as the disease normalizes, also accompanied by normalization of the barrier function (QAlb).

image

image

IgLoc is the best candidate for showing changes in intrathecal immunoglobulin synthesis over the course of a disease in the individual patient (see, e. g., Fig. 19.5).

IgIF—Intrathecal Immunoglobulin Fraction

To find out how strongly the various immunoglobulin classes are being intrathecally synthesized in an individual patient, it is advantageous to present IgLoc as the relative intrathecal fraction (IgIF). With IgLocrelative to the total immunoglobulin concentration in the CSF (IgIF = IgLoc/IgCSF) and with QIg = IgCSF/IgSerum, the intrathecal fraction is calculated in percent as follows:

image

The intrathecal fractions (in percent) can be read off directly by interpolation from the percentage lines in the quotient diagrams (Fig. 5.21). The mathematical function of the percentage line in the quotient diagrams (Fig. 5.21) for 20%, as an example, is calculated according to the formula for IgIF as (1 – QLim/QIgG) = 0.2 or QIgG = 1.25 × QLim.

image

Fig. 5.22 Time course of CSF protein data obtained from a patient with neuroborreliosis (Tumani et al., 1995). The patient underwent lumbar punctures at 3 weeks (■) and at 4, 6, 10, 16, and 83 weeks after the tick bite. Cell counts were 132/μL, 100/μL, 39/μL, 90/μL, 15/μL, and 3/μL for the respective puncture dates; Borrelia-specific antibody indices at the time of first diagnostic puncture (■): IgG AI = 31, IgG AI = 42. The intrathecal fraction of IgM (IgMIF) was constant between weeks 4 and 16 after the tick bite (punctures 2–5, Table 5.4). The IgM class response in the brain was dominant right from the start for a very long period with IgMIF > IgGIF. There was therefore no clear isotype switch in the brain, unlike the switch in the blood of the same patient. As shown in the bottom diagram, the relative concentrations of IgM and IgG in the serum depended very much on the time since infection, revealing an isotype switch between weeks 2 and 4. Since these data were evaluated as CSF/serum quotients, the protein dynamics in the blood are not relevant for assessing the dynamics of intrathecal synthesis. (Elevated blood IgM levels lead to elevated CSF IgM levels, but the quotient remains constant.)

image

In the example in Table 5.4 and in Fig. 5.22 the intrathecal fraction of IgM (IgMIF) remains constant because both IgM fractions—the blood-derived fraction (IgMSF) due to changing QAlb and the brain-derived fraction (IgMLoc)—decrease simultaneously (i. e., by chance with (IgCSF = IgSF + IgLoc) we find IF = IgMLoc/IgMCSF = constant). The consequences of independent changes in the barrier function and in intrathecal synthesis in the course of the disease are shown for the IgG data combination in Table 5.5: should QAlb = 40 × 10−3 become QAlb = 20 × 10−3, then IgGIF would become 63.4% if the amount of intrathecal IgG synthesis (IgGLoc = 370.4 mg/L) remains constant. By contrast, if IgGIF = 45% is found in the patient, IgGLoc must have decreased to 175 mg/L. As a consequence we see that IgIF is not a suitable parameter for quantitative characterization of intrathecal immunoglobulin synthesis over the course of the disease.

The relative intrathecal fraction IgIF is the best parameter by which to compare intrathecal synthesis of IgG, IgA, and IgM classes in the detection of disease-related immunoglobulin patterns (e. g., in Reibergrams).

Dominance of an Intrathecally Synthesized Immunoglobulin Class. By using the relative value for intrathecal synthesis (IgIF), account is taken of the fact that the amounts of IgG, IgA, and IgM synthesized in blood (IgSerum in Table 5.5) are different by orders of magnitude, and therefore so are the intrathecally synthesized amounts (IgLoc in Table 5.5). Thus, by this approach expressing relative values instead of absolute concentrations, it is finally possible to compare data in terms of an immunoglobulin reaction with a pattern of relative dominance. This is the basis of the disease-related patterns shown in Chap. 19. In addition, we learn from this evaluation that in the intrathecal immune response the isotype switch, typically seen in blood during systemic infections, does not take place in the brain. This is demonstrated in Fig. 5.22, which shows a long-lasting constant dominance of the IgM class reaction during the course of neuroborreliosis (IgMIF > IgGIF).

By dominance we mean the top ranking in the extent of intrathecal immunoglobulin class response, e. g., IgA dominance in the pattern IgAIF > IgGIF of the two-class immune response in tuberculosis (see Fig. 19.3 b; further examples in Chap. 19, “Evaluation”).

Statistics for Groups in Quotient Diagrams

In contrast to the diagnostic approach which takes as its reference the upper limit of the “reference range,” QLim, the statistical approach for comparison of groups must refer to the mean of the control group, QMean (Figs. 2.3 and 5.23). A new software program (Reiber and Albaum, 2008; www.comed-com.de) allows characterization of the difference between groups in quotient diagrams, e. g., multiple sclerosis (MS) patients and noninflammatory control. It is important not to confuse the two options:

Diagnostic CSF interpretation, individual patient:

• Reference is QLim (Fig. 5.23).

• IgGIF is used for pattern recognition (IgG, IgA, and/or IgM dominance).

• Pathological fraction is detected with the highest specificity with QIg > QLim = QMean + 3SD (99% level) (Fig. 5.23).

Statistical CSF interpretation, group analysis:

• Reference is QMean (Fig. 5.23).

• IgGLoc (mean) is used to quantitate mean intrathecal synthesis in a group.

• Pathological fraction is counted with the highest sensitivity with QIg > QMean + 2SD (96% level) (Fig. 5.23).

Mean intensities. The calculation of the mean amount synthesized uses the IgLoc (mean) with reference to QMean instead of IgLoc with reference to QLimQMean is calculated according to the hyperbolic function for QIg, replacing the parameters for IgLim by IgMean as given in Table 5.3. Correspondingly IgLoc (mean) is calculated with QMean replacing QLim:

image

Fig. 5.23 Characterization of the reference range for blood-derived proteins in CSF. The upper discrimination line QLim = QMean + 3SD includes 99% of the patients with noninflammatory diseases (oligoclonal IgG-negative). This is regarded as the most specific discrimination line for diagnosis. For statistical purposes, comparing groups in diagrams, it is more appropriate to refer to the discrimination line QMean + 2SD, which includes 96% of the controls, and approaches the number of MS patients positive for oligoclonal IgG (Reiber et al., 2009).

image

image

As the calculated IgLoc (mean) is independent of the QAlb of the individual patient, it is possible to calculate mean intensities of the group from all IgLoc (mean) values, even if the patients have strongly varying QAlb values. An example is shown in Table 5.6 for a group of MS patients (age group 28–29 years, shown in Fig. 5.24). For the same reason it is possible to compare different groups with different mean QAlb values without the result being biased by their mean QIg values, which are also different (Fig. 5.24). This program provides the data to perform a t-test sufficient to check the null hypothesis and calculate significances with p-values (example in Bechter et al., 2010). The dataset of the 4300 patients used for the development of reibergrams are integrated into the program as the perfect basis for a control group of noninflammatory cases—the largest control group ever available in the field.

image

Fig 5.24 Age-related change of data in MS patients. Age group 7–11 years (■); age group 28–29 years (○). (Graph created with CoMed CSF statistics tool.)

image

Table 5.8 Frequencies of blood CSF barrier dysfunctions QAlb > QAlb (ref) (see p. 254) in a group of patients (the same age group, 28–29 years, as in Fig. 5.24), calculated for the two different levels of significance with age-related threshold + 10% (1.1 x QAlb [ref]) or + 20% (1.2 x QAlb[ref]). The data were calculated with the CSF statistics tool.

 

Number

QAlb frequencies

> +10% level

n10

5

> +20% level

n20

4

Total number

N

29

Mean frequencies. As a reference for statistically significant intrathecal immunoglobulin synthesis the program uses the new threshold of QMean + 2SD instead of QLim (= QMean + 3SD) (Fig. 5.23Table 5.7). As a consequence we obtain more reliable interpretations in particular in groups containing many patients in whom intrathecal immunoglobulin synthesis is weak (Reiber et al., 2009). The data for MS patients (Table 5.7) are a good example: in a group with 98% positive oligoclonal IgG we find only 75% with IgGLoc > 0 if QLim is taken as the reference, but a 92% incidence of pathological IgG in CSF if the reference is QMean + 2SD.

Frequency of blood CSF barrier dysfunctions in a group. The software will also calculate the incidence of blood–CSF barrier dysfunction at two levels of significance for different age-related threshold values of QAlb (Table 5.8).

IgG index: not suitable for statistics in CSF analysis. As shown in Fig. 5.25, as QAlb increases the slope of the IgG index must decrease, in particular if intrathecal IgG synthesis is constant. The data are shown in detail in Table 5.9. With increasing QAlb the constant serum concentration of IgG leads to increasing QIgG, even though intrathecal IgG synthesis (IgGLoc) and serum IgG remain constant. The constant IgGLoc is not mirrored by the index, which drops from 3.1 to 1.2. This erroneous interpretation is an additional argument for leaving behind the linear IgG index as an outdated method that can lead to false diagnostic interpretations as well (Fig. 5.20). Table 5.9 also shows why the intrathecal fraction as a relative value is not suited to statistics, but helps to characterize dominance of immunoglobulin class response in the individual patient.

Oligoclonal IgG

The sensitive detection of oligoclonal bands is described in Chap. 4, “Detection of Oligoclonal IgG Bands”. The interpretations of five types of band patterns according to international consensus are presented in Figs. 4.13–4.15.

As described earlier, every immune reaction is polyspecific but also oligoclonal. The term “oligoclonal IgG” originates from a time when these interconnections were not yet clearly understood (for references, see Andersson et al., 1994). The different antibody specificities of individual bands have been demonstrated by Western blot (Sindic et al., 1994). The following applies to the detection of oligoclonal IgG:

• According to international consensus (Andersson et al., 1994), detection of oligoclonal IgG with isoelectric focusing and subsequent immunodetection is recommended (Figs. 4.134.14).

• A precondition for comparing CSF and serum is the use of identical amounts of IgG in both samples.

• For the immunoblot (Figs. 4.134.14), two bands in the CSF are sufficient to identify type 2, whereas for the direct silver stain, 2–4 bands may be necessary to identify type 2, depending on the homogeneity of the ampholyte pattern (Fig. 4.15). Only in 16% of cases examined in the immunoblot (n = 100) was an isolated individual band in the CSF associated with an inflammatory CNS reaction.

• Isoelectric focusing can detect an intrathecal IgG fraction of just 0.5% of the total IgG in CSF.

Because of its high sensitivity, the qualitative detection of oligoclonal IgG is one of the most important supplements to quantitative IgG analysis. Calculation of the intrathecal fraction (IF) of IgG refers to the upper limit of the statistically defined reference range derived from data from a large group (Fig. 2.3). In this way, a brain-derived IgG fraction could be up to 200% of the blood-derived fraction (e. g., near QLow in Fig. 2.3) before it exceeds the upper limit (QLim) of the reference range in a statistically significant manner. By contrast, isoelectric focusing compares the profiles of CSF IgG and serum IgG of an individual patient, and minor contributions from the brain (0.5%) can thus be identified as a different pattern. In multiple sclerosis, for example, an intrathecal IgG fraction (IgGIF > 0%) is found in only 75% of cases, but it is detectable as oligoclonal IgG in 98% of cases. Thus, for the diagnosis of MS, a quantitative method (IgG index or IgGIF) can not replace the demonstration of oligoclonal IgGs. For statistical evaluations, on the other hand, it is advisable to use QMean + 2SD instead of QLim as the reference for the calculation of prevalence (see “Quotient Diagrams [Reibergrams]” above).

image

Fig. 5.25 Comparison of changing IgG index with constant amount of intrathecal IgG synthesis (IgGLoc) due to independently increasing albumin quotient QAlb (barrier function). Data in Table 5.9. (Graph created with CoMed CSF statistics tool.)

image

Methods that use normal electrophoresis (i. e., without isoelectric focusing) for the separation of the immunoglobulin fraction are by definition unsuitable for demonstrating “oligoclonal IgG”.

image

Fig. 5.26 a, b Principle of interpreting intrathecal antibodies in CSF.

a The CSF/serum data of this patient with herpes zoster ganglionitis (facial nerve palsy) were QAlb = 6.0 × 10–3QIgG = 1.7 × 10–3, and the specific quotient, Qspec was Q VZV = 3.3 × 10–3. The corresponding antibody index was VZV AI = 1.9. The equation AI = Qspec/QIgGwas chosen for this case of QIgG < QLim.

b The data of a patient with MS were QAlb = 5.0 × 10–3QIgG = 11.6 × 10–3QLim = 3.5 × 10–3Q Measles = 22.4 × 10–3Q Rubella = 8.2 × 10–3Q VZV = 5.9 × 10–3, and Q HSV = 2.8 × 10–3 AI = 2.3; VZV AI = 1.8, and HSV AI = 0.8. The equation AI = Qspec/QLim was chosen for polyspecific IgG synthesis (QIgG > QLim). As a rule, a Qspec value above the 30% line in the diagram (Qspec ≥ 1.5 × QLim) represents a pathological AI value (≥ 1.5).

Antibody Index

The detection of intrathecally synthesized antibodies is at its most sensitive when the corrected antibody index (AI) is used (Reiber and Lange, 1991). Fundamentally, there are two cases to distinguish when evaluating specific antibody quotients, Qspec (Fig. 5.26):

• The total IgG quotient is within the normal range, i. e., no local IgG synthesis is detected (IgGIF ≤ 0%) (Fig. 5.26 a).

• There is intensive antibody synthesis, and the resulting IgG quotient lies above the discrimination line in the quotient diagram. In this case, QIgG > QLim or IgGIF > 0%; QLim becomes the reference calculated from the patient's albumin Quotient (Fig. 5.26 b).

Figure 5.26 shows two examples: a case of zoster ganglionitis (Fig. 5.26 a) with QIgG < QLim (no correction required), and a case of multiple sclerosis (Fig. 5.26 b) with pronounced polyspecific immune reaction (QIgG > QLim, correction for QLim is required).

• When calculating the varicella-zoster virus (VZV) AI in the MS patient with intensive intrathecal IgG synthesis (Fig. 5.26 b), the result relative to QIgG would look like this: VZV AI = Qspec/QIgG = 5.9 × 10–3/11.6 × 10–3 = 0.51. However, this value leads to a false interpretation (no synthesis).

• However, when the calculation for QIgG > QLim is based on AI = Qspec/QLim, the following result is obtained: VZV AI = 5.9 × 10–3/3.5 × 10–3 = 1.8. This value indicates intrathecal synthesis of zoster antibodies.

Correction of the AI calculation relative to QLim instead of QIgG leads to a higher sensitivity, i. e., without this correction, 40% of the results of the MRZ reaction (against measles, rubella, and varicella-zoster viruses) in MS patients would be false-negative (Quentin and Reiber, 2004).

In summary, the following two formulas are used for calculating the AI:

image

QIgG is the empirically found immunoglobulin quotient for IgG (or IgA and IgM); Qspec is the specific antibody CSF/serum quotient; and QLim is calculated as shown above.

Reference range for the antibody index (AI):

image Standard deviation depending on the method (x± SD): AI = 1.0 ± 0.16.

image Normal values (x± 2SDs; clinically evaluated): AI = 0.7–1.3.

image Pathological values (clinically evaluated): AI ≥ 1.5.

AI values of less than 0.5 are only possible if a serious analytical error has been made or there has been a sample mix-up (CSF and serum are not from the same patient).

Improving interpretation accuracy. In addition to the QIgG based reference range, one (or several) other “normal” AI value(s) may be used as a reference. This is often useful (for interpretation or the decision whether to repeat the analysis) when the data are borderline (see examples in Table 5.10):

• In case A, the rubella AI of 1.4 should be assessed as pathological.

• In case B, the rubella AI of 1.5 should be assessed as borderline/normal.

• In case C, the data clearly indicate either a serious technical error or a sample mix-up (CSF and serum do not belong together).

Analysis of small sample volumes. If not enough lumbar or ventricular CSF is available for analyzing IgG and albumin, comparison of the Qspec values of different antibody species may suffice. If one of the Qspec values is 50% higher than one (or, even better, several) of the other values, that is evidence of specific intrathecal synthesis.

image

Relative Sensitivities of Immunodetection Methods

Antibody index vs. Western blot to detect viral infections. As has been convincingly shown in cases of herpes encephalitis and zoster meningitis (Felgenhauer and Reiber, 1992), the antibody index is more sensitive than a Western blot.

Antibody index vs. Western blot to detect bacterial infections (borreliosis). Over the years there have been intensive discussions among the various schools of thought about the detection of neuroborreliosis (Fig. 5.22). Because this disease is caused by a great variety of Borrelia sp. (Wilske, 2004) and the antibodies to be detected are directed against different antigens in different patients, it makes a great deal of difference what antigen coating is used in the ELISA. If the right antigen is not there, specific intrathecal synthesis will not be detected.

To prevent this happening, a comparison with the Western blot has been introduced. The earlier concept was developed when certain antigens, such as p41 flagellin, were omitted in order to avoid cross-reaction and misinterpretations. However, the risk of cross-reactivity, between Borrelia spp. and Treponema pallidum, for example, is considered to be not so critical for the detection of neuroborreliosis as is sometimes claimed, so long as a complete CSF analysis is performed. If a positive Borrelia AI is found, a Treponema AI could still be analyzed if the differential diagnosis was not already obvious from the patient's history, clinical features, and basic CSF diagnosis. A drastic change in analytical accuracy arrived with the implementation of the VslE antigen (variable lipoprotein surface antigen of Borrelia) by most manufacturers.

Provided the antigen coating in the Borrelia ELISA is adequate (Borrelia extract and VslE antigen), there is no need for a Western blot. The ELISA has the higher sensitivity.

Antibody index vs. oligoclonal IgG to detect acute and chronic viral infections. In varicella-zoster meningitis, the antibody index is more sensitive than is detection of oligoclonal IgG (Felgenhauer and Reiber, 1992). This is also true for varicella-zoster-induced facial paresis, where oligoclonal IgG can be detected in only 50% of cases whereas the antibody index is always increased. As a general rule:

• In chronic inflammatory processes, detection of oligoclonal IgG is more often successful (98% in patients with MS) than is detection of specific antibodies (MRZ reaction in 90% of patients with MS).

• In acute inflammatory processes, detection of intrathecally produced antibodies against the causative antigen is more sensitive than is detection of oligoclonal IgG.

One reason for this is that the intensity of intrathecal IgG synthesis is up to 60 times higher in a reaction against the causative antigen than in a polyspecific co-reaction (Quentin and Reiber, 2004).

References

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

Bechter K, Reiber H, Herzog S, Fuchs D, Tumani H, Maxeiner HG. Cerebrospinal fluid analysis in affective and schizophrenic spectrum disorders: Identification of subgroups with immune responses and blood–CSF barrier dysfunction. Journal of Psychiatric Research 2010. In Press

Delpech B, Lichtblau E. Etude quantitative des immunglobulines G et de l'albumine du liquide céphalorachidien. Clin Chim Acta 1972;37:15

Dorta-Contreras AJ, Reiber H, Magraner-Tarrau ME, et al. Valor neuroimmunoepidemiológico del reibergrama en la primera epidemia de meningoencefalitis por echovirus 16 en Cuba. Rev Neurol 2002;35:517–520

Felgenhauer K, Reiber H. The diagnostic significance of antibody specificity indices in multiple sclerosis and herpes virus induced diseases of the nervous system. Clin Investig 1992;70:28–37

Ganrot K, Laurell CB. Measurement of IgG and albumin content of cerebrospinal fluid and its interpretation. Clin Chem 1974;20:571–576

Ganrot-Norlin K. Relative concentrations of albumin and IgG in cerebrospinal fluid in health and in acute meningitis. Scand J Infect Dis 1978;10:57–60

Jacobi C, Reiber H, Lange P. Quantitation of intrathecal antibodies in CSF of subacute sclerosing panencephalitis, herpes simplex encephalitis and multiple sclerosis: discrimination between microorganismdriven and polyspecific immune response. J Neuroimmunol 2007;187:139–146

Laurell CB. On the origin of major CSF proteins. In: Thompson EJ, ed. Advances in CSF protein research and diagnosis. Amsterdam: Elsevier; 1987:123–128

Lejon V, Legros D, Richer M, et al. IgM quantification in the cerebrospinal fluid of sleeping sickness patients by a latex card agglutination test. Trop Med Int Health 2002;7:685–692

Lejon V, Reiber H, Legros D, et al. The intrathecal humoral immune response pattern for improved diagnosis of central nervous system involvement in trypanosomiasis. J Infect Dis 2003;187:1475–1483

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

Luxton RW, Patel P, Keir G, Thompson EJ. A micro-method for measuring total protein in cerebrospinal fluid by using benzethonium chloride in microtiter plate wells. Clin Chem 1989;35:1731–1734

Öhman S, Ernerudh J, Forsberg P, et al. Comparison of seven formulas and isoelectrofocusing for determination of intrathecally produced IgG in neurological diseases. Ann Clin Biochem 1992;29:405–410

Quentin CD, Reiber H. Fuchs heterochromic cyclitis: rubella virus antibodies and genome in aqueous humor. Am J Ophthalmol 2004;138:46–54

Reiber H. Eine schnelle und einfache nephelometrische Bestimmungsmethode für Protein im Liquor cerebrospinalis. J Clin Chem Clin Biochem 1980 a;18:123–127

Reiber H. The discrimination between different blood–CSF barrier dysfunctions and inflammatory reactions of the CNS by a recent evaluation graph for the protein profile of CSF. J Neurol 1980 b;224:89–99

Reiber H. Kinetics of protein agglomeration. A nephelometric method for the determination of total protein in biological samples. J Biochem Biophys Meth 1983;7:153–160

Reiber H. Evaluation of blood-cerebrospinal fluid barrier dysfunctions in neurological diseases. In: Suckling AJ, Rumsby MG, Bradbury MWB, eds. The blood-brain barrier in health and disease. Chichester, UK: Ellis Horwood, 1986;132–146

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, 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, Lange P. Quantification of virus-specific antibodies in cerebrospinal fluid and serum: sensitive and specific detection of antibody synthesis in brain. Clin Chem 1991;37:1153–1160

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

Reiber H, Ungefehr S, Jacobi C. The intrathecal, polyspecific and oligoclonal immune response in multiple sclerosis. Mult Scler 1998;4:111–117

Reiber H, Thompson EJ, Grimsley G, et al. Quality assurance for CSF protein analysis: international consensus by an internet-based group discussion. Clin Chem Lab Med 2003;41:331–337

Reiber H, Albaum W. Statistical evaluation of intrathecal protein synthesis in CSF/serum quotient diagrams. Acta Neuropsychiatrica, 2008; 20 (S 1): 48–49 and download of “CSF statistics tool” from www.comed-com.de

Reiber H, Teut M, Pohl D, et al. Pediatric multiple sclerosis: age related differences and dynamics of the neuroimmunological response in CSF. Mult Scler 2009; 15:1466–1480

Sindic CJM, Delacroix DL, Vaerman JP, et al. Study of IgA in the cerebrospinal fluid of neurological patients with special reference to size, subclass and local production. J Neuroimmunol 1984;7:65–75

Sindic CJM, Monteyne P, Laterre E. The intrathecal synthesis of virusspecific oligoclonal IgG in MS. J Neuroimmunol 1994;54:75–80

Stauch C, Reiber H, Rauchenzauner M, et al. Intrathecal IgM synthesis in children with multiple sclerosis is associated with a slower progression. Arch Neurol 2010; submitted

Tourtellotte WW. On cerebrospinal fluid immunoglobulin-G (IgG) quotients in multiple sclerosis and other diseases. A review and a new formula to estimate the amount of IgG synthesized per day by the central nervous system. J Neurol Sci 1970;10:279–304

Tumani H, Nölker G, Reiber H. Relevance of cerebrospinal fluid parameters for early diagnosis in neuroborreliosis. Neurology 1995;45:1663–1667

Wilske B. Diagnosis of Lyme borreliosis in Europe. Vector-Borne Zoonot Dis 2004;3:215–227

Zane HD (ed). Immunology: theoretical and practical concepts in laboratory medicine. Philadephia: Saunders; 2001