**3. Qualitative measures of CSF IgG—method and limitations of oligoclonal band analysis**

#### **3.1. Oligoclonal bands**

underestimation may vary widely from 1 to 50 mg/L according to *Q*Alb (unpublished results). In cohort studies, *Q*IgG\_basal is advantageously replaced by *Q*mean, which provides a closer

Lastly, inter‐assay variability may directly impact *Q*IgG or the IgG index. For example, a 10% decrease in serum IgG directly means a 10% increase in IgG index, and 10% variations of the IgG index from day to day are commonly reported [7, 10]. As a consequence, minor fluctuations in IgG levels may be translated into normal or abnormal *Q*IgG results, although the intrathecal IgG synthesis rate is not really impacted. A final pitfall of ITS assessment relates to the properties of CNS‐targeting antibodies themselves, which are capable of brain adsorption that can potentially abolish low levels of specific antibodies that are synthesized locally or spill

Using formulas and normal values obtained from the literature, we simulated results of a cohort with tunable ITS level (unpublished results). This model provides the advantage of

IgGLoc estimation based on *Q*mean fitted well with the exact IgGLoc, even in small cohorts and for small ITS (<1 mg/L). On the other hand, individual or cohort estimations of IgGLoc based on

**Figure 2. Effect of plasma exchange on** *Q***IgG**. Simulated population with ITS +5 mg/L. *Q*IgG values increase to abnormal

Plasma exchange depletes both serum IgG and IgG passively transferred through the BBB. Therefore, assuming that ITS remains constant during the procedure, the contrast in locally synthesized and passively diffused IgG in CSF is dramatically tuned by plasma exchange (**Figure 2**). For example, after a 90% decrease in [IgGserum], IgG in CSF originates almost entirely

being able to compare the calculated (approximated) IgGLoc with the fixed IgGLoc.

**2.2. Predicted changes in ITS measures in response to treatments**

*Q*Lim were strongly biased in a range dependent on *Q*Alb.

range whereas ITS level remains unchanged.

estimation of the exact IgGLoc [7].

54 Trending Topics in Multiple Sclerosis

over to the CSF [11].

CSF OCB analysis is based on an isoelectric focalization (IEF) technique fractionating Ig into multiple bands according to their respective pI. Structurally diverse antibodies sharing the same pI may cofocus in a single band [12]. Studying IgM OCBs is harder owing to the dissociation of pentamers before the F fragment and the arbitrary reassociation of the separated monomers [13].

Moreover, owing to differential posttranslational modifications (PTMs) (glycosylation or amino acid modifications), each particular IgG clone may display several pKs and may run in multiple bands in IEF conditions [14, 15]. Monoclonal antibodies occurring naturally (mono‐ clonal gammopathy) or commercially available ones are resolved in multiple bands on IEF gel runs (ladder pattern) [15–17]. In a series of 20 myelomas, IEF identified 5–10 OCB in serum [18]. Commercial monoclonal antibodies produced by hybridomas (monoclonal mouse antibody) or monoclonal IgG produced in vivo by plasma cell tumors (multiple myeloma) are highly heterogeneous and present a clone‐specific profile of glycosylation [17, 19]. Lastly, each OCB is a visual optical interpretation of a local contrast of color density differentiating OCB from the surrounding polyclonal background. Apart from the relative subjectivity of the technique, faint and ambiguous OCB may be obscured by a dense polyclonal background. Capillary IEF seems to increase sensitivity and demonstrated OCB in negative patients with classical IEF [20]. Therefore, immunoblotting against known antigens, for example, in association with viral encephalitis, unmasks specific OCB in cases failing to demonstrate any OCB with conventional IEF [21]. In MS patients explored for specific antibody anti‐viral synthesis, OCB against multiple viruses may occur in the same migrating zone on IEF [22] and CSF OCB against myelin basic protein (MBP) or measles obtained with immunoblot detection does not comigrate with OCB obtained by IEF [23].

There may be several consequences of this as follows: (1) each OCB may be composed of multiple distinct IgG, which may share by chance the same isoelectric point [15]; (2) multiple OCB in MS may derive from one or a small number of cell clones [17]; (3) OCB count cannot predict the number or variety of B‐cell clones; and (4) clonally expanded B‐cell clones may precede OCB detection, thus reflecting the difficulty to detect very low levels of ITS.

ITS in MS is characterized by numerous unambiguous OCBs (≥10 OCB) that are highly specific of MS [24]. Monoclonal bands are exceptional and should be considered carefully. Repeated LP in these atypical cases demonstrates a broadening of ITS or a different diagnosis [25]. Owing to their lack of specificity [25], ≥2 OCB are required in most studies to define an oligoclonal pattern with optimal specificity.

Absence of OCB (type 1 pattern) is observed in less than 10% in recent studies, whereas most MS patients display the type 2 pattern and only some have type 3 (**Figure 3**). Among OCB+ patients, patterns 2 and 3 are observed in about 90 and 10% in relapsing‐remitting (RR) and secondary progressive (SP) patients, respectively, whereas the distribution is 40 and 60% in primary progressive (PP) patients [27], suggesting that pattern responses may be linked to pathophysiology.

**Figure 3. Types of isoelectric focusing patterns on agarose gels** [26]: Type 1, no OCB in CSF or serum (normal pat‐ tern). Type 2, OCB restricted to CSF, absent in serum. Indicates low‐level ITS. Typical pattern in MS. Type 3, Identical OCB in both serum and CSF with extra bands in CSF. Pattern seen during systemic synthesis associated with intrathe‐ cal synthesis. Seen in MS. Type 4, OCBs in CSF mirror those in serum. Indicates a systemic IgG synthesis and passive transfer of OCB from blood to CSF, without any local synthesis. From a purely theoretical point of view, a low local synthesis with similar B‐cell clones cannot be completely deciphered from this situation. Type 5, Ladder‐type identical OCB in both serum and CSF typically associated with monoclonal IgG proteins. Peripheral IgG synthesis without local synthesis.

#### **3.2. Absence of OCB in MS patients is a technical limitation**

OCB are almost always present when *Q*IgG> *Q*Lim, but are also commonly present when *Q*IgG *Q*Lim [26, 28, 29]. Nonetheless, about 5–10% of MS patients fail to demonstrate any OCB or an elevated IgG index. Nevertheless, the question remains whether ITS is really absent from such patients' CSF. Our short review argues for a probable faint ITS in the rare 'CSF‐negative' patients, making ITS the most valuable marker of MS to date.

A high CSF IgA synthesis has been demonstrated in a few patients lacking the classical IgG synthesis [30, 31]. Free light chain (FLC) sensitivity seems to be near 100% but the (expected) specificity lower than OCB makes FLC less useful for routine clinical purposes [32, 33]. Oligoclonal κ‐FLCs are detected in about 50% of MS patients without OCB [34, 35]. Isoelectric focusing with affinity blotting against known antigens overcomes the limitation induced by the background and unmasks specific ITS. OCBs against the neurotropic MRZ viruses, measles, rubella, and varicella zoster virus (VZV), which are commonly observed in MS for reasons that remain unclear, were present in the CSF of 72% of MS patients who otherwise failed to demonstrate OCB with IEF [36].

The specific antibody index (AI) is calculated by using the ratio of specific antibodies *Q*Spec=[SpecCSF]/[Specserum] in the formula, AI = *Q*Spec/*Q*IgG, where AI values >1.3–1.5 indicate the presence of ITS. An MRZ pattern, which is defined as an elevated AI against ≥2 neurotropic viruses, is observed in up to 90% of MS patients [37, 38]. However, the AI is not usually assessed in patients with *Q*IgG *Q*Lim and negative OCB, because they are thought to be MRZ‐negative since AI correlates with *Q*IgG [29]. When multiple AIs (VZV, herpes simplex virus (HSV), cytomegalovirus (CMV), measles, rubella, and Borrelia) were systematically assessed in MS patients without apparent ITS (*Q*IgG *Q*Lim and negative OCB), all of them showed ≥1 MRZ reactivity and up to 47% of patients had OCB against ≥1 MRZ [29, 36, 39–41]. Interestingly, AI results are not completely congruent with those obtained by IEF with affinity blotting, thus increasing the prevalence of ITS detected by ≥1 technique to 64% in the "CSF‐negative" subgroup of MS patients [36]. Since the MRZ reaction is common but not exclusive and because reactions against many other viruses have been confirmed in MS (see below), a larger antigenic test panel might improve the frequency of ITS detection [29], for example, by the systematic use of antigen array [42] and the systematic assessment of IgM.

Studies combining all currently available techniques should be undertaken in MS patients, especially in those with "negative CSF", in order to establish the true prevalence of ITS in MS, especially in patients of non‐Caucasian descent who are thought to harbor a lower CSF positivity [43, 44]. Future studies could combine quantitative techniques (IgGLoc, FLC, AI against MRZ) and highly sensitive qualitative techniques (OCB) until the discovery of putative antigenic targets such as anti‐CCP (cyclic citrullinated peptide) in rheumatoid arthritis. Criteria for standardizing intrathecal Ig synthesis should be based on the simultaneous normalization of all the tests. In view of the fluctuations of ITS in individual patients (up to 30% of IgGLoc [45]), the action of a drug upon ITS should be statistically demonstrated in groups. To be able to demonstrate a successful intrathecal reset, a null Ig synthesis should be confirmed by using several techniques together.
