1. In CSF diagnostics, the following principles are used that can be generalized to improve the quality of laboratory medicine:

The current development in clinical neurochemistry is critically discussed with reference to the database of the INSTAND external quality assessment schemes (EQAS) for CSF (Reiber, 1995) and the current certification practice.

2. The integration of disease pathology and current knowledge bases must be part of any quality control of medical laboratory data. The second main aspect of this article is, therefore, the presentation of the medical and biological knowledge bases relevant for the plausibility control and, finally, the correct interpretation of diagnostic data. The obvious deficits in the practice of diagnostic interpretations (Uhr, 2024) and the subsequent lack of adequate therapeutic consequences make this an important goal (Reiber, 2024).

The review of the following topics presents some essentials of clinical neurochemistry in particular and clinical chemistry in general, based on a recent publication (Reiber, 2024):

• The diffusion-flow model of blood–brain barriers

In times of AI-based big data analysis, the call for a shift from a growing number of individual analytes that lack rational argumentation to a limited amount of functionally linked data seems to be swimming against the tide. However, it is not. In medicine, there are no sufficiently large datasets for disease group statistics with deep learning approaches; conversely, the perspective for AI with machine learning is to integrate an existing secure knowledge base to ensure the reality of categorizations (Wahlster, 2021).

With the integration of current knowledge bases, CSF diagnostics shows the relevance of data coupling to obtain a disease model-based selection of diagnostic datasets. The increasing acceptance of complex system approaches in diagnostics and EQASs in CSF show the foundations of a medical-based quality control that goes beyond the usual external accuracy control of individual analytes.

These aspects could contribute to the perspectives in medical laboratory diagnostics and restore the medical competence of clinical chemists.

The blood–brain and blood–cerebrospinal fluid barriers are morphologically different and also vary in different brain areas. The molecular passage between blood and extracellular fluid (ECF), the blood–brain barrier, and the passage from blood to CSF, the blood–cerebrospinal fluid barrier, are based on two basic transfer ways, the paracellular passage with facilitated or active transfer mechanisms and the intercellular passage for proteins, which depends on passive diffusion (Reiber, 2024). The biophysical principles in both barriers are the same, but the CSF has a 10-fold faster flow (turnover) than the ECF in the brain.

Barrier function for proteins: Serum proteins pass through the endothelial cell layer of the capillaries, which are reinforced with additional brain-specific structures, different in different areas of the brain. The molecular size-dependent restriction of the diffusion of proteins into the CSF is at a steady state with the elimination by CSF outflow (bulk flow). This steady state leads to very low–normal concentration ranges in the CSF, with approximately 1/100–1/3,000 (IgM) of serum concentrations for the most common proteins with the corresponding analytical problems.

In many neurological diseases, the concentrations of serum proteins in CSF are pathologically increased, which is diagnostically characterized by the increased albumin CSF/serum concentration quotient, QAlb (Figure 1). The cause, a pathologically reduced CSF turnover, has three possible sources:

- The inflammation-related disturbance of CSF production in the ventricular plexus;

The new paradigm for the barrier dysfunctions is the biophysically derived and patho-physiologically validated diffusion-flow model of the barriers (Reiber, 2003; Reiber, 2021a; Reiber, 2021b). It provides a rationale for the correct interpretation of a barrier dysfunction. Even the age-dependent increase in the normal QAlb value can be explained by reduced CSF production due to age-related changes in the choroid plexus. The increasing QAlb value, the barrier dysfunction, thus represents a reciprocal function of the pathologically decreasing CSF flow rate (Figure 1).

Figure 1 shows a consequence of the changing paradigms. The flow-related change in the local molecular flux, dependent on a non-linear concentration gradient across the barrier (Reiber, 2003; Reiber, 2021a), leads to non-linear, hyperbolic equations of the blood–CSF barrier function instead of linear correlations (Index, I, Figure 1). QAlb is the general reference value for the individual barrier function. It is a gift of nature that albumin, the largest serum protein fraction, is synthesized only in the liver and not also in the brain in pathological processes.

The discrimination between a protein fraction (e.g., IgG) diffusing from the normal blood across the barrier into the brain and a pathological protein fraction synthesized in the brain (intrathecal IgG fraction) is characterized by a hyperbolic limit function either in quotient diagrams (Qlim in Figure 1) or as a mathematical equation (Reiber, 2020). The still frequently assumed linear relationship (Index, I, in Figure 1) leads to interpretation errors. Calculation software for numerical and graphical statistical treatment of CSF data in diseases is available (free software from www.albaum.it), as explained by Reiber (2020).

The example of the blood–brain barrier function shows that without an understanding of the physics of diffusion and the stability of biological structures through material self-organization (see below), an adequate interpretation of the empirical data could be missed.

On one hand, this example of a changed knowledge base replaces the clonal selection model of immunity an on the other hand, the idea that the brain is immunologically isolated. The immune system reacts as a network of B-cell clones, and the brain is linked to the immune and endocrine systems by pleiotropic cytokines. This leads to a new interpretation of the development of autoimmune diseases and immune system-associated pathologies such as bipolar spectrum disorders in psychiatry, which miss classical signs of immune reaction in the CSF.

Interpretations of the immune response in brain need to consider the following processes:

• All antibody-forming B cells in the brain migrate from the blood across the barrier into the brain and proliferate in the perivascular lymphocyte cuffs.

The idea of the immunological network dates back to 1975, with numerous theoretical approaches being developed in the 1980s (Varela and Coutinho, 1989; De Boer and Perelson, 1991). These theories extended the classical view that the adaptive immune system reacts to invading substances by producing antigen-specific antibodies (clonal selection) to rid the body of invaders. With the discovery of anti-idiotypic antibodies that recognize the body’s own antibodies, lymphocyte-based networks became relevant. With the discovery of the polyspecific nature of the immune response in CSF, CSF research has made a major contribution to the practical significance of these theories. Felgenhauer et al. (1985) first described measles virus antibodies in the CSF of MS patients. Meanwhile, the observation that all immune responses, whether acute or chronic, involve polyspecific antibody synthesis (Reiber et al., 1998; Reiber, 2017a) has made the network property of the immune response an obligatory knowledge base for the interpretation of antibody data. In addition to this B-cell network, the trans-organ cytokine network associated with immunocompetent brain cells has also become important for the understanding and diagnosis of diseases related to the immune system, especially chronic diseases (Reiber, 2017a; Reiber, 2017b; Reiber, 2024).

Connectivity in the immune system explains many frequently misinterpreted observations. Only through the polyspecific activation of different coupled B-cell clones can lifelong immunity be maintained, as observed with measles immunization. The short lifespan of the B cells would not allow this if the specific B-cell clones were not maintained in the system through constant polyspecific activation.

The individual network depth of the connected B-cell clones determines the duration and efficiency of immunization (wild-type infection or vaccination). Connectivity can also be the cause of an autoimmune reaction by activating an existing B-cell clone for autoantibodies. When low levels of autoantibodies that provide immune tolerance are upregulated by a factor of 50, an autoimmune response appears as an undesirable damage associated with the individual immune response.

The Danish Disease Register (Nielsen et al., 2016) shows that autoimmune diseases and symptoms such as chronic fatigue syndrome/ME can occur a few weeks after the occurrence of infectious diseases. This also corresponds to post-Lyme or post-COVID symptoms. These correlations of non-specific, delayed immune reactions also make it clear that vaccinations can act as polyspecific triggers and, thus, also make the largely suppressed connection of the Gulf War illness with the 8-fold vaccinations in the first Gulf War plausible (Reiber and Davey, 1996; Rook and Zumla, 1997; Nielsen et al., 2016; Reiber, 2017b). Post-COVID syndrome has also been observed after vaccination, albeit less frequently than after wild-type infections. These associations offer a new approach to chronic diseases associated with the immune system that were previously difficult to diagnose (Reiber, 2024).

In the brain, cytokines are produced in immunocompetent cells, astrocytes, microglia, and endothelial cells. They can have both pro-inflammatory and anti-inflammatory effects. This is an important aspect of the immune response in the brain as a self-organizing local process, which is thought to be involved in various psychiatric chronic diseases (Bechter et al., 2010; Bechter, 2020; Runge et al., 2021).

The network is formed by the three properties of the different cytokines:

- Functional pleiotropism.

Pleiotropism refers to the binding of a cytokine to receptors in different organ systems. This creates a link between the three central control systems of the organism: nervous system, endocrine (hormonal) system, and immune system. Functional redundancy means that different cytokines can have the same effect in one and the same organ.

Since one and the same cytokine can also be upregulating and downregulating depending on its concentration, a very complex network is created that is influenced by different body systems. This explains experiences that have led to the establishment of research concepts such as psychoneuroimmunology and diseases such as “stress-induced para-inflammation” (Bechter, 2020).

The involvement of cytokines in the initial processes of brain inflammation via the choroid plexus (Castellani et al., 2023) is also fundamental information for understanding the frequent barrier dysfunction in neurological diseases as a consequence of reduced cerebrospinal fluid production in the plexus.

The inclusion of cytokine analysis must become a fundamental requirement in neurology and psychiatry in order to explain pathologies associated with the immune system.

The above examples of barrier function and the immunological network have highlighted a deficit in medical development. This becomes even clearer when we consider the unsuccessful therapeutic research into chronic diseases. There is no chronic disease for which we have a causal therapy, be it high blood pressure, type 2 diabetes, multiple sclerosis, autoimmune diseases, post-infectious chronic fatigue syndrome, heart failures, or the many types of cancer. This is primarily the result of the lack of complexity of the predominantly linear disease models, which are based on simple cause–effect relationships.

Fifty years ago, glycolysis oscillation (Hess and Boiteux, 1980; Gerok et al., 1989; Reiber, 2024) described how even in the smallest metabolic process of the cell, oscillating regulatory states can spontaneously change from one rhythm to another due to non-linear functions. This phenomenon is explained by complexity science as a change between attractors.

In addition to obtaining a better understanding of complex systems (Gerok et al., 1989; Goodwin, 2001; Reiber, 2007), we need to move from Jacob and Monod’s refuted, 70-year-old model of the “genetic program” to the relevance of a phenotypic biology with a new view of epigenesis (Reiber, 2012). This also includes a fundamental recognition of the concepts of material self-organization in the context of non-equilibrium thermodynamics (Prigogine, 1997) or the non-linearity of biological processes in the context of complexity science (Mandelbrot, 1991). A short survey on these topics (Reiber, 2024) was published recently, providing more details.

There is no doubt that the improvement in medical diagnostics and therapy requires better biophysical disease models.

The daily fluctuations in the concentration of antibodies in the patient blood are shown in Figure 2. The coordinated variations indicate a common complex regulation. In patients with osteoporosis (Gerok et al., 1989), a disorder of calcium/parathyroid hormone regulation, the mean blood concentrations of calcium or parathyroid hormone remained unchanged compared to that in controls. Rather, the transitions changed from a normal, deterministic–chaotic time series of parathyroid hormone concentration in the blood to an almost constant concentration curve, indicating the loss of regulation. Analyzing a single blood sample may be useless for diagnosis, but a time series could provide it.

In contrast to analyzing individual samples, the electrocardiogram in heart disease provides a sufficiently large dataset to create time series (tachograms). The normally chaotic rhythm of the heartbeat (Reiber, 2024) changes under certain conditions to a time series, which can be analyzed for a lower complexity (change in fractal dimension) using complexity science methods (Mandelbrot, 1991; Reiber, 2024).

In general, time series (e.g., from electrocardiograms or electroencephalograms) are not available for clinical chemical diagnostics. However, these theoretical principles show that chronic diseases, in particular, can represent a stable state, i.e., have an attractor. This has consequences for diagnostics and therapy (Reiber, 2024).

This becomes clear, for example, in post-Lyme disease (Reiber et al., 2013): antibody findings must be interpreted differently, and treatment with antibiotics is pointless. The current discussion about post-COVID shares these problems.

CSF diagnostics cannot refer to the serial examination possible in blood, as a CSF puncture is rarely repeated unless for special reasons. This leads to a further concept for the integration of the complexity approach. The identification of disease-related data patterns can compensate, to a certain extent, for the complexity in the processes, as represented in the time series.

The disease-typical reaction patterns of intrathecal IgG, IgA, and IgM syntheses differ from the uniform immunoglobulin dynamics in the blood due to the lack of an isotype switch in the brain and the local character of the intrathecal immune response with a barrier passage of the immune cells originating from the blood. By calculating the relative intrathecal fractions of the total immunoglobulin in the CSF (intrathecal fractions, IF, shown as % lines of intrathecal Ig in Figure 3), disease-typical combinations of IgG, IgA, and IgM syntheses can be described in the quotient diagrams, which are also known as Reibergrams or Reiber diagrams (Figure 3).

The evaluation of combined data patterns as part of an EQAS with INSTAND had been introduced in the 1990s (Albaum and Reiber, 2024). In many surveys, we demonstrated that the quality of medical diagnostics does not depend on the smallest possible coefficient of variation of a method but on the interpretability of laboratory data by the clinical chemist and, finally, the physician. The Göttingen neurologist Klaus Felgenhauer, who emphasized the importance of laboratory analyses, said, “If a laboratory finding does not fit into my clinical diagnostics, I ignore it.” This can save lives, but it can also cause an alternative diagnosis to be missed. This aphorism became reality when a laboratory report of an isolated intrathecal IgM synthesis with a normal cell count and other normal protein concentrations was ignored as the clinicians had discarded an inflammatory process. So, the alternative interpretation as a non-Hodgkin lymphoma in the CNS was missed by the neurologists, and it needed a particular case conference to find the diagnosis with delay. To query these options of intrathecal IgM synthesis in an external quality control system contributes more to the quality of laboratory diagnostics than the correctness of absolute IgM concentration values. Some control institutions (Controllab, Brazil) cover this aspect of knowledge-based interpretations by an occasional question/answer test. INSTAND is hosting an online conference of the surveyor with the customers, in addition to the explicit medically oriented commentary, with the results reported to the participants.

The concept of an integrating laboratory report, which presents all laboratory data of a patient together with the interpretation in a quotient diagram (Reiber, 2016c; Wildemann et al., 2010; Reiber, 2016a; Albaum and Reiber, 2024), made it seem obvious to couple the absolute values measured on the nephelometer machine with software that integrates the data directly into an online laboratory data report. These software developments by Albaum (2024) for Dade Behring (now Siemens Healthcare with another software manufacturer) and by Wormek (2024) for Beckman Nephelometers contributed significantly to the acceptance of the Reibergrams. Siemens offers the “Protis” program (Siemens, 2024) as part of an advanced diagnostic concept. However, the expensive accreditation of this long-established software program currently prevents providers from offering their software, e.g., for stand-alone solutions in individual laboratories, possibly in an international context.

Test certification, originally introduced as laboratory marketing, has developed over the decades into a profit-orientated industry in its own right. This has had devastating consequences, particularly for the isolated solutions developed and offered for sale by smaller IT companies. Waiting several years for certification, e.g., by the TÜV (Official technical surveyance of cars in Germany), and, above all, the annual costs of 30,000–50,000 Euros are a deterrent. The additional personnel costs of the software manufacturer for the administrative requirements make, for example, the certification of a simple CSF software program that has been functioning for 30 years prohibitively expensive as these costs cannot be passed on to the user.

The fact that a specialist in metallurgy is sent to the institute for quality assurance in medical diagnostics (INSTAND eV) for accreditation is one of the symptomatic problems of this type of pseudo-inspection. This does not contribute to diagnostic accuracy. Instead, it results in the explosion of costs in the healthcare system. Ultimately, it also means that development in small IT companies is no longer financially viable, and the programs are therefore not even offered. Therefore, this certification practice is an obstruction to quality in medicine.