The logos or logic of events postsubarachnoid hemorrhage is that the oxygen-rich arterial blood and activated lymphocytes, macrophages, microglia, etc., are combining to contribute to oxidative stress. With oxidative stress, there are oxygen-free radicals and reactive oxygen species. Radicals and reactive oxygen species are not the same. An oxygen-free radical is an ionized form of oxygen that can often occur by the addition of an extra electron. However, that molecule will not want to “share” that electron in an ionic bond, it will tend to form covalent bonds, which means that new molecules are formed. Reactive oxygen species are not necessarily ionized, but are oxygen containing molecules that are chemically active, such as hydrogen peroxide. These molecules as representatives for their molecular families can chemically modify numerous molecules susceptible to their nucleophilic attack. The result is a host of covalently modified molecules.

Lipid oxidations are a common occurrence when there is oxidative stress because the brain and all cell membranes contain lipids that can be oxidized. Typically unsaturated lipids are more susceptible to oxidation because their double bonds produce an electron cloud in the pi orbital that forms a partial negative charge, the constitutive positive charge caused by electron withdraw is prone to covalent modification from negatively charged oxygen-free radicals. The result is that poly unsaturated fatty acids, such as arachidonic acid, are covalently modified to produce compounds, such as the HETEs and EETs (2–4). The observation of HETEs and EETs in blood and spinal fluid of subarachnoid hemorrhage patients is indicative of lipid oxidation. The type of lipids oxidized contain information concerning the pathology/damage being done and a growing body of evidence suggests that the products of such oxidations may be biologically active and contribute to complications postsubarachnoid hemorrhage (2, 5–8).

Lipids, even oxidized lipids, such as HETEs and EETs, are good candidates for markers that can be assessed in the spinal fluid as well as blood markers. Further several HETEs and EETs are known to activate the immune system, so concomitant activation of inflammatory markers is expected (9–11). A lack of apparent response may indicate a threshold for HETEs and/or EETs or that the duration of their levels may be insufficient for an immune response. Thus, this introduces an important component to the ethos or theme for this part of the discussion. One marker being evaluated could impact on other markers and thus a physician learning to evaluate spinal fluid and blood markers will need to learn how they interact. Similar to when physicians are learned to read pH markers in a CBC and have an understanding of renal versus respiratory compensation for pH, the physician will need to understand how markers interact as well.

Our lab has been studying the structure, time course, and biological activity of blood products in the spinal fluid of the subarachnoid hemorrhage patient (1, 6, 7, 12–18). Historically, bilirubin was implicated in the pathogenesis seen in subarachnoid hemorrhage patients and complications seen therein (19–24), but the cause and effect role for bilirubin was unclear. The time course for bilirubin appearance in the spinal fluid was concomitant with complications postsubarachnoid hemorrhage, such as vasospasm, but bilirubin in experimental settings did not cause vasospasm or neurologic deficits.

Interestingly, there is a large body of evidence demonstrating that bilirubin has antioxidant activity. While the mechanisms of many antioxidants is known to be an electron acceptor or transporter to other electron acceptors bilirubin appeared/appears to be a direct electron acceptor as an antioxidant. However, prior to the year 2000, there was relatively little characterization of the chemical changes that occurred when bilirubin was oxidized (25).

Bilirubin oxidation products (BOXes) are a relatively new family of oxidized bilirubin that appear to be produced postsubarachnoid hemorrhage and associated with neurologic decline and vasospasm (6). Recent mechanistic studies have found that BOXes may act in vascular smooth muscle by manipulating phosphorylation and dephosphorylation pathways (26, 27). These signaling pathway changes are consistent with several of the clinical observations seen in patients postsubarachnoid hemorrhage, thus the presence, time course, and activity of BOXes can be employed to better evaluate the events occurring in the subarachnoid hemorrhage patient.

Proteomics is a field where proteins are determined and correlated to conditions (28). Proteomic detection can detect thousands of proteins and peptides in a sample of blood or spinal fluid. There are enormous problems when analyzing blood because of the large amount of albumin, globins, hemoglobin, and immunoglobulins in the sample. These overload many systems and can mask important proteomic signals. It is also important to have control samples to compare these against such that changes or differences can be assessed. The field has not yet been established long enough to have a wide range of normal values for all the possible protein hits that might occur. The technology is also lacking sophistication to precisely determine subtle concentration differences, so gross presence or absence (or orders of magnitude change) tends to be reported. It is not uncommon for a proteomic analysis on a patient to show hundreds of differences from a baseline or control database. The vast amount of data thrown at a physician or scientist becomes unwieldy and unmanageable. The result is the research and clinical communities are slow to adopt these methods. However, and importantly, there are ways to simplify the proteomic message and make it more useful.

Recent advances in microfluidics and mass spectrometry have enabled the latest level of technology advancement to focus proteomic studies. For example, recent reports on spinal fluid from subarachnoid hemorrhage patients (29, 30) have found that metalo proteins or phosphorylated proteins can be specifically detected and analyzed (31).

In 2005, the first commercially available microfludic (lab on a chip type of technology) nano mass spectroscopy (nano-LC-ESI-MS) instrument was introduced for proteomics analysis (32–34). This system can obtain qualitative information from complex biological samples (35–38). From these studies, it has been demonstrated that nano-LC-ESI-MS when enhanced by the CHIP™ technology has excellent resolution from complex systems, such as biologic fluids as well as good sensitivity and reproducibility. The newer systems can perform a wide range of useful peptide identification routines for metalo proteins and phospho proteins using the public access databases, such as Spectrum Mill (Agilent, Santa Clara, CA, USA) and MASCOT (Matrix Science, London, England).

Importantly, as new technologies become more prevalent new markers may be eligible for evaluating and assessing the subarachnoid hemorrhage patient. With technologies, such as genomics, genetics, proteomics, metabolomics, immunomics, and physiomics, it is difficult to continuously modify diagnostic strategy and criteria. However, if the new technologies produce results that can fit into the families and/or with additional families of pathophysiological responses, then an evaluation of the subarachnoid hemorrhage patient can progress quickly. For example, if proteomics produces results concerning proteins, enzymes, phosphoproteions, metaloproteins, and coenzymes, then these can be categorized according to the families of responses described above as well as new families. Genomics is the study of the mRNA produced by cells in response to their environment. Sharp et al. (40–42) has shown that genomics has characteristic and familial responses to pathophysiologic stresses following stroke. This work has shown that not only can ischemic stroke be diagnosed but also the cause of the ischemic stroke can be characterized.

While perhaps a bit puerile, let me clearly differentiate “state of the art” versus “cutting edge” for this discussion. State of the art is what is done now. When a cutting edge technology is adopted by the medical community, it is no longer cutting edge and becomes state of the art. So we might talk about a cutting edge technology that is paradigm shifting. For this discussion that would be a brand new technology that will change the way we do business.

The state of the art in diagnostic and assessment technologies especially associated with protein and genetic technologies requires extensive sample handling and substantial delays in producing information of hours to days. For example, if a family of genes were to be required for evaluating subarachnoid hemorrhage and this evaluation required amplification, the results would require skilled personal and substantial time to obtain results. Costs and time could make the results prohibitive and impractical. However, current technology of a lab on a chip and point of care is making it such that some results can be achieved relatively quickly. When this state of the art is applied to the subarachnoid hemorrhage patient improvements in care can follow. Unfortunately, there are still signal resolution issues that mean detecting low levels of analytes is difficult and not yet achievable. What is exciting is that there is cutting edge technology called metal enhance florescence that would produce a paradigm shifting way business is done with assessing subarachnoid hemorrhage patients. Metal-enhanced florescence (43–45) can be coupled to almost any fluorescent assay methodology to enhance the florescence signal. The result is 10,000-fold increase in signal. So the tests discussed above that might take hours or days could be coupled with lab on a chip technology and/or metal-enhanced florescence to provide a quick, easy, and quantitative result in minutes. As cutting edge technology, it will take time to become state of the art and now is the time to direct molecular and biochemical assessments that will eventually adopt and apply these technologies.