Changes in Water Properties in Human Tissue after Double Filtration Plasmapheresis—A Case Study

Double-filtration plasmapheresis (DFPP) is a blood cleaning technique that enables the removal of unwanted substances from the blood. In our case study, we performed near-infrared (NIR) spectroscopy measurements on the human hand tissue before and after a specific DFPP treatment (INUSpheresis with a TKM58 filter), along with NIR measurements of the substances extracted via DFPP (eluate). The spectral data were analyzed using the aquaphotomics approach. The analysis showed that the water properties in the tissue change after DFPP treatment, i.e., an increase in small water clusters, free water molecules and a decrease in hydroxylated water as well as superoxide in hydration shells was noted. The opposite effect was observed in the eluates of both DFPP treatments. Our study is the first that documents changes in water spectral properties after DFPP treatments in human tissue. The changes in tissue water demonstrated by our case study suggest that the positive physiological effects of DFPP in general, and of INUSpheresis with the TKM58 filter in particular, may be associated with improvements in water quality in blood and tissues.


Introduction
Introduced in the early 1980s by Agishi et al. [1], double-filtration plasmapheresis (DFPP) allows the removal particles in blood plasma having sizes between the pore size of the first (plasma separator) and second filter membrane (plasma fractionator). This is realized by first separating the blood into plasma and blood cells (with the plasma separator) and then fractioning the separated plasma into large and small molecular weight components. Although the blood cells and plasma with small molecular weight components are transfused back to the patient, the large molecular weight components (eluate) are filtered out. DFPP is an effective, efficient and patient-friendly blood purification procedure.
Since the physicochemical properties of water can change when passing through a filter or when the concentration of substances in the water change, we hypothesized that such changes in the water of the blood plasma would also have to occur with blood washing using DFPP. In order to verify this experimentally, we carried out corresponding measurements using near-infrared (NIR) spectroscopy before and after a specific DFPP treatment (INUSpheresis with the TKM58 filter) in one subject as a case study. It has not previously been investigated how DFPP affects the water in the tissue and what water properties the eluate has.

Double-Filtration Plasmapheresis Treatments and Spectroscopic Measurements
DFPP INUSpheresis (with TKM58 filter) treatments were performed on a 39-yearold man (first author, FS) at a private clinic in Switzerland in February 2022 on two different days, one day apart. Each treatment lasted about 2.5 h. No official ethical approval was necessary to conduct the measurements and report the results since it is a case report (Kantonale Ethikkommission, Kanton Zürich) and measurements were conducted by and on the first author (FS). The DFPP treatment was performed as part of a routine medical treatment.
A portable and ultra-compact NIR spectrometer (MicroNIR 1700, Viavi, Milpitas, CA, USA) was used to measure the diffuse reflectance spectra of the hand palm of the left hand and of the eluate. Measurements were performed in the spectral range 950-1650 nm with a nominal spectral resolution of 6.25 nm, an integration time of 11.7 ms and an average of 1000 scans for each spectrum to ensure an optimal signal-to-noise ratio. 15 consecutive measurements were performed for each measurement. Measurements were conducted in the morning one day before the first DFPP treatment (day 1), in the morning on the day of the first DFPP treatment (day 2), 1 h after the first DFPP treatment (day 2), in the morning on the day of the second DFPP treatment (day 4), 1 h after the second treatment (day 4) and in the morning one day after the second DFPP treatment (day 5). For each measurement it was ensured that the hand had subjectively the same temperature and the NIR spectrometer had also always the same temperature (T = 31.5 • C). Furthermore, the eluate of the first and second DFPP treatment was also measured by placing the NIR spectrometer on the plastic bag with the eluate inside and also measuring the spectrum of the plastic bag as a reference. This measurement was performed at an instrument temperature of T = 35.4, 35.5 and 35.6 • C.

Data Analysis
First, the spectra of the hand tissue and eluate measurements were pre-processed applying the SNV (standard normal variate) algorithm for baseline correction. SNV normalization was performed on both datasets separately.
For the analysis of the hand tissue measurements, the difference spectra (X Diff ) were calculated for the first and second DFPP treatment according to while the indices A 1 and A 2 (and B 1 and B 2 , respectively) refer to the calculation of the difference spectra for the 1 st DFPP (2 nd DFPP, respectively) based on the data measured on the same day (A 1 , B 1 ) and 24 h apart (A 2 , B 2 ). With this approach, the spectroscopic measurement after the DFPP treatments were compared to two baselines (i.e., at the same day and before/after 24 h).
For the analysis of the eluate measurements, the spectrum of the empty plastic bag containing the eluate was removed from the spectrum of the eluate measured through the plastic bag. With this, the spectra of the eluate itself was obtained.
To analyze the water spectral changes, the aquaphotomics approach was used [76][77][78][79][80][81]. 12 spectral regions of particular interest in the region of the 1st overtone of water (Ci, i = 1-12; Water Matrix Absorbance Coordinates, WAMACS) were analyzed in particular by calculating the absorbance of the difference spectra in these 12 spectral regions and visualizing the results on aquagrams to determine the specific water absorbance spectral pattern [76,77]. A listing of the WAMACS with the corresponding water properties can be found in Table 1 in Muncan et al. [79].

Results
The spectroscopic analysis of the hand tissue before and after the DFPP treatments revealed clear differences in the spectral features in the first overtone spectral region of water, i.e., in the region of the 12 WAMACS ( Figure 1). The corresponding aquagram (Figure 3a) shows the strongest increase in C6 (1421-1430 nm), corresponding to the water hydration band [79], the H-O-H bending mode, as well as the O-H stretch vibration mode, linked to the hydrogen bound strength of the water molecules [82]. The strongest decrease was found to be in C4 (1380-1388 nm), corresponding to water hydration shells (OH-(H 2 O), 1 , 4 ), hydrated superoxide water clusters (O 2 -(H 2 O 4 )) and the H 2 O symmetrical stretch vibration (2ν 1 ) [79]. In general, there was a decrease in C2 (1360-1366 nm) to C4 (1380-1388 nm) (linked to weaker H-bounded water) and an increase in C5 (1392-1412 nm) to C11 (1492-1494 nm) (linked to free water molecules and small water clusters). Interestingly, this specific change in spectral properties was evident after the first as well as the second DFPP treatment, whereas the first treatment caused the most pronounced effect. After the first DFPP treatment, the number of small and large water clusters increased, whereas after the second DFPP treatment, only the number of small water clusters increased while the larger ones decreased slightly.  Table 1 in Muncan et al. [79].
The spectroscopic analysis of the eluate obtained after the first and second DFPP treatment also revealed clear differences in the spectral features in the first overtone spectral region of water ( Figure 2). The corresponding aquagram (Figure 3b) shows the strongest increase in absorbance at C2 (1360-1366 nm), corresponding to water salvation shells (OH-(H2O)1,2,4) and C3 (1370-1379 nm), corresponding to symmetrical and asymmetrical stretching vibration of water molecules (ν1 + ν3) [79]. The strongest decrease in absorbance was at C6 (1421-1430) associated with the water hydration band, as well as the H-O-H bending mode and O-H stretch vibration mode. Fascinatingly, the specific water absorbance spectral pattern of the eluate is thus complementary to that measured in the tissue.  Table 1 in Muncan et al. [79].
The spectroscopic analysis of the eluate obtained after the first and second DFPP treatment also revealed clear differences in the spectral features in the first overtone spectral region of water ( Figure 2). The corresponding aquagram (Figure 3b) shows the strongest increase in absorbance at C2 (1360-1366 nm), corresponding to water salvation shells (OH-(H 2 O) 1,2,4 ) and C3 (1370-1379 nm), corresponding to symmetrical and asymmetrical stretching vibration of water molecules (ν 1 + ν 3 ) [79]. The strongest decrease in absorbance was at C6 (1421-1430) associated with the water hydration band, as well as the H-O-H bending mode and O-H stretch vibration mode. Fascinatingly, the specific water absorbance spectral pattern of the eluate is thus complementary to that measured in the tissue.  To complement the aquaphotomics-based NIR spectral analysis, the eluate obtained from both DFPP treatment was also analyzed for toxic ingredients (IGL Labor GmbH, Wittbek, Germany). The analysis revealed the presence of several toxins (Figure 4). The ten highest detected concentrations were from aflatoxin B1 (596.   To complement the aquaphotomics-based NIR spectral analysis, the eluate obtained from both DFPP treatment was also analyzed for toxic ingredients (IGL Labor GmbH, Wittbek, Germany). The analysis revealed the presence of several toxins (Figure 4). The ten highest detected concentrations were from aflatoxin B1 (596.  To complement the aquaphotomics-based NIR spectral analysis, the eluate obtained from both DFPP treatment was also analyzed for toxic ingredients (IGL Labor GmbH, Wittbek, Germany). The analysis revealed the presence of several toxins (Figure 4). The ten highest detected concentrations were from aflatoxin B1 (596.2 nmol/L), chromium-VI (591.0 nmol/L), lead (571.6 nmol/L), cadmium (558.1 nmol/L), arsenic (556.1 nmol/L), lindane (516.3 nmol/L), cobalt (509.5 nmol/L), polycyclic-aromatic-hydrocarbons (493.1 nmol/L), disulfoton (489.2 nmol/L) and aluminum (429.3 nmol/L). The concentration of toxins was generally significantly lower in the eluate from the second DFPP treatment compared to the first one. The concentration of some toxins increased after the second DFPP treatment (DDT, mercury, vinyl chloride), indicating that the first DFPP treatment most probably caused a diffusion gradient from the tissue to the blood, releasing these toxins from the tissue into the blood. The detoxification with DFPP is a multi-stage process whereby different compartments in the human organism are cleaned.
toxins was generally significantly lower in the eluate from the second DFPP treatment compared to the first one. The concentration of some toxins increased after the second DFPP treatment (DDT, mercury, vinyl chloride), indicating that the first DFPP treatment most probably caused a diffusion gradient from the tissue to the blood, releasing these toxins from the tissue into the blood. The detoxification with DFPP is a multi-stage process whereby different compartments in the human organism are cleaned.

Discussion and Conclusions
In this study, we have shown that the water properties in the tissue change after DFPP treatment (INUSpheresis with TKM58 filter). DFPP caused an increase in free water molecules, small water clusters and a decrease in hydroxylated water clusters, superoxides of water solvation shells and weaker H-bonded water. The opposite effect was observed in the eluates of both DFPP treatments.
In our opinion, these observations can be caused by two processes involved. First, the treatment filters out many molecules from the blood plasma to which water is bound in the form of hydration shells. This is the fraction of rather weakly H-bound water (which was reduced in the tissue after DFPP and was enriched in the eluates). Second, the process of filtration will cause water passing through the fine filter pores in a hydrophilic material to be structurally altered [83,84]. The formation of water clusters with stronger H-bonds is favored.
Our study is the first to date to investigate changes in tissue water after a DFPP treatment and also the first to perform a spectroscopic investigation of the eluates obtained by DFPP. In particular, it is also the first analysis of this kind concerning this specific type of DFPP, i.e. INUSpheresis with the TKM58 filter. We know of only one comparable study that investigated the dialysate after dialysis using NIR spectroscopy and the aquaphotomics approach [85]. This study showed that the absorption of 1398 nm and 1410 nm increased during dialysis. This finding is in line with our observation of an increase in this wavelength range after DFPP.

Discussion and Conclusions
In this study, we have shown that the water properties in the tissue change after DFPP treatment (INUSpheresis with TKM58 filter). DFPP caused an increase in free water molecules, small water clusters and a decrease in hydroxylated water clusters, superoxides of water solvation shells and weaker H-bonded water. The opposite effect was observed in the eluates of both DFPP treatments.
In our opinion, these observations can be caused by two processes involved. First, the treatment filters out many molecules from the blood plasma to which water is bound in the form of hydration shells. This is the fraction of rather weakly H-bound water (which was reduced in the tissue after DFPP and was enriched in the eluates). Second, the process of filtration will cause water passing through the fine filter pores in a hydrophilic material to be structurally altered [83,84]. The formation of water clusters with stronger H-bonds is favored.
Our study is the first to date to investigate changes in tissue water after a DFPP treatment and also the first to perform a spectroscopic investigation of the eluates obtained by DFPP. In particular, it is also the first analysis of this kind concerning this specific type of DFPP, i.e., INUSpheresis with the TKM58 filter. We know of only one comparable study that investigated the dialysate after dialysis using NIR spectroscopy and the aquaphotomics approach [85]. This study showed that the absorption of 1398 nm and 1410 nm increased during dialysis. This finding is in line with our observation of an increase in this wavelength range after DFPP.
The changes in tissue water demonstrated by our case study suggest that the positive physiological effects of DFPP in general and of INUSpheresis with the TKM58 filter in particular, may be associated with improvements in water quality in blood and tissues related to the respective water molecular structures. Such an improvement in water quality could, for example, be associated later on with improved blood circulation and optimized metabolic processes. Our small study should serve as a stimulus to explore these possibilities through further, larger and more comprehensive studies.
It should be noted that our study is based on the measurements of a single person and two eluates. Generalizations of our results are only possible to a limited extent. As noted, our case study serves to stimulate further research on the interesting results and to show how an aquaphotomics-based analysis of NIRS data can be performed to investigate the effects of a DFPP. Institutional Review Board Statement: Ethical review and approval were waived for this study since it is a case report and measurements were conducted by and on the first author (FS). The DFPP treatment was performed as part of a routine medical treatment.

Informed Consent Statement: Not applicable.
Data Availability Statement: The data will be made available by the corresponding author, upon reasonable request.