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10.3390/bios7010014


**1. Introduction**

Immunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis [1]. According to a systematic review of 40 worldwide studies, the incidence of IgAN is reportedly 2.5/100,000/year [2]. If left untreated, IgAN has a poor prognosis, developing into end-stage renal failure in approximately 20% to 40% of cases within 20 years after onset [3].

There are two IgA isotypes in humans, IgA1 and IgA2 [4]. Galactose-deficient IgA1 (Gd-IgA1), which lacks galactose (Gal) in the O-glycan side chains in the hinge region and exposes N-acetylgalactosamine (GalNAc), has been identified as one of the key molecules in the pathogenesis of IgAN and is increased in the sera of patients with IgAN [5]. According to the multi-hit hypothesis [6], Gd-IgA1 is recognized by anti-glycan autoantibodies, resulting in the formation of pathogenic immune complexes. These immune complexes are deposited in the kidneys, activate mesangial cells, and induce glomerular injury.

Serum Gd-IgA1 levels can predict IgAN progression [7,8]. In contrast, most relatives of IgAN patients with abnormal IgA1 glycoforms do not develop IgAN [9], and serum

**Citation:** Fukao, Y.; Suzuki, H.; Kim, J.S.; Jeong, K.H.; Makita, Y.; Kano, T.; Nihei, Y.; Nakayama, M.; Lee, M.; Kato, R.; et al. Galactose-Deficient IgA1 as a Candidate Urinary Marker of IgA Nephropathy. *J. Clin. Med.* **2022**, *11*, 3173. https://doi.org/ 10.3390/jcm11113173

Academic Editors: Christos Argyropoulos and Giacomo Garibotto

Received: 31 March 2022 Accepted: 30 May 2022 Published: 2 June 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Gd-IgA1 levels do not correlate with proteinuria [5], suggesting that serum Gd-IgA1 may not be a useful biomarker.

We previously developed a monoclonal antibody (KM55 mAb) that specifically recognizes Gd-IgA1, and demonstrated that glomerular Gd-IgA1 was specifically detected in IgAN and IgA vasculitis by immunohistochemical analysis using KM55 mAb [10,11].

In a mouse model, injection of purified nephritogenic IgA from IgAN-prone mice led to deposition in the glomeruli in nude mice. Parts of the injected IgA passed through into the bladder, suggesting that some parts of such glomerular IgA were cleared into the urine. In addition, the nephritogenic IgA had strong affinity not only to the glomerular mesangium, but also to the subepithelium [12]. On the other hand, several studies have supported that epithelial cells have the potential to clear matrix material and epithelial deposits into the cavity of Bowman's capsule [13]. Therefore, we hypothesized that urinary Gd-IgA1 could be a disease-specific marker. Indeed, an enzyme-linked immunosorbent assay (ELISA) using Helix aspersa agglutinin (HAA), a GalNAc-specific lectin, could detect urinary Gd-IgA1 and differentiate patients with IgAN from patients with other renal diseases [14]. However, lectin-dependent assays are not suitable for large-scale, multi-specimen testing because of the instability of glycan recognition. Thus, a novel and stable assay is required for the early detection of IgAN.

Many studies have shown that the degree of proteinuria is an outcome predictor in IgAN [15,16]. However, it is difficult to determine whether urinary protein excretion is due to active lesions triggered by glomerular immune deposition or chronic lesions represented by glomerulosclerosis and nephron reduction [17]. Therefore, proteinuria may not always reflect the disease activity, and its assessment is insufficient to determine the indications for treatment.

Several reports have indicated a high remission rate of tonsillectomy combined with steroid pulse therapy in the early stages of IgAN [18,19]. In addition, the renal biopsy findings of 56 patients with hematuria without overt proteinuria revealed that IgAN was common in their pathological diagnoses, and 31% of the patients with IgAN had crescentic lesions [20]. Thus, early diagnosis and treatment of IgAN are important for remission, and a useful biomarker for the indication of renal biopsy is desired.

There are no established disease-specific biomarkers for IgAN. Furthermore, repeated renal biopsies are difficult because of the invasiveness. We established a stable and simple ELISA for Gd-IgA1 using the KM55 mAb in 2015 [11]. In this study, we investigated the usefulness of urinary Gd-IgA1 as a disease-specific marker for IgAN from Japanese cohorts and further verified this using non-Japanese Asian cohorts.

#### **2. Materials and Methods**

#### *2.1. Patients and Samples*

We recruited 338 Japanese adults ( ≥18 years old) with biopsy-proven IgAN and 120 patients with other renal diseases (disease controls) diagnosed at Juntendo University Hospital, Tokyo, Japan from 2015 to 2018. In addition, to validate the use of the urinary Gd-IgA1 level, we recruited 69 Korean and 35 Taiwanese biopsy-proven IgAN patients, as well as 39 Korean disease control patients.

Clinical and laboratory data were collected at the time of the renal biopsy. The laboratory parameters included serum creatinine (Cr) levels, serum Gd-IgA1 levels, urinary protein-to-creatinine ratios (UPCR), and urinary Gd-IgA1 levels.

## *2.2. Pathological Parameters*

The histological samples were classified according to the clinical guidelines for IgAN from the Japanese Society of Nephrology (JSN) [21] or the Oxford classification [22,23]. Briefly, the histological grade (H-grade) of the JSN criteria was defined as 1 (0–24.9%), 2 (25–49.9%), 3 (50–74.9%), and 4 (75–100%) based on the percentage of glomeruli with pathological features, such as crescents, global sclerosis, and segmental sclerosis, which predict the progression to end-stage renal disease.

#### *2.3. Measurement of Gd-IgA1*

Serum and urinary Gd-IgA1 levels were determined using the KM55 mAb according to the manufacturer's instructions (Immuno-Biological Laboratories, Fujioka, Japan), and logarithmically transformed (log10 basis).

#### *2.4. Statistical Analyses*

Data are expressed as mean ± standard error. Comparisons between groups were performed using the Mann–Whitney U test. Spearman's correlation analysis was used to analyze the correlation between two variables. Statistical significance was defined as *p* < 0.05. Statistical analyses were performed using GraphPad Prism software ver.8.0 (GraphPad Software, San Diego, CA, USA).


**3. Prevention**

Acanthamoeba keratitis is a potentially blinding condition of the eye, and any steps toward prevention of this disease are encouraged. It is recommended contact lens wearers to use daily disposable contact lenses to eliminate the contact lens case as a reservoir for Acanthamoeba. If daily disposable contact lens can not be used, then hydrogen peroxide cleaning solutions are preferred over the more common multipurpose solutions because of the increased efficacy of hydrogen peroxide against Acanthamoeba cysts. Planned replacement of contact lens storage cases may also help reduce contamination. It is important for eye care professionals not only to emphasize proper contact lens wear and hygiene in the prevention of infectious keratitis but also to maintain a high clinical suspicion for Acanthamoeba keratitis because an early diagnosis may lead to a better visual outcome (Tanhehco & Colby, 2010). The use of surfactant cleaner has been shown to be an effective way of removing Acanthamoeba trophozoites and cysts (Kilvington, 1993). In the light of these findings further research into the types of disinfectant used by patients with Acanthamoeba keratitis is required, with a view to calculating the relative risks of different lens type and disinfectant combinations. This series also show the importance of the fact that contact lens practitioners should ensure at the time of prescribing lenses that patients have understood all the disinfectant procedures (Illingworth et al., 1995). Gray et al. suggest leaving contact lens case open to dry air after heat disinfection. If hydrogen peroxide disinfection is the preferred solution, one has to use a two step hydrogen peroxide system. It is necessary to call one's attention to wash hands properly before handling contact lens cases and what is more important is to avoid homemade saline. The contact lens case should be replaced regularly. Nowadays the use of more frequently disposable contact lenses is highly recommended (Gray et al., 1995). Also the rub and rinse step should be mentioned as the critical part of the disinfection that may reduce significantly the microbiological load (Shih et al., 1985). Detailed education of contact lens wearers concerning of the entire disinfection procedure could reduce the incidence of Acanthamoeba keratitis further on. There is a persistent need to educate contact lens wearers continuously about the possible risk factors of Acanthamoeba keratitis, and since the frequency of Acanthamoeba keratitis appears to be largely determined by the ever changing trends in contact lens use, continued monitoring is indicated (Radford et al., 1998). All of the eye care practitioners have to call attention of all contact lens users to aware the wearing of soft contact lenses in case of swimming so as to reduce the number of patients with Acanthamoeba keratitis (Kaji et al., 2005; Kettesy et al., 2010). If even a soft contact lens wearer wants to swim, then daily disposable contact lens and swimming spectacles should be used.


**2. General aspects of bowel anastomoses and modern variations that impacted the outcome**

Barbed sutures are available in a variety of both absorbable and nonabsorbable monofilament materials. Specifically, currently available bidirectional and unidirectional barbed suture materials include PDO, polyglyconate, poliglecaprone 25, glycomer 631, nylon, and polypropylene. A study performed by Wiggins [1]

#### **Figure 1.** *Intraoperative aspect of an anastomosis performed manually at the level of the small bowel.*

**Figure 2.** *Intraoperative aspect of an anastomosis performed mechanically at the level of the small bowel.*

searched through a systematic review and meta-analysis for the benefits of barbed suture utilization in gastrointestinal anastomosis. The conclusion was that the use of barbed sutures for gastrointestinal anastomosis appears to be associated with shorter overall operative times. There was no difference in rates of complications (including anastomotic leak, bleeding, or stricture) compared with standard suture materials.

The study included consecutive CD patients with ileal/ileocolonic strictures who had SWE shear wave elastography within one week of surgical resection.

The SWE of the stenotic bowel wall was compared to the biofragmentable anastomosis ring used for gastrointestinal anastomoses in a literature review conducted by Bobkiewicz and coauthors [2].

The theoretical idea was that a biofragmentable anastomosis ring (BAR) could be used instead of manual and stapled anastomoses in the upper and lower GI tracts.

The aim of this study was to see how effective BAR was for bowel anastomoses using our own content. Methodologies: Between 2004 and 2014, a retrospective study was conducted on a total of 203 patients who underwent bowel surgery with *The Problem of the Colorectal Anastomosis DOI: http://dx.doi.org/10.5772/intechopen.100302*

**Figure 3.** *Foreign body reaction at the level of the tissues containing suture material.*

**Figure 4.** *Colorful surgical nylon monofilament suture with a curved needle.*

BAR anastomosis in the upper and lower gastrointestinal tract. The study concluded that using BAR for GI tract anastomoses is an easy and quick procedure with a low rate of perioperative mortality (0.5%) and complication rates (**Figures 3** and **4**).


**1. Introduction**

Hyperspectral images have been widely applied to remote sensing image applications, such as land cover classification [1], target detection [2], anomaly detection [3], spectral unmixing [4] and others. Each pixel in HSI has hundreds of narrow contiguous bands, spanning from visible to infrared spectrum [5], which makes it possible to detect and distinguish various objects with higher accuracy [6]. However, increasing the number of spectral bands or features of an HSI pixel does not always help to increase the classification accuracy. Therefore, how to make full use of the information in HSIs is a problem in practical applications.

Many algorithms have been developed for the classification of HSIs. Among these, there are some well-known pixelwise classifiers, such as the support vector machine (SVM) [7–9], support vector conditional random classifier [10], multinomial logistic regression [11], neural network [12] and adaptive artificial immune network [13]. These pixelwise classifiers can make full use of the spectral information of HSIs, but the classification results are often noisy because the spatial information is not considered.

Therefore, some recent researches incorporated the spatial information in HSI classification to enhance the classification performance. The basic way to use spatial information is to assume that the pixels within a local region usually represent the same material and have similar spectral

characteristics [1]. Various researches [14–25] have been done based on this assumption. Besides these researches, Sparse representation (SR), which is based on the observation that spectral pixels of a particular class should lie in a low-dimensional subspace spanned by dictionary atoms (training pixels) from the same class, is also employed. In [26], a Joint Sparse Representation Classification (JSRC) method has been proposed to incorporate spectral information and spatial information. The spatial information is expressed by a fixed-size local square window centered with the test pixel. Then all pixels in the window are simultaneously joint represented by a few common atoms in the specified dictionary. The JSRC can achieve a good performance but the optimal size of the window cannot be determined easily. In [27], a stepwise Markov random field (MRF) optimization was proposed to exploit spatial information based on the result of multitask joint sparse representation. In [28], MASR was proposed to release the difficulty in choosing region size. Instead of choosing a single scale, this method extends the spatial information to several scales to take advantage of correlations among multiple region scales for HSI classification. But the multiscale regions used in MASR refer to multiscale patches which may contain noise pixels. Better than patch region, shape-adaptive superpixel can provide more accurate spatial information. In [29], the superpixel was introduced to replace the patch region. Then a shape-adaptive local smooth region was generated for each test pixel by a shape-adaptive algorithm in [30]. The latest research proposed a Multiscale Superpixel-Based Sparse Representation [31]. In this research, multiscale superpixels were generated and then each scale was represented by JSRC. Finally, a fusion result was gotten from multiscale results by majority voting. But the selection of scales for superpixels is still a problem. Although it uses multiscale to release the difficulty of selecting segmentation scale, it still needs a fundamental number of superpixels determined empirically.

In fact, patch and superpixel both have their own advantages and shortages. The patch can include all nearest neighbors but it also may contain noise pixels. Shape-adaptive superpixel can exploit more accurate spatial information but there are still some mixed superpixels when the scale is not optimal. In a mixed superpixel, there must be wrong representation because all pixels in the superpixel share the same representation. Inspired by merits and demerits of patch and superpixel, we propose to use a union region to replace the patch and superpixel. Union region refers to the overlap of patch and superpixel. Compared with patch, union region includes more similar pixels for the test pixel aiming at decreasing the effect of noise pixels. Compared with superpixel, union region provides more direct neighbors for the test pixel to enhance the representation of pixels located in the wrong superpixel. In addition, the required superpixels for generating union regions don't need empirical scale. The scales are determined by the size of the image and the corresponding patch sizes. By replacing patch in MASR with union region, we get a new algorithm called Multiscale Union Regions Adaptive Sparse Representation (MURASR). MURASR also adopts a probability majority voting method to optimize the classification result generated from the sparse representation. Experiment results show that the union region based algorithms always perform better than patch region based algorithms and the proposed MURASR outperforms other algorithms in terms of quantitative metrics and visual quality on the classification maps.

The rest parts of the paper are organized as follows. The JSRC and MASR are briefly introduced in Section 2. The details of proposed MURASR method are described in Section 3. The experimental results and discussions are presented in Section 4. Finally, Section 5 summarizes the paper and future works are suggested. The outline of the MURASR is illustrated in Figure 1.

**Figure 1.** Outline of the proposed MURASR framework.


For protein analysis, microglial cells were seeded on 12 well plates (40 × 104 cells) and treated with LPS (100 ng/ml) for 24 h and the day after with CX3CL1 (100 nM) for further 24 h; cells were

#### TABLE 1 | Sequences of the primers used for RT-qPCR.

fncel-13-00414 September 11, 2019 Time: 16:22 # 5

washed with PBS and lysed in hot 2× Laemmli buffer, boiled 5 min and sonicated. The same amount of protein samples was separated on 12% SDS-polyacrylamide gel electrophoresis and analyzed by western immunoblot using the following primary antibodies: Arg-1 (1:200, Santa Cruz Biotechnology) PKM2 (1:2000, Cell Signaling), Actin (1:2000 Sigma-Aldrich), HRPtagged goat anti-mouse and anti-rabbit IgG were used as a secondary antibody (1:2000; Dako). For protein analysis we used four different primary microglial culture preparations. Detection was performed through the chemiluminescent assay Immun-Star Western C Kit (Bio-Rad, CA) and densitometric analysis was carried out with Quantity One software (Bio-Rad, CA).

#### Statistical Analysis

Data are expressed as the means ± SEM. Student's t-test, paired t-test, one-way analysis of variance (ANOVA) was performed. A value of P < 0.05 was considered significant. All statistical analyses were carried out using the Sigma Plot 11.0 Software (Systat Software GmbH, Erkrath, Germany).

#### RESULTS

#### CX3CL1 Induces Change in the Polarization State of Microglia in Culture

Since CX3CL1 is neuroprotective in ischemia (Cipriani et al., 2011) and it is known that neuro-inflammation plays a role in brain damage following ischemic insult (Iadecola and Alexander, 2001; Cheon et al., 2017), we wanted to verify the hypothesis that the neuroprotective effect of CX3CL1 was due to its ability to modulate the phenotype of microglia. For this reason, we used primary murine microglial cells, treated them with CX3CL1 (100 nM) for 24 h and analyzed the expression levels of different anti- (ym1, arg1, fizz, tgfβ1, tgdβ1r) and pro- (inos, il1β, tnfα, tlr4, cd86, il6) inflammatory genes (Michelucci et al., 2009; Gabrusiewicz et al., 2011) by quantitative real time PCR (RT-qPCR). Data reported in **Figure 1** show that cells exposure to CX3CL1 increased the expression of antiinflammatory (**Figure 1A**, n = 8 ∗∗p < 0.001, ∗p < 0.05; Student's t-test) and lowered the expression of pro-inflammatory genes (**Figure 1B**, n = 8 ∗∗p < 0.001, ∗p < 0.05; Student's t-test). Since numerous evidence suggest that upon different activation stages microglial cells switch from the oxidative phosphorylation to the anaerobic glycolysis in order to increase ATP production (Voloboueva et al., 2013; Gimeno-Bayón et al., 2014) we decided to investigate whether CX3CL1 treatment could modify the metabolic repertoire of microglia. Data obtained indicate that CX3CL1 increased the transcription of some genes involved in the oxidative pathway (pgc1α, pgc1β, prc, sirt3, slc25a15, pparγ) (**Figure 1C**, n = 8 ∗∗p < 0.001, ∗p < 0.05; Student's t-test) and reduced the expression of others related to the glycolytic pathway (g6pdh, aldoA, eno1, hk1, ldha, pklr) (**Figure 1D**, n = 8 ∗∗p < 0.001; Student's t-test).

#### CX3CL1 Induces Shape Changes of Microglia in Culture

The activation state of microglia changes their shape, even if it is not possible to uniquely correlate a specific morphology to a specific phenotype (Kettenmann et al., 2011). We investigated the morphological changes of microglia in culture, upon CX3CL1 stimulation, calculating the "form factor" of single cells, that was calculated as described in the Method section. The form factor is 1 for round cells and correspondingly < 1 when the morphology deviates from the spherical shape. Microglia in culture displayed ramified processes with a small cell body and

∗∗p < 0.001, ∗p < 0.05, Student's t-test.

several long processes (**Figure 2**, not stimulated condition, ns). The results in **Figure 2** show that microglia stimulated with LPS (100 ng/ml, 24 h), significantly change their form factor from 0.26 ± 0.06 (ns) to 0.7 ± 0.05; upon interleukine-4, (IL-4) treatment (20 ng/ml, 24 h) this parameter is similar to not stimulated cells (0.27 ± 0.08) in analogy with what previously reported (Grimaldi et al., 2016). The form factor of cells stimulated with CX3CL1 (0.28 ± 0.05) was similar to untreated cells and CX3CL1/LPS co-treatment significantly reverts the effect of LPS on cell shape variations (form factor: 0.41 ± 0.07) confirming that CX3CL1 efficiently contrasts the effects of inflammatory stimuli on microglial cells (n = 120 cells in total for each different conditions, ∗∗p < 0.001; one-way ANOVA followed by Mann–Whitney Rank Sum post hoc test).

### CX3CL1 Modulates Microglia Polarization in Pro-inflammatory Conditions in vitro

Arginine metabolism via nitric oxide synthase (NOS) or arginase is at the crossroad of the pro- and anti-inflammatory microglia phenotypes (Orihuela et al., 2016). To verify the hypothesis

that CX3CL1 could modulate microglia phenotype also under pro-inflammatory conditions, we stimulated cells with LPS (100 ng/ml, 24 h) and then with CX3CL1 (100 nM) for additional 24 h, and measured the release of nitric oxide (NO), the production of lactate and the arginase activity in microglia 24 h later. As shown in **Figure 3A**, LPS-induced NO release was significantly reduced by treatment with CX3CL1, bringing it back to the control level (n = 5, ∗p < 0.05; One- way ANOVA followed by Holm–Sidak post hoc test). In the same conditions we measured the lactate concentration in the medium of cultured microglia: **Figure 3B** shows that the LPS stimulation induced an increase in the lactate production that is reduced after CX3CL1 treatment (n = 3, ∗p < 0.05; One- way ANOVA followed by Tukey post hoc test). We also decided to analyze the expression of the Pyruvate Kinase isozymes M2 (PKM2) which is an enzyme involved in glycolysis that converts phosphoenolpyruvate to pyruvate and generates ATP. Data obtained by western blot assays reported in **Figure 3D** showed that PKM2 protein levels in microglia markedly increased after LPS exposure while they returned to the control level after CX3CL1 treatment (n = 3, ∗p < 0.05; One- way ANOVA followed by Holm–Sidak post hoc test). Regarding the arginase activity and arginase 1 (Arg-1) protein expression in microglia, cells were treated as described above and, as shown in **Figure 3C**, CX3CL1 increased arginase activity and reverted the reduction induced by LPS treatment (n = 6, ∗p < 0.05; One-way ANOVA followed by Tukey post hoc test). Moreover data reported in **Figure 3E** demonstrated that the stimulation with CX3CL1 (100 nM, 24 h) induced an increase in Arg-1 protein in microglia vs. not treated cells (n = 4, ∗p < 0.05, one-way ANOVA followed by Tukey post hoc test) and that the low expression level observed after LPS treatment is back at control level upon CX3CL1 stimulation. To further analyze the effect of fractalkine on the central metabolism, the energetic profile of microglia was assessed through the Cell Mito Stress Test (Agilent) using the extracellular flux analyzer XFe96 (Seahorse Bioscience, Houston, TX, United States). Briefly, this platform allows the determination of the Oxygen Consumption Rate (OCR) under both basal and stressed conditions which is indicative of the mitochondrial function; at the same time the extracellular acidification rate (ECAR) was also measured as an indirect quantification of the glycolytic process. The drugs used to promote the stressed condition target (according to the addition order) (i) complex V, i.e., ATP production; (ii) membrane potential, i.e., by uncoupling electron transfer and proton translocation: (iii) electron transfer chain, i.e., complex I and III. As expected (Voloboueva et al., 2013; Orihuela et al., 2016), LPS treatment (10 ng/ml, 48 h) promoted a deep metabolic re-programing as compared to the untreated sample: the OCR was dramatically reduced (∼65% lower; **Figure 3F**), while ECAR (and therefore glycolysis) was doubled (see **Supplementary Figure S1A**), shifting the aerobic profile of microglia to a glycolytic phenotype upon LPS treatment (see **Supplementary Figure S1B**). In light of this, we investigated whether CX3CL1 is able to attenuate the effect of LPS on the metabolic profile of microglia. Both untreated and LPS-treated samples were incubated with 100 nM CX3CL1 24 h after LPS treatment, and Seahorse analysis was then performed. While CX3CL1 did not affect the energetic profile of the untreated sample (see **Supplementary Figure S2**), in the LPS sample this chemokine promoted a significantly decrease of glycolysis and of OCR even though for the latter to a lower extent (**Figure 3G** and **Supplementary Figure S3**). The same trend was observed also at 100 ng/ml of LPS (data not shown).

# CX3CL1 Attenuates Microglial Activation After Ischemia in vivo

We have previously demonstrated that exogenous CX3CL1 has a long-lasting neuroprotective action in vivo against pMCAO in rodents reducing neurological deficits and infarct size related with cerebral ischemia (Cipriani et al., 2011). We have now decided to investigate how CX3CL1 affects microglia phenotype during ischemia: to this aim mice were subjected to pMCAO,

FIGURE 3 | CX3CL1 modulates microglia phenotype in the context of pro-inflammatory conditions in vitro. (A) Release of NO by microglia cells not stimulated (C) or stimulated with LPS or CX3CL1 or LPS + CX3CL1; data are expressed as NO concentration (µM). (B) Lactate production by microglia cells not stimulated (C) or stimulated with LPS or CX3CL1 or LPS + CX3CL1; data are expressed as lactate concentration (ng/µl). (C) Arginase activity in microglia cells not stimulated (C) or stimulated with LPS or CX3CL1 or LPS + CX3CL1; data are expresses as units for liter. (D) Western-blot analysis of PKM2 protein expression in microglia cells incubated with vehicle (C) LPS, CX3CL1 or LPS + CX3CL1. At the top, representative image; at the bottom, histogram bar of the quantification of PKM2 expression (data are expressed as PKM2 signal normalized to Actin signal). (E) Western-blot analysis of Arg-1 protein expression in microglia cells incubated with vehicle (C) LPS, CX3CL1 or LPS + CX3CL1. At the top, representative image; at the bottom histogram bar of the quantification of Arg1 expression (data are expressed as Arg1 signal normalized to Actin signal). (F) Bioenergetic profile of microglial cells: the mitochondrial respiration of microglia, obtained by means of OCR by seahorse experiments, ± 10 ng/ml of LPS (48 h of treatment; triangle and circle, respectively). Arrows indicates the addiction of drugs used to specifically target the mitochondrial function (i.e., oligomycin, FCCP, rotenone + antimycin, in order of addiction). In principle, the first and the third drug lead to a dramatic OCR drop, while FCCP, as uncoupler agent, leads to a maximal oxygen consumption. While this behavior is observed in the untreated sample, LPS treatment abolished this kind of response. In the figure a representative experiment; values reported in the plot are the means of at least 8 replicates ± SD. (G) Energetic profile of LPS-treated microglia ± CX3CL1 treatment: the phenotype plot, where both ECAR and OCR are reported on the X and Y-axis, respectively, indicates that the LPS sample (10 ng/ml, open triangle) is more glycolytic than the LPS + CX3CL1-treated one (10 ng/ml + 100 nM, open square). This trend is maintained also under stressed conditions (gray symbols), where FCCP is not able to trigger a maximal respiratory capacity, as expected. In the figure a representative experiment is shown; values reported in the plot are the means of at least 8 replicates ± SD. Statistical analysis: Data are expressed as the mean (± SEM.) (A) n = 5, ∗∗p < 0.001 ∗p < 0.05, one-way ANOVA followed by Holm–Sidak post hoc test; (C) n = 6, ∗p < 0.05, one-way ANOVA followed by Tukey post hoc test; (E) n = 4,∗p < 0.05, one-way ANOVA followed by Tukey post hoc test. (G) n = 2, ∗∗p < 0.001 ∗p < 0.05, Student's t-test.

we verified that CX3CL1-treated mice had a reduced ischemic volume as previously shown (Cipriani et al., 2011), then CD11b+ cells were isolated from the ipsi- and contra-lateral brain hemispheres 24 or 72 h after ischemia and gene expression was analyzed by RT-qPCR. As reported in **Figure 4A**, at 24 h after ischemia CD11b+ cells increased their expression of antiinflammatory genes (arg1, ym1, fizz, tgfβ1, tgfβ1r) in the ipsilateral hemisphere, upon CX3CL1 treatment (n = 6 mice per group, ∗p > 0.05; Student's t-test).

Instead the expression pattern of the pro-inflammatory genes analyzed (inos, il1β, tnfα, tlr4, cd86, il6) is less clear: in particular, we observed an increase in the expression of tnfα, cd86 e tlr4 in CX3CL1 treated mice, while CX3CL1 reduced il1β expression (n = 6 mice per group, ∗∗p < 0.001 ∗p > 0.05; Student's t-test). Since it was demonstrated that microglia phenotype changes from anti- to pro-inflammatory with the progression of cerebral ischemia (Fumagalli et al., 2015; Ma et al., 2017) we isolated CD11b+ cells from the ipsi- and contra-lateral brain hemispheres of mice at 72 h after ischemia and analyzed by RT-qPCR the same set of genes.

Data reported in **Figure 4C** show that CD11b+ cells isolated from the ipsilateral hemisphere 72 h after ischemia induction, express higher level of pro-inflammatory genes that undergo a significant reduction of expression upon CX3CL1 treatment (n = 6 mice per group, ∗p > 0.05; Student's t-test). Interestingly, most of the anti-inflammatory genes considered (**Figure 4C**) are still increased in the ipsilateral hemisphere of CX3CL1 treated mice (n = 6 mice per group, ∗∗p < 0.001 ∗p > 0.05; Student's t-test). The MACS column system does not permit to distinguish microglia from other myeloid resident cells and from infiltrating immune cells during the ischemic insult, because they can express CD11b as well as CX3CR1. For this reason, we used a new animal cohort and performed gene analysis only on microglia after pMCAO, taking advantage of the fluorescence-activated cell sorting. Starting from CD45 positive low population, microglial cells were isolated as CD11b positive Ly6C/Ly6G double negative cells from both the ipsi- and contra-lateral brain hemispheres, 24 or 72 h after ischemia and gene expression was analyzed by qRT-PCR. With this gating strategy we uncovered a microglia fraction of 80–90% among CD45+ cells at all time points and an immune cell infiltrate (CD45high among CD45+ cells) of 16.37 ± 6.63% saline- versus 19.42 ± 5.77% CX3CL1-treated mice at 24 h after ischemia and of 10.87 ± 1.87% saline- versus 20.07 ± 2.71% CX3CL1-treated mice at 72 h after ischemia, confirming the chemotactic role of CX3CL1. FACS analysis allowed us to recover an average of 20 × 103 microglial cells per hemisphere, that is not enough to analyze the expression of all the genes taken into consideration in the previous panels; we have therefore chosen only a few of them. Data reported in **Figures 4E,F** confirmed that CX3CL1 treatment increased the expression of the anti-inflammatory genes arg1 and ym1 and decreased that of pro-inflammatory gene inos on microglial cells isolated from brain hemispheres both 24 and 72 h after ischemia. Regarding the CX3CL1 effect on il1β expression in microglial cells after brain ischemia, it induced an increase in the expression of this cytokine 24 h after ischemia while it had an opposite effect after 72 h from ischemic damage (n = 4 mice per group, ∗∗p < 0.001 ∗p > 0.05; Student'st-test), contrary to what observed for CD11B+ cells (see **Figures 4B,D**).

#### CX3CL1 Modulates Microglial Metabolic State After Ischemia in vivo

Several evidence suggest a role of metabolic reprograming in the regulation of the innate inflammatory response of microglia (Voloboueva et al., 2013; Gimeno-Bayón et al., 2014). We analyzed the expression profiles of genes related to the glycolytic and oxidative pathways in CD11b+ cells extracted from the ipsi- and contra-lateral brain hemispheres of mice treated with CX3CL1, at 24 and 72 h after ischemia induction.

The analysis of some genes involved in the oxidative pathway (such as pgc1β, sirt3, prc, slc25a15 and pparγ) shows that CX3CL1 increases their expression in the ipsilateral region at 24 h and 72 h after ischemia with the exception of pparγ , whose expression is reduced at 24 h (**Figure 5A**, n = 6 mice per group, ∗∗p < 0.001; Student's t-test) and of prc, that decreased at 72 h (**Figure 5C**, n = 6 mice per group, ∗∗p < 0.001 ∗ p > 0.05; Student's t-test). On the other hand, among the genes associated with the glycolytic pathway (such as aldoA, g6pdh, eno1, and ldha), (**Figure 5B**) at 24 h only aldoA was reduced (n = 6 mice per group,

FIGURE 4 | Effects of CX3CL1 in modulating microglia polarization state after ischemia. Expression analysis by RT-qPCR for mRNAs of anti-inflammatory (arg1, ym1, fizz, tgfβ1, tgfβ1r), and pro-inflammatory (inos, il1β, tnfα, tlr4, cd86, il6) related genes in Cd11b+ cells extracted from contra- and ipsi-lateral brain hemispheres of mice subjected to pMCAO 24 h (A,B) and 72 h (C,D) after the insult. Expression analysis by RT-qPCR for mRNAs of anti-inflammatory (arg1 and ym1; (E) and pro-inflammatory (inos and il1β; F) related genes in microglial cells sorted by FACS from contra- and ipsi-lateral brain hemisphere cell suspensions of mice subjected to pMCAO 24 h and 72 h after the insult (E,F). For each gene data are expressed as specific mRNA fold increase in the ipsilateral hemisphere of vehicle (black bars) or CX3CL1 treated (gray bars) mice normalized to the mRNA expression for the respective contralateral brain hemisphere. Statistical analysis: Data are expressed as the mean (± SEM.) of n = 6 (A–D) and n = 4 (E,F) mice per group, ∗∗p < 0.001, ∗p < 0.05, Student's t-test.

∗∗p < 0.001; Student's t-test). In contrast 72 h after ischemia they were all reduced upon CX3CL1 treatment (**Figure 5D**; n = 6 mice per group, ∗∗p < 0.001 ∗p > 0.05; Student's t-test). We performed a similar analysis on microglial cells isolated from brain hemispheres by fluorescence-activated cell sorting 24 and 72 h after ischemia and what we observed is an increase in the expression of pgc1β and slc25a15 in CX3CL1 treated mice both 24 and 72 h after ischemia (**Figure 5E**, n = 4 mice per group, ∗∗p < 0.001 ∗p > 0.05; Student's t-test), while CX3CL1 reduced pkm2 expression 24 and 72 h after brain damage and ldhα expression 72 h after ischemia insult (**Figure 5F**, n = 4 mice per group, ∗∗p < 0.001 ∗p > 0.05; Student's t-test).

# DISCUSSION

#### Microglia and Neuroinflammation

Neuroinflammation is associated with the pathophysiology of neurodegenerative disorders (Leszek et al., 2016), and glial activation during neuroinflammation is a common feature in disease progression, resulting in the production of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), reactive oxygen species, and nitric oxide, which can ultimately lead to neuronal loss (Meraz-Ríos et al., 2013). As CNS-resident immune cells, microglia play a key role in maintaining tissue homeostasis, as well as in inducing neurotoxicity (Liddelow et al., 2017). Under normal oxygen conditions, cells usually acquire energy via two mechanisms: glucose is converted to pyruvate via glycolysis which moves to mitochondria to produce ATP through oxidative phosphorylation (Dashty, 2013). Under hypoxic conditions, anaerobic glycolysis converts pyruvate into lactate (Murray, 2006; Hardie, 2007). Immune cells switch from oxidative phosphorylation to aerobic glycolysis, as described in other cell types (Warburg effect) (Warburg, 1956; Vander Heiden et al., 2009), in order to produce metabolic resources necessary to satisfy the request of cell proliferation and activation. This metabolic reprograming plays a key role in the process of the innate inflammatory response; in particular, the proinflammatory state is correlated with a shift of energy production from oxidative phosphorylation to aerobic glycolysis (Krawczyk et al., 2010; O'Neill and Hardie, 2013; Orihuela et al., 2016). Moreover, glycolysis inhibition reduces the pro-inflammatory polarization of immune cells, indicating a possible regulatory role in inflammatory cell function (Cheng et al., 2014). When stimulated with LPS/IFN-γ, microglia switch toward a proinflammatory phenotype, undergo aerobic glycolysis and release pro-inflammatory cytokines; when stimulated with IL-4/IL-13,

they acquire an anti-inflammatory phenotype, with inflammation resolution and tissue repair (Kelly and O'Neill, 2015). In this view, energy demand would be associated with specific functional activities such as change in morphology, phagocytosis and translocation to the injured site of microglial cells (Stence et al., 2001; Koizumi et al., 2007; Fu et al., 2014), and thus may influence the contribution of microglia activation to various neurodegenerative condition. There are evidence of both harmful and protective roles of microglia in stroke (Nakajima and Kohsaka, 2004; Neumann et al., 2006; Neher et al., 2013), and so the precise role and timing of microglia in stroke and cerebral ischemia is debated. Activated microglia produce pro-inflammatory cytokines, nitric oxide and reactive oxygen species inducing an acute and chronic inflammatory response which exacerbates neuronal injury in stroke (Block et al., 2007). Nevertheless, both exogenous and resident proliferating microglia are able to exert neuroprotective action in brain ischemia, through the production of neurotrophic factors (Imai et al., 2007; Lalancette-Hébert et al., 2007; Narantuya et al., 2010). This dual activity of microglia might be explained by the differences in the parenchymal milieu composition produced in injuries of diverse entity and/or at distinct stages of the pathology, which might have various effects on microglia activation (Lai and Todd, 2008). In this contest, the molecular mechanisms through which microglia activate different responses have become an important area of ischemia research. CX3CL1 has been reported to inhibit the release of inflammatory cytokines (Zujovic et al., 2000; Mizuno et al., 2003; Cardona et al., 2006) and reduce microglia activation, keeping these cells in what (before the deep use of transcriptome analysis) has been defined an "off " state (Biber et al., 2007). We previously demonstrated that CX3CL1 is protective against cerebral ischemia (Cipriani et al., 2011). Since it was demonstrated that during the progression of cerebral ischemia microglia phenotype changes from antito pro-inflammatory (Fumagalli et al., 2015; Ma et al., 2017) and microglia express CX3CR1, we investigated whether the neuroprotective effect of CX3CL1 in ischemia is due to its ability to change the phenotype of microglia. For this reason, we analyzed the expression profiles of anti- and pro-inflammatory genes and those related to the metabolic reprograming, in the regulation of the inflammatory response both in vitro and in vivo.

# CX3CL1 Modulates the Inflammatory Phenotype of Microglia in vitro

In this paper we demonstrate that CX3CL1 drives microglia toward an anti-inflammatory phenotype reducing the expression of pro-inflammatory genes and increasing the expression of

FIGURE 5 | Effects of CX3CL1 in modulating microglia metabolic state after ischemia. Expression analysis by RT-qPCR for mRNAs of oxidative pathway (pgc1β, sirt3, prc, slc25a15 pparγ) and glycolytic pathway (aldoA, g6pdh, eno1, ldha, pkm2) related genes in Cd11b+ cells extracted from contra- and ipsi-lateral brain hemispheres of mice subjected to pMCAO 24 h (A,B) and 72 h (C,D) after the insult. Expression analysis by RT-qPCR for mRNAs of oxidative (pgc1β and slc25a15; (E) and glycolytic pathway (ldha and pkm2; (F) related genes in microglial cells sorted by FACS from contra-and ipsi-lateral brain hemisphere cell suspensions of mice subjected to pMCAO 24 h and 72 h after the insult (E,F). For each gene data are expressed as specific mRNA fold increase in the ipsilateral hemisphere of vehicle (black bars) or CX3CL1 treated (gray bars) mice normalized to the mRNA expression for the respective contralateral brain hemisphere. Statistical analysis: Data are expressed as the mean (± SEM.) of n = 6 (A–D) and n = 4 (E,F) mice per group, ∗∗p < 0.001, ∗p < 0.05, Student's t-test.

those related to an anti-inflammatory state. Moreover, CX3CL1 switched the metabolic state of microglia from glycolysis to oxidative pathway, increasing the expression of gene related to oxidative phosphorylation and decreasing those involved in glycolytic metabolism of glucose. These effects are in line with what was observed in vitro with NO, lactate and arginase activity in primary microglia in the context of inflammatory environment (induced by LPS stimulation), where CX3CL1 counteracts the acquisition of a pro-inflammatory phenotype. To provide specific experimental data on the bioenergetics profile of microglia following pro-inflammatory activation and CX3CL1 treatment, primary microglia cells were seeded in a Seahorse XF plate in order to detect the cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). First, we observed a normal response pattern for unstimulated primary microglia and a decrease in basal respiration in LPS-treated cells that were unresponsive to the mitochondrial stressors, suggesting an impairment of mitochondrial function. More interesting, while microglia exposed to CX3CL1 maintained a bioenergetics profile similar to control condition, CX3CL1 promoted a significant decrease of glycolysis and OCR in LPSstimulated microglia, confirming that the different phenotypes of microglia induced by CX3CL1 treatment are associated with different metabolic programing.

### CX3CL1 Modulates the Inflammatory Phenotype of Microglia in vivo

It is known that injured neurons respond to toxic insults by releasing CX3CL1, which is upregulated, cleaved, and released upon ischemia or excitotoxic insult (Chapman et al., 2000; Tarozzo et al., 2002; Limatola et al., 2005; Noda et al., 2011). The neuroprotective or neurotoxic action of CX3CL1 likely depends on the activation (priming) state of microglia in the different phases of acute and chronic brain diseases (Dénes et al., 2008; Cho et al., 2011; Cipriani et al., 2011; Liu et al., 2011; Pabon et al., 2011; Shan et al., 2011; Fumagalli et al., 2013; Lauro et al., 2015). In this view, we hypothesized that CX3CL1 is neuroprotective, in permanent focal cerebral ischemia, due to its ability to modulate microglia polarization and activation state toward a protective one. To confirm its role in modulating microglia polarization also during ischemia in vivo, we isolated CD11b+ cells from the ipsi- and contra-lateral cerebral hemispheres of mice subjected to pMCAO, at 24 and 72 h after ischemic insult, and analyzed the expression of the same genes taken in consideration in the in vitro experiments.  However, over time, microglia acquire a proinflammatory phenotype releasing pro-inflammatory cytokines, chemokines, and inducible nitric oxide synthase, which results in the exacerbation of brain damage (Shi and Pamer, 2011). We observed that CX3CL1 changes the phenotype of CD11b+ cells isolated in the pMCAO mice model and that this effect is time dependent, according with the appearance of anti-inflammatory (at 24 h) and pro-inflammatory microglia (at 72 h) (Schroeter et al., 1997; Hu et al., 2012; Fumagalli et al., 2015). In fact, CX3CL1 induced an increase in the expression of several antiinflammatory and oxidative pathway-related genes already 24 h after ischemia and this effect remained up to 72 h. On the contrary, it down-modulated the expression of several proinflammatory and glycolytic pathway markers only at 72 h after the ischemic insult, suggesting that in a way CX3CL1 acts potentiating the function of the anti-inflammatory microglia, trying to prolong this population phenotype. Since CD11b+ cells contain a mixture of myeloid cells, we specifically sorted microglial cells (24 or 72 h after ischemia) from both the ipsi- and contra-lateral brain hemisphere by flow cytometry to specifically analyze their gene expression. With this gating strategy we uncovered a microglia fraction of 80–90% among CD45+ cells and we repeated on these cells the expression analysis for some of the genes evaluated on CD11b+ cells. In this way we confirmed that CX3CL1 induces an increase in the expression of antiinflammatory and oxidative pathway related genes and decreases that of pro-inflammatory and glycolytic pathway markers on microglia. Since in our experimental condition the immune cell infiltrate (CD45high among CD45+ cells) in CX3CL1-treated mice is about 20%, both at 24 and 72 h after ischemia, we cannot ruled out that what we observed for the CD11b+ cells included the contribution of the infiltrating immune cells. Furthermore, it is possible that CX3CL1 modulates the activation state of these cells too in order to recover the ischemic damage. For example, this would explain the observed differences regarding the CX3CL1 effect on il1β expression. It would be interesting to analyze the infiltrating population and the activation state of these cells in the presence and in the absence of the CX3CL1 stimulus and this will probably be the subject of a subsequent study. Moreover, we interpret that CX3CL1 does not modulate all the markers taken in consideration, because the injury-induced inflammatory processes are dynamic, with spatial and temporal heterogeneity, and/or because some microglial cells have intermediate phenotypes, with the simultaneous expression of pro- and anti-inflammatory genes (Ito et al., 2001; Taylor and Sansing, 2013; Morrison and Filosa, 2013). More interestingly, the ability of CX3CL1 to induce a metabolic switch toward oxidative metabolism in microglia might be necessary to accommodate the beneficial role of anti-inflammatory microglia

and results in the generation of metabolites, which are harmful for neuronal survival. Indeed, CX3CL1 can act in concert with other chemokines, growth factors and metabolites to counteract glutamate excitotoxic damage with a mechanism that involves neurons, microglia and astrocytes (Lauro et al., 2010; Rosito et al., 2014). Considering all these data, we hypothesize that, in addition to limit neuronal damage counteracting excitotoxicity, the release of CX3CL1 in response to ischemic insult might be a physiological response of brain tissue to trigger neuroprotection, modulating the activation state of microglia and its metabolism in order to limit neuro-inflammation and give more time to the cells of the brain parenchyma to organize a neuroprotective response. In this view CX3CL1 may be an important messenger molecule that plays a part in microglia response to extracellular signals during ischemic injuries, linking anti-inflammatory microglia to reduced injury and potential repair and could represent a possible target to modulate microglia phenotype to limit inflammation upon ischemia.

#### ETHICS STATEMENT

The experiments described in the present work, were approved by the Italian Ministry of Health in accordance with the guidelines on the ethical use of animals from the European Community Council Directive of September 22, 2010 (2010/63/EU).

#### AUTHOR CONTRIBUTIONS

CLa and CLi contributed to the conception and design of the study. CLa designed and performed all the experiments and statistical analysis and wrote the manuscript. GC performed the in vivo experiments. LM performed the real time PCR analysis. FA and GP designed and performed isolation of microglia cells by cell sorting. AP and SR ran and calculated the seahorse experiments. FC supervised the Hyp-ACB facility and data analysis. All authors contributed to the manuscript revision, and read and approved the submitted version.

#### FUNDING

This work was supported by a grants from the Italian Ministry of Health (RF 2018) and from the Italian Ministry of University

# REFERENCES

and Research (PRIN 2015 E8EMCM\_001) to CLi and from Sapienza University of Rome (Italy) for the Hyp-ACB Platform (GA116154C8A94E3D) to FC.

#### ACKNOWLEDGMENTS

Dr. Giovanna Boumis and Dr. Mariachiara Magnifico are acknowledged for skillful technical support for the experiments performed with the Hyp-ACB Platform.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fncel. 2019.00414/full#supplementary-material

FIGURE S1 | Bioenergetic profile of microglial cells. (A) The glycolysis of microglia ± 10 ng/ml of LPS as ECAR parameter of the seahorse experiment reported in Figure 3F, indicates that the LPS treatment doubled the basal value. (B) The effect of LPS on microglia is reported as phenotype plot. This plot, where both ECAR and OCR are reported on the X and Y-axis, respectively, indicates that the LPS sample (open triangle) is more glycolytic and less aerobic than the untreated one (open circle). Interestingly, while the untreated responds to stressors as expected (gray circles), by increasing both OCR and ECAR, the LPS sample is quite insensitive to drugs treatment (gray triangles). This could be ascribed to a poor and probably maximal basal mitochondrial activity. In the figure a representative experiment; values reported in the plot are the means of at least 8 replicates ± SD.

FIGURE S2 | Bioenergetic profile of microglial cells. The mitochondrial respiration of microglia, obtained by means of OCR by seahorse experiments, ± 100 nM of CX3CL1 (24 h of treatment; black and empty symbols, respectively). Arrows indicate the addiction of drugs used to specifically target the mitochondrial function (i.e., oligomycin, FCCP, rotenone + antimycin, in order of addiction). Chemokine treatment does not affect the energetic profile. In the figure a representative experiment; values reported in the plot are the means of at least 8 replicates ± SD.

FIGURE S3 | Energetic profile of LPS-treated microglia ± CX3CL1 treatment. LPS sample (10 ng/ml, open triangle) and LPS + CX3CL1-treated one (10 ng/ml + 100 nM, open square). Arrows indicates the addiction of drugs used to specifically target the mitochondrial function (i.e., oligomycin, FCCP, rotenone + antimycin, in order of addiction). In the figure a representative experiment is shown as OCR (A) and ECAR (B) measurements. Histograms from panels (C–H) (LPS sample, black square and LPS + CX3CL1-treated sample, open square) are belong to panels (A,B) data, according to Agilent Mito Stress Report generator. Values reported in the plot are the means of at least 8 replicates ± SD.

Chapman, G. A., Moores, K., Harrison, D., Campbell, C. A., Stewart, B. R., and Strijbos, P. J. (2000). Fractalkine cleavage from neuronal membranes represents an acute event in the inflammatory response to excitotoxic brain damage. J. Neurosci. 20:RC87. doi: 10.1523/JNEUROSCI.20-15-j0004.2000

Chaudhary, N., Gupta, M. M., Shrestha, S., Pathak, S., Kurmi, O. P., Bhatia, B. D., et al. (2017). clinicodemographic profile of children with seizures in a

tertiary care hospital: a cross-sectional observational study. Neurol. Res. Int. 2017:1524548. doi: 10.1155/2017/1524548

neurons, microglia and astrocytes to counteract pMCAO and excitotoxic neuronal death. Front. Cell. Neurosci. 8:193. doi: 10.3389/fncel.2014.00193

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Lauro, Chece, Monaco, Antonangeli, Peruzzi, Rinaldo, Paone, Cutruzzolà and Limatola. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fncel-13-00414 September 11, 2019 Time: 16:22 # 16

digital media

of impactful research

article's readership


**2. Processing**

MaxCell CB, and patients receiving MaxCell CB showed superior engraftment, survival,

In addition, the three main post-thaw manipulation methods are reviewed. Comparison data for some of the thaw methods are presented, and matched-pair analysis was used to confirm the superiority of direct infusion thaw method over post-thaw wash

**Keywords:** cord blood banking, cord blood processing, MaxCell cord blood processing, MaxCB cord blood processing, stem cell processing, cord blood thawing, cord blood cryopreservation, cord blood transplantation, 2nd and 3rd generation cord

Today, hematopoietic stem cell transplantation (HSCT) can be performed using stem cells derived from three sources: bone marrow (BM), peripheral blood (PB), and umbilical cord blood (CB) from autologous, related allogeneic, or unrelated allogeneic donors. Autologous HSCT is not indicated for most indications, and only about one-third of patients in need of HSCT have a suitable related donor. Although there are currently tens of millions of adult volunteer donors registered worldwide, about 60% of patients will not find a suitably HLAmatched unrelated adult donor, and thus cannot access HSCT [1]. For minority patients, the probability of finding an HLA-matched unrelated adult donor is further reduced. Since the search process for an adult donor often takes several months, a significant proportion of patients will become higher risk or ineligible for HSCT or even die while waiting for the donor search [2]. In contrast, without the possibility of last minute donor refusal, CB products are available upon request and can be shipped to any transplant center in the world immediately. Importantly, cord blood transplantation (CBT) is associated with a lower incidence and severity of graft-versus-host disease (GvHD), and a partial HLA match between the donor and recipient is tolerable [3], making it an ideal donor source for minority patients without

Compared with historical controls of BM or PB HSCT, CBT has shown favorable clinical outcome despite significantly worse HLA match [3]. In the pediatric setting, CBT can be now considered established practice. For adults, CBT adoption has been slower because of cell dose limitation. Many strategies focused on overcoming this limitation are being developed, including double CBT, the utilization of two CB unit grafts [4–6], the combination of an unrelated CB graft with haplo-identical donor stem cells [7], and foregoing the post-thaw wash with direct infusion or reconstitution/dilution, which have shown promising results [8, 9]. Engraftment and survival appear to be at least equivalent to or may even be superior to historical data using post-thaw wash for CB products versus when CB was not washed. One

and transplant-related mortality, confirming pre-match observations.

4 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

for MaxCell CB products.

blood technologies

**1. Introduction**

large adult donor registries.

The world's first CB banks were established at the New York Blood Center, Düsseldorf and Milan in 1992 [13]. Initially, CB was processed and cryopreserved as whole cord blood, which we refer to in this chapter as zero generation process [19]; however, in 1995, Rubinstein et al. pointed out that "the first problem is that stockpiling ("banking") a sufficiently large number of cryoprotected Whole PCB (placental cord blood) units requires vast amounts of costly storage space in liquid nitrogen (LN). To establish an adequate panel, therefore, the hematopoietic cells of PCB units need to be concentrated into units of much smaller volume."[13] It was for this reason that volume reduction processing of CB units was developed, with associated reduction of the 'bulk of the erythrocytes and plasma'. It was reported that the red blood cell (RBC) reduction processing method increased the number of units that could be stored in the same freezer space by as much as 10-fold, and thus provided significant economic advantage [13].

Rubinstein et al. reported in 1998 the first comparison of engraftment rates using RBC-replete units (whole CB) and RBC-reduced (RCR) units [19]. In their **Table 2**, they indicated that the majority of patients in their series (337 of 546 patients) were transplanted with whole CB that were RBC-replete, and of the 365 patients (out of 546 patients) who had myeloid engraftment, the Kaplan-Meier engraftment rate was 80% [95% confidence interval (CI) 75–85%] among the RBC-replete CB transplant recipients, and 82% with RCR units (95% CI 76–88%). Whether the unit used for the transplants was RBC-replete or RCR was not considered among the variables associated with the engraftment- and transplantation-related events.

Many volume reduction methods of processing umbilical CB which reduce RBCs prior to the cryopreservation have been considered since then (**Table 1**); however, the most common method for processing CB units today is the hetastarch or HES RBC reduction (RCR), whether manual or automated. For the sake of convenience, we will call these first generation (1st Gen) cord blood processing techniques. It should be noted that these methods are often erroneously referred to as red cell depletion when all of these techniques retain considerable RBCs and do not deplete, but only reduce the number of RBCs.

Unfortunately, all 1st Gen RCR processing methods lose significant numbers of nucleated cells, stem cells, and progenitor cells as measured by CD34+ cells or colony forming units (CFU) enumeration, with an approximately 25% nucleated cell loss on average among various reports in the literature [10–17] and a range of 14–42% for the various methods reported by Takahashi et al. [10]. This is of concern because it is well-known that the success of CBT is critically dependent on cell dose, and insufficient cell dose is widely regarded as the most important limitation for umbilical CB transplantation, especially for adults and large children [1, 3]. **Table 1** lists some of the most common 1st Gen CB processing techniques.

\* Proprietary Technologies: SEPAX from BioSafe; AXP-Express from Thermogenesis; MaxCell 2nd Gen from StemCyte; MaxCell 3rd Gen from CyteTherapeutics, Inc.

**Table 1.** Technical comparison of some of the most popular 1st Gen CB processing techniques and the proprietary 2nd and 3rd MaxCell CB processing technologies.

To maximize CB cell dose, R. Chow has developed two proprietary alternative CB volume reduction technologies (2nd Gen and 3rd Gen) which he calls MaxCell CB processing technologies, because both share the common characteristics of (1) depleting or reducing plasma, (2) maximizing stem cell, progenitor, nucleated and mononuclear cell recovery after processing and most importantly, and (3) having identical cellular composition upon infusion for one version of the third generation technology (**Tables 1**–**3**).

Rubinstein et al. reported in 1998 the first comparison of engraftment rates using RBC-replete units (whole CB) and RBC-reduced (RCR) units [19]. In their **Table 2**, they indicated that the majority of patients in their series (337 of 546 patients) were transplanted with whole CB that were RBC-replete, and of the 365 patients (out of 546 patients) who had myeloid engraftment, the Kaplan-Meier engraftment rate was 80% [95% confidence interval (CI) 75–85%] among the RBC-replete CB transplant recipients, and 82% with RCR units (95% CI 76–88%). Whether the unit used for the transplants was RBC-replete or RCR was not considered among the variables

Many volume reduction methods of processing umbilical CB which reduce RBCs prior to the cryopreservation have been considered since then (**Table 1**); however, the most common method for processing CB units today is the hetastarch or HES RBC reduction (RCR), whether manual or automated. For the sake of convenience, we will call these first generation (1st Gen) cord blood processing techniques. It should be noted that these methods are often erroneously referred to as red cell depletion when all of these techniques retain considerable RBCs and do

Unfortunately, all 1st Gen RCR processing methods lose significant numbers of nucleated cells, stem cells, and progenitor cells as measured by CD34+ cells or colony forming units (CFU) enumeration, with an approximately 25% nucleated cell loss on average among various reports in the literature [10–17] and a range of 14–42% for the various methods reported by Takahashi et al. [10]. This is of concern because it is well-known that the success of CBT is critically dependent on cell dose, and insufficient cell dose is widely regarded as the most important limitation for umbilical CB transplantation, especially for adults and large children [1, 3]. **Table 1** lists some of the most common 1st Gen CB processing techniques.

Zero generation Whole cord blood Manual None None

First generation PrepaCyte Red cell reduced Manual Yes Yes First generation Top & bottom Optipress II Red cell reduced Manual Yes Yes First generation Ficoll Red cell reduced Manual Yes Yes

reduced

Replete

RBC Reduced + RBC

\* Proprietary Technologies: SEPAX from BioSafe; AXP-Express from Thermogenesis; MaxCell 2nd Gen from StemCyte;

**Table 1.** Technical comparison of some of the most popular 1st Gen CB processing techniques and the proprietary 2nd

First generation hetastarch Red cell reduced Manual SEPAX\* or

**Technique Manual/automated**

**processing**

AXP-Express\*

Manual SEPAX

Manual SEPAX AXP-Express **RBC reduction**

Yes Yes

No Yes

Yes/No Yes

**Plasma depletion/ reduction**

associated with the engraftment- and transplantation-related events.

6 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

not deplete, but only reduce the number of RBCs.

Second generation Plasma depleted/

Third generation MaxCord

MaxCell 3rd Gen from CyteTherapeutics, Inc.

and 3rd MaxCell CB processing technologies.

MaxCell (MC) Technologies

1st generation RBC-reduced CB Products—Manual: Hetastarch most commonly used; however, also include PrepaCyte, Optipress, Ficoll Hypaque; Automated: Sepax & AXP-AutoXpress.

MaxCell: 2nd generation plasma depleted/reduced CB products and 3rd generation MaxCord CB products.

**Table 2.** A comparison of the volumes and amount of some of the various components of first (hetastarch), second and third generation CB technologies.

The MaxCell second generation (2nd Gen) technology or plasma depletion/reduction reduces or depletes plasma without reducing RBCs. Plasma is removed or reduced from the product prior to the cryopreservation [18]. Although the resulting volume of CB products processed by such a method is two to three times larger, 2nd Gen results in greater than 99.9% processing recovery of nucleated cells and all critical cell types prior to the testing and archival sampling. True nucleated cell loss was less than 0.01% from 2nd Gen CB processing, as evidenced by complete blood count (CBC) enumeration of the discarded plasma fraction. In practice, because of the testing and archival sampling, the actual loss will be raised to around 5–10% of the collected CB cell dose. Such minimal loss was reproducible from validation to an actual MaxCell inventory of more than 12,000 CB products that had pre- and post- processing CBC available. Even after thawing, in 188 post-thaw segment samples, a median of 90.33% *postsampling/post-thaw* TNC recovery and a median of 91.84% *post-sampling/post-thaw* CD34 cell recovery of preprocessing values were achieved for 2nd Gen CB products. The resultant volume, DMSO load, RBC, WBC and RBC lysate content of 2nd Gen CB products are similar to cryopreserved peripheral blood products (**Tables 1**–**3**).

The newest MaxCell technology, MaxCord processing technology, referred to as third generation (3rd Gen) CB processing technology in this chapter, combines the advantages of ease and convenience of thawing for the 1st Gen products and maximum stem (VSEL), progenitor (CFU and CD34+), nucleated, and mononuclear cell recovery of the 2nd Gen, without any of the associated disadvantages. While the processing methods and cryopreserved products are distinctly different, with the preferred embodiment of 3rd Gen technology, the cellular compositions of the final infused products are virtually identical between the two MaxCell technologies. **Table 2** outlines some of the component differences in volume, RBC content, and amount of additional components, such as DMSO, dextran-40, HES, and anticoagulant CPD.

Comparisons between HES 1st Gen and MaxCell 2nd and 3rd Gen are performed using parallel processing comparison using one half of the same cord blood unit for first generation hetastarch and the other half for MaxCell CB Products [18]. Cell types which exhibit significant differences are highlighted by boldfaced fonts. Second and third generation cord blood products have similar yield for all cell lineages, and only differ in the final RBC yield depending on the embodiment of 3rd Gen used. Third generation is similar to first generation in RBC reduction in RBC reduction. Sample size n = 10 for all the cell types, except for VSEL, which had a sample size n = 3. P values are calculated using the paired-sample *t*-test.

**Table 3.** Stem cell (VSEL), progenitor cell (CFU and CD34+), nucleated cell, mononuclear cell, nucleated RBC, RBC, and viability recovery of 1st (hetastarch method) MaxCell 2nd and MaxCell 3rd generation CB processing.

Third generation CB processing technology is perhaps the most flexible CB processing technology developed to date, with the additional option to reduce RBCs without losing as much stem, progenitor, nucleated, and mononuclear cells as 1st Gen RCR techniques (**Table 3**). **Table 1** highlights some of the processing technical differences among various popular 1st Gen techniques versus 2nd Gen and 3rd Gen CB processing technologies. Unlike Chow's 2nd Gen CB technology which is only amenable to SEPAX-automated processing and not accessible to AXP-Express, Chow's latest MaxCell technology can be automated using both SEPAX or AXP-Express. **Table 3** shows the differences in CB stem cell (VSEL), hematopoietic progenitor cell (CD34+ and CFUs), total nucleated cells (TNC), mononuclear cells, nucleated RBCs (nRBC), RBCs, and viability between the 1st Gen HES technique and the proprietary MaxCell 2nd/3rd Gen technologies. While the processing methods and cryopreserved products are distinctly different, the cellular composition of the final infused product is identical between the two MaxCell technologies using the preferred embodiment of 3rd Gen (**Table 3**) technology, while other embodiments can be adapted to reduce RBC, free hemoglobin, DMSO, and WBC lysate load for certain situations to better suit the needs of the individual patients (**Table 2**).

and CD34+), nucleated, and mononuclear cell recovery of the 2nd Gen, without any of the associated disadvantages. While the processing methods and cryopreserved products are distinctly different, with the preferred embodiment of 3rd Gen technology, the cellular compositions of the final infused products are virtually identical between the two MaxCell technologies. **Table 2** outlines some of the component differences in volume, RBC content, and amount of additional components, such as DMSO, dextran-40, HES, and anticoagulant CPD.

> **CD34+ cells (×106 )**

1.16 84.0%

1.42 99.9% **Viability (%)**

96% 98.3%

98% 99.3% **CFU-GM (×105 )**

3.5 42.1%

7.8 78.1%

118% 117% **124% 121%** 102% **225%** 128% **202% 186% 181%**

0.18 0.27 **0.002 0.003** 0.24 **0.05** 0.50 **0.001 0.01 0.007**

**CFU-E (×105 )**

3.6 48.5%

4.7 111.2% **CFU-GEMM (×105 )**

4.6 50.4%

9.3 88.4% **Total CFU (×105 )**

11.7 47.1%

21.8 88.1% **Total VSEL (×103 )**

4.434 54.6%

8.134 100.1%

**Post-processing Recovery Cell # Recovery %**

RCR Gen 1- HES

MaxCell Gen 2/3

Gen 2 and/or 3 MaxCellGen/ RCR

Recovery Ratio

P value **Gen 2**

**RBC (×1010) Total**

8 32.9%

Gen 2–24 100.2% Gen 3–8 33%

**Gen 2 312%** Gen 3 100–312%

**<0.0001** Gen 3 Variable

values are calculated using the paired-sample *t*-test.

**nRBC (×107 )**

8 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

2.1 78.1%

2.5 91.5% **Total MNC (×107 )**

26 80.7%

31 92.4% **TNC (×107 )**

65.77 78.7%

81.56 96.4%

Comparisons between HES 1st Gen and MaxCell 2nd and 3rd Gen are performed using parallel processing

Products [18]. Cell types which exhibit significant differences are highlighted by boldfaced fonts.

viability recovery of 1st (hetastarch method) MaxCell 2nd and MaxCell 3rd generation CB processing.

comparison using one half of the same cord blood unit for first generation hetastarch and the other half for MaxCell CB

**Table 3.** Stem cell (VSEL), progenitor cell (CFU and CD34+), nucleated cell, mononuclear cell, nucleated RBC, RBC, and

Third generation CB processing technology is perhaps the most flexible CB processing technology developed to date, with the additional option to reduce RBCs without losing as much stem, progenitor, nucleated, and mononuclear cells as 1st Gen RCR techniques (**Table 3**). **Table 1** highlights some of the processing technical differences among various popular 1st Gen techniques versus 2nd Gen and 3rd Gen CB processing technologies. Unlike Chow's 2nd Gen CB technology which is only amenable to SEPAX-automated processing and not accessible to AXP-Express, Chow's latest MaxCell technology can be

Second and third generation cord blood products have similar yield for all cell lineages, and only differ in the final RBC yield depending on the embodiment of 3rd Gen used. Third generation is similar to first generation in RBC reduction in RBC reduction. Sample size n = 10 for all the cell types, except for VSEL, which had a sample size n = 3. P Barker et al. [20] have questioned whether the MaxCell data [8] indicating significantly higher TNC doses with red cell-replete MaxCell CB products are meaningful. Indeed, it was speculated that a 'correction factor' of 0.75 should be applied to the reported TNC content of RBCreplete products, arguing that such products contain the noncritical nucleated RBC (nRBC) and neutrophils, and implying that critical stem and progenitor cells are somehow not retained to a similar extent as TNC. Unfortunately, no hard data accompanied this implication or their recommendation and the authors ignored the data on tens of thousands of CB units presented by Chow et al. [18] in **Table I**–**IV** of their paper, which showed significantly higher recovery of stem (VSEL), progenitor (CD34+ and CFU), nucleated and mononuclear cells with parallel processing of the two halves of the same CB units using MaxCell 2nd Gen technologies versus RCR techniques. More importantly, the superior outcome data for the MaxCell CB products [8] were similarly disregarded. Petz and Chow [21] pointed out that implementation of Barker's recommendation, if incorrect, would underestimate the progenitor cell content of RBC-replete CB products, resulting in the inappropriate rejection of certain optimal units by transplant physicians in favor of RCR products with an apparently higher TNC dose after application of the 'correction factor' for RBC-replete products.

This outcome assumes that large cord blood banks have normal and similar distribution of patients, disease indications, and disease stage.

**Table 4.** Published overall clinical outcome of CBT patients receiving RCR CB products from NYBC, COBLT, NMDP, and CIBMTR [19, 23–34] versus MaxCell CB products from CIBMTR audited outcome data [8].

To study this issue, Chow et al. divided 10 CB units in half and processed one half by RCR using the most commonly used hetastarch method, and the other half by MaxCell in parallel [18]. As indicated in **Table 3**, the average TNC of MaxCell CB products after processing was 124% of that in RCR units (*p* = 0.002). Moreover, MaxCell caused virtually no loss of CD34 cells compared to RCR produced CB, with the mean post-processing CD34 cell count in MaxCell products being 121% of that in RCR units (*p* = 0.003). Page et al. [22] have reported on their experience with 435 CB transplants and concluded that total CFU is the best predictor of engraftment. Of major importance is that the mean recoveries of CFUs were also higher for MaxCell products [11], as shown in **Table 3**, 225% for CFU-GM (*p* = 0.05), 202% for CFU-GEMM (*p* = 0.001) and 186% for total CFU (*p* = 0.01). Taken together, this means that CFU, the most important cellular predictors of engraftment success, showed an even bigger difference than TNC between RCR and MaxCell products, in contrast to the speculation by Barker and NYBC [20].

In fact, in clinical studies of MaxCell CB products, outstanding clinical outcome in terms of engraftment and patient survival have been achieved. **Table 4** showed published engraftment and survival outcome for transplantation using 1st Gen RCR CB products from diverse CB banks (NYBC, EuroCord, COBLT CBB, London CBB, French CBB) or large outcome data registries, such as CIBMTR or NMDP [19, 23–34] versus 2nd/3rd Gen Max Cell CB products [8].

Ballen et al. [34] studied the outcome of manually processed RCR CB products, automated processed RCR CB products, and manually processed MaxCell 2nd Gen CB products. While acknowledging some of the significant design flaws in their study, the authors concluded that automated processing systems resulted in higher day-28 neutrophil recovery than manually processed plasma-reduced products; however, overall survival by day-100 was not different among the three groups. Many study decisions and design issues were not clearly explained or delineated in this study, such as patient selection or study end point selection. For patients receiving Max Cell 2nd Gen CB products and manually processed RCR CB products, there were only 133 and 279 patients, respectively, when hundreds to thousands more were available fitting the study criteria during the study period for both of these groups. For example, even in a 2008 study with just pediatric hematological malignancy patients from one transplant center (U. Arizona) and one CBB (StemCyte), Graham et al. presented data for 105 matched pairs of patients from 128 patients transplanted with PDR 2nd Gen CB and 112 patients transplanted with RCR 1st Gen CB [35]. In fact, it is obvious that of the 16 public CB banks in the study, at least four of the banks each alone had more patients fitting the exact criteria for patients transplanted with manually processed CB in the study, so the reasons why these other patients were not selected and the reasons for inclusion of the particular patients in the study were not clear. The decision to use only 100-day survival, and not 1 year, 3-year, or 5-year overall survival, was also quite unconventional. All three patient groups had approximately 80% 100-day overall survival, which should not give an erroneous impression that long-term survival is that high or that long-term survival may be the same for all three groups. Previous studies with far larger patient populations not restricted to acute leukemia/MDS from NYBC, COBLT, CIBMTR, and NMDP have confirmed that transplants with RCR CB yielded around 40–45% 1-year survival and much higher 100-day transplant-related mortality [19, 23–33]. Indeed, long-term survival data on many more patients fitting the study criteria were available from CIBMTR, NMDP, and the various banks when the study was analyzed, so it was odd that 100-day overall survival (OS) was chosen as one of the study's primary end points, when it would have been far more meaningful and just as easy to calculate and show longer term survival and transplant-related mortality data, as is typical for such CIBMTR studies. In fact, our experience of a large MaxCell CB bank

with thousands of patients transplanted with its products have shown that survival or transplant-related mortality during the first 100 days is often not predictive for long-term survival or patient mortality [8, 35–43].

of engraftment. Of major importance is that the mean recoveries of CFUs were also higher for MaxCell products [11], as shown in **Table 3**, 225% for CFU-GM (*p* = 0.05), 202% for CFU-GEMM (*p* = 0.001) and 186% for total CFU (*p* = 0.01). Taken together, this means that CFU, the most important cellular predictors of engraftment success, showed an even bigger difference than TNC between RCR and MaxCell products, in contrast to the speculation by

10 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

In fact, in clinical studies of MaxCell CB products, outstanding clinical outcome in terms of engraftment and patient survival have been achieved. **Table 4** showed published engraftment and survival outcome for transplantation using 1st Gen RCR CB products from diverse CB banks (NYBC, EuroCord, COBLT CBB, London CBB, French CBB) or large outcome data registries, such as CIBMTR or NMDP [19, 23–34] versus 2nd/3rd Gen Max

Ballen et al. [34] studied the outcome of manually processed RCR CB products, automated processed RCR CB products, and manually processed MaxCell 2nd Gen CB products. While acknowledging some of the significant design flaws in their study, the authors concluded that automated processing systems resulted in higher day-28 neutrophil recovery than manually processed plasma-reduced products; however, overall survival by day-100 was not different among the three groups. Many study decisions and design issues were not clearly explained or delineated in this study, such as patient selection or study end point selection. For patients receiving Max Cell 2nd Gen CB products and manually processed RCR CB products, there were only 133 and 279 patients, respectively, when hundreds to thousands more were available fitting the study criteria during the study period for both of these groups. For example, even in a 2008 study with just pediatric hematological malignancy patients from one transplant center (U. Arizona) and one CBB (StemCyte), Graham et al. presented data for 105 matched pairs of patients from 128 patients transplanted with PDR 2nd Gen CB and 112 patients transplanted with RCR 1st Gen CB [35]. In fact, it is obvious that of the 16 public CB banks in the study, at least four of the banks each alone had more patients fitting the exact criteria for patients transplanted with manually processed CB in the study, so the reasons why these other patients were not selected and the reasons for inclusion of the particular patients in the study were not clear. The decision to use only 100-day survival, and not 1 year, 3-year, or 5-year overall survival, was also quite unconventional. All three patient groups had approximately 80% 100-day overall survival, which should not give an erroneous impression that long-term survival is that high or that long-term survival may be the same for all three groups. Previous studies with far larger patient populations not restricted to acute leukemia/MDS from NYBC, COBLT, CIBMTR, and NMDP have confirmed that transplants with RCR CB yielded around 40–45% 1-year survival and much higher 100-day transplant-related mortality [19, 23–33]. Indeed, long-term survival data on many more patients fitting the study criteria were available from CIBMTR, NMDP, and the various banks when the study was analyzed, so it was odd that 100-day overall survival (OS) was chosen as one of the study's primary end points, when it would have been far more meaningful and just as easy to calculate and show longer term survival and transplant-related mortality data, as is typical for such CIBMTR studies. In fact, our experience of a large MaxCell CB bank

Barker and NYBC [20].

Cell CB products [8].

The most rigorous method to address clinical outcome differences between different types of CB processing methods would be to conduct prospective double-blind placebo-controlled clinical trials; however, in HSCT, this is largely not feasible. Instead, matched-pair analyses allow for comparisons that are reasonably free from extraneous factors. To investigate rigorously whether clinical outcome differences exist between RCR and MaxCell CB products, Chow and collaborators performed matched-pair analysis using retrospective outcome data. Moreover, they secured an independent third party's assistance from CIBMTR to audit the outcome data with patient chart review on site at transplant centers using an audit design proposed by CIBMTR and finding the MaxCell CB outcome data to be 97.3% accurate, with no errors in survival, mortality, or engraftment. For both studies, all thalassemia and pediatric hematological malignancy patients were included and outcome were similar before and after matched-pair comparisons. A logistic regression model was used to find patients with similar characteristics to form pairs. For both studies, cumulative incidence was used for ANC500 neutrophil and platelet 20K and 50K engraftment. Kaplan–Meier was used for overall survival, disease-free survival, transplanted-related mortality, and relapse or autologous recovery. Cox regression analysis, log-rank test, univariate comparisons and the paired Prentice-Wilcoxon method were performed to compare the two matched-pair groups. Using the above methodologies, two rigorous retrospective matched-pair analysis of patients using RCR versus MaxCell CB products were conducted for thalassemia as well as for pediatric hematological malignancy patients [35–38].

Relative Risk calculated by Cox Regression for Univariate Analysis, with Red Cell Reduced CB products as reference. PPW = Paired Prentice-Wilcoxon test used in comparing matched-pair patients [**35–38**]. Absolute Neutrophil Count (ANC 500) is the first day of 3 consecutive days of an absolute neutrophil count equal to or greater than 0.5 × 109 /L prior to day 60 after transplantation (ANC 500)) engraftment. Platelet 20K engraftment is the first day of 7 consecutive days of a platelet count equal to or greater than 20 × 109 /L prior to the day 180 after transplantation.

**Table 5a.** Matched-Pair Comparison of Clinical Outcome of Patients transplanted with First Generation Red Cell Reduced CB Products versus MaxCell 2nd/3rd Generation CB products—30 pairs of thalassemia patients [36, 38] (Jaing et al. 2008) and 105 pairs of pediatric malignancy patients [35, 37, 38] (Jaing et al. 2008).

Relative Risk calculated by Cox Regression for Univariate Analysis, with Red Cell Reduced CB products as reference. PPW = Paired Prentice-Wilcoxon Test used in comparing matched-pair patients [**35–38**]. Absolute Neutrophil Count (ANC 500) is the first day of 3 consecutive days of an absolute neutrophil count equal to or greater than 0.5 × 109 /L prior to day 60 after transplantation (ANC 500)) engraftment. Platelet 20K engraftment is the first day of 7 consecutive days of a platelet count equal to or greater than 20 × 109 /L prior to the day 180 after transplantation.

**Table 5b.** Matched-Pair Comparison of Clinical Outcome of Patients transplanted with First Generation Red Cell Reduced CB Products versus MaxCell 2nd/3rd Generation CB products—105 pairs of pediatric malignancy patients [35, 37, 38].

For the thalassemia matched-pair study, 48 patients and 10 patients transplanted with MaxCell CB and RCR CB, respectively, and 3 MaxCell CBT patients were matched to each RCR CBT patients to form 30 pairs [36, 38]. Outcome comparisons of the two patient groups pre-match showed superiority in overall survival, disease-free survival, and transplant-related mortality for patients transplanted with MaxCell CB. Factors matched between the two groups were age, weight, #HLA matches, TNC dose, and transplant center experience. As the patients are mostly pediatric, there were no differences in median TNC between the two patient groups before (MaxCell 9.1 versus RCR 8.9 × 107 /kg) or after the match (MaxCell 9.1 versus RCR 8.9 × 107 /kg). **Table 5** showed significant improvement in 1- to 3-year overall survival and disease-free survival and 1-year and 3-year transplant-related mortality with the use of MaxCell CB for thalassemia patients [36, 38]. Interestingly, neutrophil engraftment, and short-term (100-day) survival or transplant-related mortality were not significantly different between MaxCell and RCR CB products.

**RCR 1st Generation CB MaxCell 2nd/3rd Generation CB**

89 ± 6%

PPW p=NS

**69 ± 6 %**

**65 ± 5 %**

**61 ± 4 %**

54 ± 4 %

**23 ± 4 %**

/L prior to the day 180 after transplantation.

Univ. RR = 0.81, *P* = 0.06 Log-Rank Test *p* = 0.12 Matched Pair 91 ± 7%

Univ. RR = 1.60; *p* = 0.007 Log-Rank test *p* = 0.006 **Matched Pair 71 ± 7 %** Matched-Pair PPW *p* = 0.001

Univ. RR = 1.41; *p* = 0.06 Log-Rank Test *p* = 0.05 **Matched Pair 68 ± 7 %** Matched-Pair PPW *p* = 0.007

Univ. RR (death) = 0.74; *p* = 0.01

Univ. RR (death) = 0.86; *p* = 0.11

Log-Rank Test *p* = 0.11 **Matched Pair 68 ± 5%** Matched-Pair PPW *p* = 0.005

Log-Rank Test *p* = 0.41 Matched Pair 61 ± 6% Matched Pair PPW *p* = 0.07

Univ. RR = 0.49; *p* < 0.001 Log-Rank Test *p* = 0.002 **Matched Pair 17 ± 4%** Matched-Pair PPW *p* = 0.0001

/L

ANC500 Neutrophil Engraftment Pediatric Leukemia Matched Pair

Platelet 20K Engraftment Pediatric Leukemia Matched Pair

Platelet 50K Engraftment Pediatric Leukemia Matched Pair

1-Yr Overall Survival

Pediatric Leukemia Matched Pair

1-Yr Disease-Free Survival Pediatric Leukemia Matched Pair

1-yr Transplant-Related Mortality Pediatric Leukemia Matched Pair

37, 38].

days of a platelet count equal to or greater than 20 × 109

85 ± 6 %

12 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

PPW p=NS

**55 ± 6 %**

**51 ± 6 %**

**48 ± 5 %**

46 ± 5 %

**45 ± 5 %**

Univ. RR = 0.81; *p* = 0.06 Log-Rank Test *p* = 0.12 Matched Pair 87 ± 6 %

Univ. RR = 1.60; *p* = 0.007 Log-Rank Test *p* = 0.006 **Matched Pair 56 ± 6 %** Matched Pair PPW *p* = 0.001

Univ. RR = 1.41; *p* = 0.06 Log-Rank Test *p* = 0.05 **Matched Pair 52 ± 6%** Matched-Pair PPW *p* = 0.007

Univ. RR (death) = 0.74; *p* = 0.01

Univ. RR (death) = 0.86; *p* = 0.11

Log-Rank Test *p* = 0.11 **Matched Pair 50 ± 5%** Matched-Pair PPW *p* = 0.005

Log-Rank Test *p* = 0.41 Matched Pair 47 ± 5% Matched Pair PPW *p* = 0.07

Univ. RR = 0.49; *p* < 0.001 Log-Rank Test *p* = 0.002 **Matched Pair 44 ± 5%** Matched-Pair PPW *p* = 0.0001

Relative Risk calculated by Cox Regression for Univariate Analysis, with Red Cell Reduced CB products as reference. PPW = Paired Prentice-Wilcoxon Test used in comparing matched-pair patients [**35–38**]. Absolute Neutrophil Count (ANC 500) is the first day of 3 consecutive days of an absolute neutrophil count equal to or greater than 0.5 × 109

prior to day 60 after transplantation (ANC 500)) engraftment. Platelet 20K engraftment is the first day of 7 consecutive

**Table 5b.** Matched-Pair Comparison of Clinical Outcome of Patients transplanted with First Generation Red Cell Reduced CB Products versus MaxCell 2nd/3rd Generation CB products—105 pairs of pediatric malignancy patients [35,

92 pairs

For the pediatric hematological malignancy matched-pair study, factors matched between the two groups were age, weight, #HLA matches, TNC dose, disease, and disease status [35]. Combining audited outcome data from one CBB (StemCyte) and data from one transplant center (U. Arizona), Graham et al. presented data for 105 matched pair of patients (paired from 128 patients transplanted with MaxCell 2nd Gen CB and 112 patients transplanted with RCR 1st Gen CB). **Table 5** shows that for the 105 pairs of pediatric hematological malignancy patients, 1- and 3-year overall survival, 100-day, 1-year and 3-year transplant-related mortality, and platelet (20K and 50K) engraftment were significantly improved with the use of MaxCell CB, while disease-free survival trended towards improvement [35, 37, 38]. Superior outcome of the MaxCell CB patient group in pre-match comparisons were confirmed by the results seen in the matched-pair analysis. Again, neutrophil engraftment and short-term (100-day) survival or transplant-related mortality were not significantly different between MaxCell and RCR CB products despite significant advantages in platelet engraftment, overall survival and transplant-related mortality.

Using data supplied by the NMDP for its large CB inventory of its CBB network (as of June 30, 2006) derived from almost 50,000 units (10,912 MaxCell and 38,819 RCR), Chow et al. [8] showed that a 24% superior nucleated cell recovery amplified into a 200% increase for MaxCell over RCR for the proportion of the inventories with products that had TNC counts higher than 150 × 107 (20% versus 10% of the inventory; test for difference in proportions, *p* < 0.0001) and a threefold difference for the proportion of products that had TNC counts higher than 200 × 107 (6% versus 2% of the inventory; test for difference in proportions, *p* < 0.0001). Therefore, the effectiveness of MaxCell CB processing is supported by data derived from the NMDP CB inventories of almost 50,000 units, which proves that the MaxCell CB inventories have significantly higher proportions of products with high TNC doses than the RCR CB inventories. Indeed, the MaxCell CB inventories had two to three times the proportion of high-cell dose products with TNC number of 150 × 107 or above and 200 × 107 or above (*p* < 0.0001) than the inventories of RCR units. Thus, MaxCell CB processing provides more efficient utilization of this valuable resource. This would seem to be particularly significant for those patients who participate in directed CB donation and private banking, because of the uniqueness of the cellular content and the importance of cell dose in outcome of HSCT.

#### **3. Thawing of cryopreserved CB products**

Unlike cryopreservation, thawing should be performed as quickly as possible by immersing the body (but not the ports) of cord blood bag in a 37°C water bath to an icy slush mixture. After thawing, there will be invariably some cell lysis, including 5–20% of the RBCs [44], a certain amount of WBC, principally neutrophils which do not survive freezing and thawing well, and occasional cell clumping and viscosity due to the release of chromosomal DNA. **Table 6** shows the expected cell loss for the various 1st Gen methods [44] as well as for the MaxCell 2nd/3rd Gen technologies [8]. The published cell loss and death associated with the CB processing method is listed below in **Table 6**, which showed the least TNC, CD34, and CFU loss for MaxCell CB products, followed by PrepaCyte, and with AXP-Express coming in last of the four techniques tested by Akel et al. [44]. Screnci et al. [45] independently confirmed that 42 un-manipulated RBC-replete CB products had significantly better post-thaw and wash recovery of TNC than 36 RCR CB, 95.2 ± 14.7% versus 85 ± 15.4% (*p* = 0.004).

**Table 6.** Post-thaw cell loss vs. pre-freeze cell count [11\*\*, 44\*, 45\*\*\*].