Open-tubular capillary cell affinity chromatography: single and tandem blood cell separation

In this paper, an open-tubular capillary cell affinity chromatography (OT-CAC) method to enrich and separate target cells is described. Open tubular capillaries coated with anti-CD4, anti-CD14, or anti-CD19 antibodies were used as affinity chromatography columns to separate target blood cells. Cells were eluted using either shear force or bubbles. Bubbles were used to elute the captured cells without diluting the captured cells appreciably, while maintaining viability (the viability of the recovered cells was 85.83 +/- 7.34%; the viability of the cells was 90.41 +/- 3.49% before separation). Several aspects of the OT-CAC method were studied, such as the affinity of one antibody between two different cell lines, the effect of shear force, and the recovery of captured cells. Single- and multicell type separations were demonstrated by isolating CD4+ cells with antiCD4 coated capillary and isolating CD4+ and CD19+ cells with two capillaries in tandem from blood samples. In the one cell type isolation test, an average of 87.7% of the recovered cells from antiCD4 capillary were lymphocytes and an average of 97.7% of those lymphocytes were CD4+ cells. In the original blood sample, only 14.2% of the leukocytes were CD4+ cells. Two capillary columns were also run in tandem, separating two blood cell types from a single sample with high purity. The use of different elution shear forces was demonstrated to selectively elute one cell type. This method is an inexpensive, rapid, and effective method to separate target cells from blood samples.     

CD34~+造血祖细胞及其亚群（CD34~+CD38~+和CD34~+CD38~-

分离纯化造血干祖细胞具有十分重要的理论和应用价值。本文利用吸附免疫微球的单克隆抗体分离系统，对不同来源造血组织的ＣＤ３４＋细胞进行纯化分离，经流式细胞仪检测，其纯度可达９５—９９％。在外源性生长因子的刺激下，ＣＤ３４＋细胞可形成大量各系造血集落，而ＣＤ３４－组分则几乎不含造血集落形成细胞。进一步的研究则是利用免疫荧光激活的流式细胞分选系统，将ＣＤ３４＋细胞群分为ＣＤ３４＋ＣＤ３８＋和ＣＤ３４＋ＣＤ３８－两个亚群，并比较正常人骨髓、脐带血、外周血来源的亚群细胞造血性能。结果表明，不同细胞亚群造血性能不均一，同一亚群不同来源的细胞也同样具有不均一性，从而为造血干细胞的基因治疗、建库、移植等提供了理论依据。     

Magnetic cell separation using antibody binding with protein a expressed on bacterial magnetic particles

Bacterial magnetic particles (BacMPs) are efficient platforms of proteins for surface display systems. In this study, mononuclear cells from peripheral blood were separated using BacMPs expressing protein A on the BacMP membrane surface (protein A-BacMPs), which were complexed with the Fc fragment of anti-mouse IgG antibody. The procedure of positive selection involves incubation of mononuclear cells and mouse monoclonal antibodies against different cell surface antigens (CD8, CD14, CD19, CD20) prior to treatment with protein A-BacMP binding with rabbit anti-mouse IgG secondary antibodies. Flow cytometric analysis showed that approximately 97.5 +/- 1.7% of CD19(+) and CD20(+) cells were involved in the positive fraction after magnetic separation. The ratio of the negative cells in the negative fraction was approximately 97.6 +/-1.4%. This indicates that CD19(+) and CD20(+) cells can be efficiently separated from mononuclear cells. Stem cell marker (CD34) positive cells were also separated using protein A-BacMP binding with antibody. May-Grunwald Giemsa stain showed a high nuclear/cytoplasm ratio, which indicates a typical staining pattern of stem cells. The separated cells had the capability of colony formation as hematopoietic stem cells. Furthermore, the inhibitory effect of magnetic cell separation on CD14(+) cells was evaluated by measurement of cytokine in the culture supernatant by ELISA when the cells were cultured with or without lipopolysaccharide (LPS). The induction of IL1-beta, TNFalpha, and IL6 was observed in the presence of 1 ng/mL LPS in all fractions. On the other hand, in the absence of LPS, BacMPs had little immunopotentiation to CD14(+) cells as well as that of artificial magnetic particles, although TNFalpha and IL6 were slightly induced in the absence of LPS in the positive fraction.     

Cell separation by dielectrophoretic field-flow-fractionation

Dielectrophoretic field-flow-fractionation (DEP-FFF) was applied to several clinically relevant cell separation problems, including the purging of human breast cancer cells from normal T-lymphocytes and from CD34+ hematopoietic stem cells, the separation of the major leukocyte subpopulations, and the enrichment of leukocytes from blood. Cell separations were achieved in a thin chamber equipped with a microfabricated, interdigitated electrode array on its bottom wall that was energized with AC electric signals. Cells were levitated by the balance between DEP and sedimentation forces to different equilibrium heights and were transported at differing velocities and thereby separated when a velocity profile was established in the chamber. This bulk-separation technique adds cell intrinsic dielectric properties to the catalog of physical characteristics that can be applied to cell discrimination. The separation process and performance can be controlled through electronic means. Cell labeling is unnecessary, and separated cells may be cultured and further analyzed. It can be scaled up for routine laboratory cell separation or implemented on a miniaturized scale.     

Multitarget dielectrophoresis activated cell sorter

The ability to rapidly and efficiently isolate specific viruses, bacteria, or mammalian cells from complex mixtures lies at the heart of biomedical applications ranging from in vitro diagnostics to cell transplantation therapies. Unfortunately, many current selection methods for cell separation, such as magnetic activated cell sorting (MACS), only allow the binary separation of target cells that have been labeled via a single parameter (e.g., magnetization). This limitation makes it challenging to simultaneously enrich multiple, distinct target cell types from a multicomponent sample. We describe here a novel approach to specifically label multiple cell types with unique synthetic dielectrophoretic tags that modulate the complex permittivities of the labeled cells, allowing them to be sorted with high purity using the multitarget dielectrophoresis activated cell sorter (MT-DACS) chip. Here we describe the underlying physics and design of the MT-DACS microfluidic device and demonstrate approximately 1000-fold enrichment of multiple bacterial target cell types in a single-pass separation.     

Microinflammation in hemodialysis is related to a preactivated subset of monocytes

Increased percentage of monocytes with low CD14 expression and that co-express CD16 (CD14+/CD16+) have been reported in hemodialysis (HD) patients. We sought to determine whether CD14+/CD16+ monocytes in HD therapy are sensibilized cells to a proinflammatory activity. Cells from 32 HD patients, and from 9 Systemic Lupus Erythematosus (SLE), 9 individuals with human immunodeficiency virus (HIV)-1- and 15 healthy controls were studied. Cells were analyzed by means of flow cytometry for CD14/CD16 expression and immune function (cytokine, chemokines, and sialoadhesin expression), and phagocytosis. Increased percentage of CD14+/CD16+ monocytes was observed in HD patients. Compared with CD14++ monocytes, the CD14+/CD16+ monocytes exhibited increased expression of proinflammatory cytokines and markers of differentiated cells. In addition, these monocytes showed an increased phagocytic activity. Similarly, CD14+/CD16+ monocytes from SLE and HIV patients showed increased inflammatory activity as compared with CD14++ cells. These results support that CD14+/CD16+ monocytes from HD patients evidence characteristics of primed prestimulated proinflammatory cells, similar to data observed in SLE and HIV.     

Size effects of magnetic beads in circulating tumour cells magnetic capture based on streptavidin–biotin complexation

Circulating tumour cells (CTCs) draw significant attention as a promising biomarker for cancer prognosis, status monitoring, and metastasis diagnosis. However, the concentration of CTCs in peripheral blood is usually extremely low, thereby requiring enrichment followed by isolation of CTCs prior to detection. An immunomagnetic separation is a promising tool for CTCs enrichment. In this study, a cost‐effective magnetic separation method, based on streptavidin–biotin complexation, was developed and the effects of magnetic beads’ size in CTCs capture were compared. Magnetic nanobeads which were 25 nm in diameter lead to highest capture efficiency (82.2%) compared with 150 nm magnetic beads and 1 µm microbeads. Based on the streptavidin–biotin system, 25 nm magnetic nanobeads could capture model CTCs over 80% efficiency even at concentrations as low as ∼25 cells/mL that may represent the actual level of CTCs in peripheral blood of cancer patients. Furthermore, the isolated cells remained robust and healthy showing insignificant changes in morphology and behaviour when cultured for 24 h immediately after capture and isolation. The magnetic nanobeads based on streptavidin–biotin complexation showed promise for the easy and efficient capture and isolation of healthy CTCs for further diagnosis and analysis.Inspec keywords: cancer, magnetic separation, nanomedicine, nanomagnetics, proteins, biomagnetism, tumours, cellular biophysics, magnetic particles, molecular biophysics, blood, nanoparticlesOther keywords: streptavidin–biotin complexation, cancer prognosis, peripheral blood, immunomagnetic separation, CTCs capture, streptavidin–biotin system, circulating tumour cells, CTC enrichment, magnetic separation method, magnetic nanobeads, magnetic capture, size 25.0 nm, size 150.0 nm, time 24.0 hour     

Ex vivo expansion of haematopoietic progenitors on an endothelialized hydroxyapatite matrix

Autologous haematopoietic progenitor cell (HPC) transplantation is increasingly used to restore haematopoiesis after high-dose chemotherapy treatments. The present study was designed to analyse the ability of hydroxyapatite (HAP) seeded with endothelial cells (EC) to support the proliferation and differentiation of CD34+ HPC in static culture conditions. HAP is endothelializable as assessed by scanning electron microscopy and time-course DNA synthesis analysis using tritiated thymidine incorporated in EC isolated from human umbilical vein cord. Short-term coculture experiments in which CD34+ cells isolated from human cord blood were seeded on endothelialized HAP, were performed. Results show that endothelialized HAP is permissive to CD34+ cell expansion with a maximum expansion obtained between days 7 and 14 of coculture in the presence of IL-1 and IL-3 when compared with other experiments omitting either EC or interleukins. From morphological analyses, the expanded cell population mainly belonged to the myelocytic lineage with 33% mature cells (polymorphonuclear neutrophils and monocytes) at day 14 of coculture. The immature HPC could remain trapped within HAP while giving rise to a more mature progeny that exit from HAP microenvironment.     

Clinically relevant microfluidic magnetophoretic isolation of rare-cell populations for diagnostic and therapeutic monitoring applications

Cells of biomedical interest are, despite their functional significance, often present in very small numbers. Therefore the analysis and isolation of previously inaccessible rare cells, such as peripheral hematopoietic stem cells, endothelial progenitor cells, or circulating tumor cells, require efficient, sensitive, and specific procedures that do not compromise the viability of the cells. The current study builds on previous work on a rationally designed microfluidic magnetophoretic cell separation platform capable of throughputs of 240 μL min(-1). Proof-of-concept was first conducted using MCF-7 (1-1000 total cells) as the target rare cell spiked into high concentrations of Raji B-lymphocyte nontarget cells (~10(6) total cells). These experiments lead to the establishment of a magnet-based separation for the isolation of 50 MCF-7 cells directly from whole blood. Results show an efficiency of collection greater than 85%, with a purity of over 90%. Next, resident endothelial progenitor cells and hematopoietic stem cells are directly isolated from whole human blood in a rapid and efficient fashion (>96%). Both cell populations could be simultaneously isolated and, via immunofluorescent staining, individually identified and enumerated. Overall, the presented device illustrates a viable separation platform for high purity, efficient, and rapid collection of rare cell populations directly from whole blood samples.     

Bacterial DNA sample preparation from whole blood using surface-modified Si pillar arrays

A novel bacterial DNA sample preparation device for molecular diagnostics has been developed. On the basis of optimized conditions for bacterial adhesion, surface-modified silicon pillar arrays for bacterial cell capture were fabricated, and their ability to capture bacterial cells was demonstrated. The capture efficiency for bacterial cells such as Escherichia coli, Staphylococcus epidermidis, and Streptococcus mutans in buffer solution was over 75% with a flow rate of 400 microL/min. Moreover, the proposed method captured E. coli cells present in 50% whole blood effectively. The captured cells from whole blood were then in- situ lyzed on the surface of the microchip, and the eluted DNA was successfully amplified by qPCR. These results demonstrate that the full process of pathogen capture to DNA isolation from whole blood could be automated in a single microchip.     

Fast Capturing on Micromagnetic Cell Sorter

A new micro immune-magnetophoretic cell sorter (micro IMP cell sorter) has been developed to isolate T lymphocytes from biological suspensions such as whole blood. By using two permanent magnets, the developed micro IMP cell sorter is designed to isolate target cells continuously and automatically without a preliminary labeling process which is often acting as a bottleneck in automated microcell sorters. By using the capture probability analysis and the binding kinetic analysis, fast capturing of the target cells was achieved in the straight microfluidic channel between the magnets within 20 s of flow time. Multiphysical simulations including fluidic and electromagnetic models were carried out describing the trajectory of the magnetic particle, also. The fabricated microcell sorter was tested for sorting of CD3+ T lymphocytes with Dynabead CD3 from a mixture of T lymphocytes and erythrocytes.      

Computational analysis of microfluidic immunomagnetic rare cell separation from a particulate blood flow

We describe a computational analysis method to evaluate the efficacy of immunomagnetic rare cell separation from non-Newtonian particulate blood flow. The core procedure proposed here is calculation of local viscosity distributions induced by red blood cell (RBC) sedimentation. Numerical calculation methods have previously been introduced to simulate particulate behavior of individual RBCs. However, due to the limitation of the computational power, those studies are typically capable of calculating only a very small number (less than 100) of RBCs and are not suitable to analyze many practical separation methods for rare cells such as circulating tumor cells (CTCs). We introduce a sedimentation and viscosity model based on our experimental measurements. The computational field is divided into small unit control volumes, where the local viscosity distribution is dynamically calculated based on the experimentally found sedimentation model. For analysis of rare cell separation, the local viscosity distribution is calculated as a function of the volume RBC rate. The direction of gravity has an important role in such a sedimentation-involved cell separation system. We evaluated the separation efficacy with multiple design parameters including the channel design, channel operational orientations (inverted and upright), and flow rates. The results showed excellent agreement with real experiments to demonstrate the effectiveness of our computational analytical method. We demonstrated higher capture efficiency with the inverted microchannel configuration.We conclude that proper direction of blood sedimentation significantly enhances separation efficiency in microfluidic devices.     

Surface-modified hyaluronic acid hydrogels to capture endothelial progenitor cells

A major challenge to the effective treatment of injured cardiovascular tissues is the promotion of endothelialization of damaged tissues and implanted devices. For this reason, there is a need for new biomaterials that promote endothelialization to enhance vascular repair. The goal of this work was to develop antibody-modified polysaccharide-based hydrogels that could selectively capture endothelial progenitor cells (EPCs). We showed that CD34 antibody immobilization on hyaluronic acid (HA) hydrogels provides a suitable surface to capture EPCs. The effect of CD34 antibody immobilization on EPC adhesion was found to be dependent on antibody concentration. The highest level of EPC attachment was found to be 52.2 cells per mm(2) on 1% HA gels modified with 25 μg mL(-1) antibody concentration. Macrophages did not exhibit significant attachment on these modified hydrogel surfaces compared to the EPCs, demonstrating the selectivity of the system. Hydrogels containing only HA, with or without immobilized CD34, did not allow for spreading of EPCs 48 h after cell seeding, even though the cells were adhered to the hydrogel surface. To promote spreading of EPCs, 2% (w/v) gelatin methacrylate (GelMA) containing HA hydrogels were synthesized and shown to improve cell spreading and elongation. This strategy could potentially be useful to enhance the biocompatibility of implants such as artificial heart valves or in other tissue engineering applications where formation of vascular structures is required.     

Fast and selective cell isolation from blood sample by microfiber fabric system with vacuum aspiration

Since circulating tumor cells (CTCs) are tumor cells which are found in the blood of cancer patients, CTCs are potential tumor markers, so a rapid isolation of CTCs is desirable for clinical applications. In this paper, a three-dimensional polystyrene (PS) microfiber fabric with vacuum aspiration system was developed for capturing CTCs within a short time. Various microfiber fabrics with different diameters were prepared by the electrospinning method and optimized for contact frequency with cells. Vacuum aspiration utilizing these microfiber fabrics could filter all cells within seconds without mechanical damage. The microfiber fabric with immobilized anti-EpCAM antibodies was able to specifically capture MCF-7 cells that express EpCAM on their surfaces. The specificity of the system was confirmed by monitoring the ability to isolate MCF-7 cells from a mixture containing CCRF-CEM cells that do not express EpCAM. Furthermore, the selective capture ability of the microfiber was retained even when the microfiber was exposed to the whole blood of pigs spiked with MCF-7 cells. The specific cell capture ratio of the vacuum aspiration system utilizing microfiber fabric could be improved by increasing the thickness of the microfiber fabric through electrospinning time.     

Targeting Mucin Protein Enables Rapid and Efficient Ovarian Cancer Cell Capture: Role of Nanoparticle Properties in Efficient Capture and Culture

The development of specific and sensitive immunomagnetic cell separation nanotechnologies is central to enhancing the diagnostic relevance of circulating tumor cells (CTCs) and improving cancer patient outcomes. The limited number of specific biomarkers used to enrich a phenotypically diverse set of CTCs from liquid biopsies has limited CTC yields and purity. The ultra-high molecular weight mucin, mucin16 (MUC16) is shown to physically shield key membrane proteins responsible for activating immune responses against ovarian cancer cells and may interfere with the binding of magnetic nanoparticles to popular immunomagnetic cell capture antigens. MUC16 is expressed in ≈90% of ovarian cancers and is almost universal in High Grade Serous Epithelial Ovarian Cancer. This work demonstrates that cell bound MUC16 is an effective target for rapid immunomagnetic extraction of expressor cells with near quantitative yield, high purity and viability from serum. The results provide a mechanistic insight into the effects of nanoparticle physical properties and immunomagnetic labeling on the efficiency of immunomagnetic cell isolation. The growth of these cells has also been studied after separation, demonstrating that nanoparticle size impacts cell-particle behavior and growth rate. These results present the successful isolation of “masked” CTCs enabling new strategies for the detection of cancer recurrence and select and monitor chemotherapy.     

Continuous Flow Deformability‐Based Separation of Circulating Tumor Cells Using Microfluidic Ratchets

Circulating tumor cells (CTCs) offer tremendous potential for the detection and characterization of cancer. A key challenge for their isolation and subsequent analysis is the extreme rarity of these cells in circulation. Here, a novel label‐free method is described to enrich viable CTCs directly from whole blood based on their distinct deformability relative to hematological cells. This mechanism leverages the deformation of single cells through tapered micrometer scale constrictions using oscillatory flow in order to generate a ratcheting effect that produces distinct flow paths for CTCs, leukocytes, and erythrocytes. A label‐free separation of circulating tumor cells from whole blood is demonstrated, where target cells can be separated from background cells based on deformability despite their nearly identical size. In doping experiments, this microfluidic device is able to capture >90% of cancer cells from unprocessed whole blood to achieve 10⁴‐fold enrichment of target cells relative to leukocytes. In patients with metastatic castration‐resistant prostate cancer, where CTCs are not significantly larger than leukocytes, CTCs can be captured based on deformability at 25× greater yield than with the conventional CellSearch system. Finally, the CTCs separated using this approach are collected in suspension and are available for downstream molecular characterization.     

Simultaneous expansion and harvest of hematopoietic stem cells and mesenchymal stem cells derived from umbilical cord blood

The simultaneous expansion and harvest of hematopoietic stem cells and mesenchymal stem cells derived from umbilical cord blood were carried out using bioreactors. The co-culture of umbilical cord blood (UCB)-derived hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) was performed within spinner flasks and a rotating wall vessel (RWV) bioreactor using glass-coated styrene copolymer (GCSC) microcarriers. The medium used was composed of serum-free IMDM containing a cocktail of SCF 15 ng·mL^?1, FL 5 ng·mL^?1, TPO 6 ng·mL^?1, IL-3 15 ng·mL^?1, G-CSF 1 ng·mL^?1 and GM-CSF 5 ng·mL^?1. Accessory stromal cells derived from normal allogeneic adipose tissue were encapsulated in alginate-chitosan (AC) beads and used as feeding cells. The quality of the harvested UCB-HSCs and MSCs was assessed by immunophenotype analysis, methylcellulose colony and multi-lineage differentiation assays. After 12 days of culture, the fold-expansion of total cell numbers, colony-forming units (CFU-C), CD34⁺/CD45⁺/CD105^? (HSCs) cells and CD34^?/CD45^?/CD105⁺ (MSCs) cells using the RWV bioreactor were (3.7 ± 0.3)- , (5.1 ± 1.2)- , (5.2 ± 0.4)- , and (13.9 ± 1.2)-fold respectively, significantly better than those obtained using spinner flasks. Moreover, UCB-HSCs and UCB-MSCs could be easily separated by gravity sedimentation after the co-culture period as only UCB-MSCs adhered on to the microcarriers. Simultaneously, we found that the fibroblast-like cells growing on the surface of the GCSC microcarriers could be induced and differentiated towards the osteoblastic, chondrocytic and adipocytic lineages. Phenotypically, these cells were very similarly to the MSCs derived from bone marrow positively expressing the MSCs-related markers CD13, CD44, CD73 and CD105, while negatively expressing the HSCs-related markers CD34, CD45 and HLA-DR. It was thus demonstrated that the simultaneous expansion and harvest of UCB-HSCs and UCB-MSCs is possible to be accomplished using a feasible bioreactor culture system such as the RWV bioreactor with the support of GCSC microcarriers.     

Human endothelial progenitor cell attachment to polysaccharide-based hydrogels: A pre-requisite for vascular tissue engineering

A hydrogel was prepared from polysaccharides (pullulan/dextran/fucoidan) and evaluated as a novel biomaterial for Endothelial Progenitor Cell (EPC) culture. Using a cross-linking process with sodium trimetaphosphate in aqueous solution, homogeneous, transparent and easy to handle gels were obtained with a water content higher than 90%. Circular scaffolds (6 mm diameter and 2 mm thickness discs) were used for cell culture. Different types of EPCs were used: CD34+ derived ECs from cord blood and two sorts of CD133+ derived ECs from human bone marrow, old (30 days) and young (4 days) cells. EPCs were characterised as endothelial cells by immunofluorescent stainings for CD31 and Dil-Ac-LDL. CD133+ derived ECs from bone marrow were characterized by RT-PCR for CD31, VE-cadherin and KDR. HSVECs (Human Saphenous Vein Endothelial Cells) were used as control cells. We evaluated whether different kinds of EPCs could adhere on this novel hydrogel 4 h and 24 h after seeding, by a colorimetric quantitative test. EPCs adhered to hydrogels in serum- free conditions with values being over than 80% for young CD133+ cells at 4 h and 24 h. This pullulan-based hydrogel could constitute a suitable support for vascular cell adhesion as a pre-requisite for vascular tissue engineering.     

Negative enrichment of target cells by microfluidic affinity chromatography

A three-dimensional microfluidic channel was developed for high-purity cell separations. This system featured high capture affinity using multiple vertical inlets to an affinity surface. In cell separations, positive selection (capture of the target cell) is usually employed. Negative enrichment, the capture of nontarget cells and elution of target cells, has distinct advantages over positive selection. In negative enrichment, target cells are not labeled and are not subjected to strenuous elution conditions or dilution. As a result, negative enrichment systems are amenable to multistep processes in microfluidic systems. In previous work (Li, P.; Tian, Y.; Pappas, D. Anal. Chem.2011, 83, 774-781), we reported cell capture enhancement effects at vertical inlets to the affinity surface. In this study, we designed a chip that has multiple vertical and horizontal channels, forming a three-dimensional separation system. Enrichment of target cells showed separation purities of 92-96%, compared with straight-channel systems (77% purity). A parallelized chip was also developed for increased sample throughput. A two-channel system showed similar separation purity with twice the sample flow rate. This microfluidic system, featuring high separation purity and ease of fabrication and use is suitable for cell separations when subsequent analysis of target cells is required.     

Biopolymer system for cell recovery from microfluidic cell capture devices

Microfluidic systems for affinity-based cell isolation have emerged as a promising approach for the isolation of specific cells from complex matrices (i.e., circulating tumor cells in whole blood). However, these technologies remain limited by the lack of reliable methods for the innocuous recovery of surface captured cells. Here, we present a biofunctional sacrificial hydrogel coating for microfluidic chips that enables the highly efficient release of isolated cells (99% ± 1%) following gel dissolution. This covalently cross-linked alginate biopolymer system is stable in a wide variety of physiologic solutions (including EDTA treated whole blood) and may be rapidly degraded via backbone cleavage with alginate lyase. The capture and release of EpCAM expressing cancer cells using this approach was found to have no significant effect on cell viability or proliferative potential, and recovered cells were demonstrated to be compatible with downstream immunostaining and FISH analysis.