A Novel Technology Allowing Immunohistochemical Staining of a Tissue Section with 50 Different Antibodies in a Single Experiment

Abstract

Immunohistochemical (IHC) examination is frequently necessary for a histological differential diagnosis of tumors. To simplify IHC examination, we have developed a novel device called a “multiplex-immunostain chip (MI chip).” The chip is a panel of antibodies contained in a silicon rubber plate that consists of 50 2-mm-diameter wells. A tissue section slide is placed on the plate and is fastened tightly with a specially designed clamp. The plate with the slide is then turned upside down, which applies the antibodies to the section. This technology allows IHC staining of a tissue section with 50 different antibodies in a single experiment, reducing the time, effort, and expense of IHC analysis. In addition, it enables pathologists to compare expression of multiple antigens on a tissue section simply by changing microscopic fields on a single slide. These features are unique to the MI chip technology. The method requires no expensive instruments. This device can be used in various applications in differential diagnosis of tumors and the field of cell biology.

Effective treatment of tumors depends primarily on the histological type. However, it is not always easy, even for trained pathologists, to diagnose accurately poorly differentiated tumors, such as round-cell and spindle-cell tumors, with conventional hematoxylin-eosin-stained tissue sections. In general, histopathological diagnosis and classification of tumors are based on cell phenotypes (Baars et al. 1999; Jones and Dorfman 2001). However, pathologists sometimes encounter tumors that are difficult to subclassify on the basis of conventional morphological observation alone. Immunohistochemical (IHC) studies are useful for determining cell phenotypes in tumors. At present, various kinds of antibodies are available for characterization and classification of tumors (Singer et al. 2000; Yaziji and Gown 2001; Chi 2002; Li 2002), and new antibodies are being developed for analysis of antigen expression. Multiple antibodies are used for differential diagnosis of poorly differentiated malignant tumors. Consequently, multiple tissue slides must be prepared for IHC, even though an automated immunostaining system is available. Furthermore, to compare immunoreactivities of different antigens in a single case, pathologists must change slides repeatedly, which can be tedious. Therefore, IHC examination for multiple antigens on a single specimen can be cumbersome, time-consuming, and costly. Development of a method that enables immunostaining of a tissue section with a panel of antibodies for a single slide would be greatly advantageous.

Tissue microarray (Kononen et al. 1998) and cell array (Oode et al. 2000) systems, which are high-throughput technologies, allow comprehensive assessment of expression of a single antigen across many tissues and cells, respectively, in a single experiment. However, pathologists want to survey expression of multiple antigens for a tumor at one time rather than the expression status of a single antigen for many tumors. Here, we report development of a novel high-throughput technology that allows IHC examination of a tissue section with 50 different antibodies in a single experiment.

Materials and Methods

Multiplex-immunostain Chip (MI chip)

MI chips, which are the same size as glass slides, are fabricated from 5-mm-thick silicon rubber. Each chip contains 50 (5 × 10) wells that are 2 mm in diameter and 3 mm in depth (Figure 1A). The wells are arranged at 1-mm intervals and contain 4 μl of optimally diluted antibody solution. When each well is allotted different antibodies, the device allows simultaneous examination of 50 different antigens on a single tissue section.

Tissue Sections

The MI chip technology was applied to 4-μm-thick tissue sections of formalin-fixed, paraffin-embedded diffuse B-cell lymphoma and advanced gastric adenocarcinoma.

Cultured Cells

PC-14 cells, a human lung adenocarcinoma cell line (purchased from Riken Cell Bank; Tsukuba, Japan), were grown on a glass slide in Dulbecco's modified minimum essential Eagle's medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% fetal calf serum (FBS) in an incubator containing 5% CO₂ at 37C. The slides were rinsed in PBS, dried well, and then fixed in 70% ethanol at room temperature (RT) for IHC.

Figure 1

Multiplex-immunostain chip. (A) A specially designed chip made of silicon rubber in which 50 2-mm-diameter wells are arranged at intervals of 1 mm. Each well (3 mm in depth) contains 4 μl of antibody. This chip enables study of the expression of 50 different antigens with a single tissue section. (B) A tissue section is placed on the chip with a wide antibody panel, and the chip and slide are tightly fastened with a specially designed clamp. (C) The slide and MI chip are inverted, allowing antibodies to contact the tissue section. (D) With the MI chip, 50 different antibodies can be applied simultaneously to a single section. (E-G) Device for clamping the MI chip and glass slide together. The clamp prevents the antibody solution from spreading to the surrounding wells. Upper (E) and lateral (G) views of the device. (F) A set of the MI chip and glass slide is put into the device but is not still clamped. In this figure, the glass slide used is printed with circles and figures for identifying each antibody at microscopic observation.

Immunohistochemistry

Each deparaffinized tissue section was treated with 0.3% H₂O₂ in 100% methanol to inhibit endogenous peroxidase activity and then incubated with normal goat serum for 20 min at RT. In the first experiment, one well in the center of the MI chip contained normal mouse serum; all remaining wells contained the antibody (4 μl) against leukocyte common antigen (CD45; DakoCytomation, Glostrup, Denmark) (Figure 2A). In brief, the slide was placed on the silicon rubber plate containing the serum and antibody and fastened tightly with the specially designed clamp (Figures 1E-1G). The slide and MI chip were then inverted (Figures 1B and 1C), allowing the antibody to contact the specimen, and incubated for 1 hr at RT. The section was incubated with universal immunoperoxidase (or alkaline phosphatase) polymer, anti-mouse and anti-rabbit (Histofine Simplestain Max PO or Histofine Simplestain Max AP; Nichirei, Tokyo, Japan) for 30 min at RT. Immunoreactivity was visualized with 3–3′-diaminobenzidine (DAB; Nichirei) and Fast Red (Nichirei) for peroxidase and alkaline phosphatase, respectively. Finally, sections were counterstained with Mayer's hematoxylin for nuclei. In the other experiments, two different antibody panels were applied to sections of malignant tumors (Tables 1–3). The IHC procedure was performed as described above.

Figure 2

(A) IHC staining of CD45 in malignant lymphoma (B-cell type) with the MI chip. Brown staining indicates CD45 immunoreactivity. A center well contains normal mouse serum, whereas all remaining 49 wells contain antibody against CD45. Each well contains 4 μl of antibody. (B) The center of the tissue, a dotted line circle, is devoid of a brown spot, corresponding to the well without the antibody. (C) Microscopically, lymphoid cells are stained with the antibody but other components are negative for the antibody. Nuclei are visible by hematoxylin staining. Original magnification ×100.

Panels of Antibodies

Three different antibody panels were used to assess the usefulness of the MI chip. The first panel contained a single antibody against CD45 (DAKO), as mentioned above. This panel was applied to a section of B-cell lymphoma. The second panel consisted of nine different antibodies against lymphocyte markers, including CD45 (DAKO), CD20 (DAKO), CD45RO (DAKO), TdT (Novocastra; Newcastle upon Tyne, UK), CD22 (Novocastra), CD5 (Novocastra), CD10 (Novocastra), CyD1 (Novocastra), and CD3 (DAKO), and was also applied to a section of B-cell lymphoma for subclassification of the lymphoma (Tables 1 and 2). The third panel contained 11 different antibodies for detection of epithelial membrane antigen (EMA; DAKO), cytokeratin (AE1/3; DAKO), cytokeratin (MNF116; DAKO), vimentin (Nichirei), desmin (Nichirei), α-smooth muscle actin (α-SMA; Novocastra), CD45 (DAKO), neuron-specific enolase (NSE; Novocastra), S-100 (DAKO), HMB-45 (Enzo; Farmingdale, NY), and CD34 (Nichirei) (Tables 1 and 3). This panel was used to stain the gastric adenocarcinoma section and the cultured cell preparation.

Table 1

Antigens examined in this study a

Marker (antigen)	Clone	Source
CD45	2B11	DAKO
CD20	L26	DAKO
CD45RO	UCHL-1	DAKO
TdT	SEN28	Novocastra
CD22	FPC1	Novocastra
CD5	4C7	Novocastra
CD10	56C6	Novocastra
CyD1	P2D11F11	Novocastra
CD3	PC3/188A	DAKO
EMA	E29	DAKO
Cytokeratin	AE1/3	DAKO
Cytokeratin	MNF116	DAKO
α-SMA	αSm-1	Novocastra
Desmin	ZC18	Nichirei
Vimentin	V9	Nichirei
NSE	5E2	Novocastra
S100	Polyclonal, rabbit	DAKO
CD34	NU-4A1	Nichirei
HMB45	HMB-45	Enzo

^aEMA, epithelial membrane antigen; NSE, neuron specific enolase; α-SMA, α-smooth muscle actin.

Antigen Retrieval

Microwave exposure was done before IHC. The MI chip technology was also applied to sections of gastric adenocarcinoma that were heated at 100C for 20 min in 10 mM sodium citrate buffer (pH 6.0) in a plastic pressure cooker with a microwave oven (650 W). This method is generally effective for antigen retrieval for the antibodies used in this study (data not shown). Immunohistochemical staining of the sections exposed to microwaving was performed as described for the sections without microwave exposure.

Results

Immunostaining of Tissue Sections with an Antibody Against CD45

There were 49 macroscopically visible 2-mm brown spots corresponding to the wells containing the anti-CD45 antibody on the tissue section (Figure 2A). No brown DAB staining was visible on tissue surrounding the wells (Figures 2B and 2C). Microscopically, immunoreactivity was present in both normal and neoplastic lymphoid cells (Figure 2C). No other tissue components displayed immunoreactivity. The centrally located spot corresponding to the well without the antibody to CD45 and the surrounding tissue were indistinguishable (Figure 2B). Application of the MI chip did not affect specimen morphology.

Immunostaining of Tissue Sections with Panels of Different Antibodies

The lymphoma tissue section stained with the panel of nine different antibodies showed distinct spots, including lymphoma cells reactive with antibodies against CD45 and CD20. In contrast, spots corresponding to other antibodies were light in color macroscopically (Figure 3A). Microscopically, neoplastic lymphoid cells were positive for CD45 and CD20 but negative for other antibodies (Figures 3B and 3C). Antibodies other than CD45 and CD20 reacted with infiltrating non-neoplastic lymphocytes but did not react with tumor cells (Figures 3D and 3E). These IHC findings led to a diagnosis of B-cell lymphoma for this case.

Sections from a gastric adenocarcinoma specimen stained with 11 different antibodies showed spots containing cancer cells reactive with antibodies against EMA, AE1/3, and MNF116, all of which are epithelial markers (Figure 4A). In contrast, immunoreactivities for α-SMA, vimentin, desmin, and NSE were detected in original stromal tissue elements but not in cancer cells. CD45 was expressed exclusively by infiltrating leukocytes.

Influence of Antigen Retrieval

Antigen retrieval had no adverse effect on IHC. However, the tissue occasionally detached from the slide at areas in direct contact with the silicon rubber plate (data not shown).

Application to Cultured Cells

We applied the MI chip to cultured PC14 lung cancer cells grown on glass slides. The panel of antibodies applied to the cultured cells was the same as that used for analysis of gastric adenocarcinoma (Table 2). PC14 cells showed immunoreactivity only with the epithelial markers EMA, AE1/3, and MNF116 (Figures 5A and 5B).

Table 2

Layout of antibodies (to antigens) on MI Chip for lymphoma a

	1	2	3	4	5	6	7	8	9	10
A	CD45	CD20	CD45RO	TdT	CD22	CD5	CD10	CyD1	CD3
B	CD3	CD45	CD20	CD45RO	TdT	CD22	CD5	CD10	CyD1
C	CyD1	CD3	CD45	CD20	CD45RO	TdT	CD22	CD5	CD10
D	CD10	CyD1	CD3	CD45	CD20	CD45RO	TdT	CD22	CD5
E	CD5	CD10	CyD1	CD3	CD45	CD20	CD45RO	TdT	CD22

^aCyD1, cyclin D1; CDs, lymphocyte surface markers; TdT, terminal deoxynucleotidyl transferase.

Table 3

Layout of antibodies (to antigens) on MI Chip for gastric adenocarcinoma and PC14 cells a

	1	2	3	4	5	6	7	8	9	10
A	EMA	AE1/3	MNF	α-SMA	Des	Vim	NSE	S100	CD45	CD34
B	HMB	EMA	AE1/3	MNF	α-SMA	Des	Vim	NSE	S100	CD45
C	CD34	HMB	EMA	AE1/3	MNF	α-SMA	Des	Vim	NSE	S100
D	CD45	CD34	HMB	EMA	AE1/3	MNF	α-SMA	Des	Vim	NSE
E	S100	CD45	CD34	HMB	EMA	AE1/3	MNF	α-SMA	Des	Vim

^aEMA, epithelial membrane antigen; AE1/3, cytokeratin AE1/3; α-SMA, α-smooth muscle actin; NSE, neuron-specific enolase; Des, desmin; Vim, vimentin.

Discussion

MI chip technology is ideal for rapid and efficient tumor classification. With a single chip, a tissue specimen can be tested simultaneously for immunoreactivity with 50 different antibodies. Although the MI chip technology appears similar to tissue microarray (Kononen et al. 1998) and cell array (Oode et al. 2000) systems, it is very different. MI chip technology permits analysis of expression of as many as 50 antigens by a single tissue, whereas the other technologies permit analysis of a single antigen across a number of different tissues. Therefore, the MI chip is useful for the histopathological differential diagnosis of tumors that is done routinely in a typical pathology department. Cell arrays, tissue arrays, and MI chips can be used together for detailed analysis of antigen expression.

Both patient prognoses and treatment paradigms for different types of cancer depend on identification of specific antigens (Baars et al. 1999; Jones and Dorfman 2001; Chi 2002; Li 2002). Therefore, histopathological diagnosis is a prerequisite for optimal treatment of cancer patients. However, it is not always easy for trained pathologists to diagnose poorly differentiated tumors precisely in conventional tissue sections (Singer et al. 2000). Pathologists occasionally encounter tumors in which differentiation between carcinoma and sarcoma is difficult. The MI chip permits screening of such tissue specimens by IHC with multiple diagnostically relevant antibodies, leading to a more precise diagnosis. For example, the wells in a single MI chip can contain antibodies against both epithelial and non-epithelial markers for differentiation between carcinoma and sarcoma. Another panel of antibodies can be prepared for typing hematopoietic tumors.

Whether or not MI chip technology can adequately address heterogeneity in levels of antigen expression in tumors may be of concern because the area of specimen treated with antibody is small in comparison to that with conventional IHC methods. With the MI chip, one spot covers 3.1 mm², which is large enough to evaluate the immunoreactivity of tumor cells. The specimen area in tissue microarray analysis is smaller than that in MI chip analysis. When two or three spots for the same antibody are distributed randomly on a chip, the problem of intratumor variation in immunoreactivity is reduced. In the clinical setting, pathologists make histological diagnoses from small biopsy specimens as well as large ones. With small tissue specimens, it is recommended to mount several sections from the same specimen on the same glass slide to approximate the size of a large section.

As shown in the present study, MI chip technology is an efficient and effective tool for evaluating expression of multiple antigens and is valuable for both routine histopathological examination and basic biological research.

Figure 3

IHC staining of a tissue section from malignant lymphoma (B-cell type) with a panel of nine different antibodies in the MI chip (Table 2). Every five wells are allotted the same antibody. (A) Circles corresponding to antibodies against CD45 and CD20 are distinct, whereas circles corresponding to other antibodies are faint or indiscernible. (B-E) Microscopically, neoplastic lymphoid cells are positive for antibodies to CD45 (B) and CD20 (C) and negative for other antibodies (D,E). In contrast, antigens other than CD45 and CD20 are detected in infiltrating non-neoplastic lymphocytes but are not expressed in tumor cells (D,E). Section counterstained with Mayer's hematoxylin for nuclei.

Figure 4

Immunostaining of a section of advanced gastric adenocarcinoma with MI chip containing 11 different antibodies and normal serum in quintuplicate (Tables 1 and 3). (A) Immunostained section. Cancer cells express epithelial markers EMA, AE1/3, and MNF116. Vimentin, α-SMA, desmin, NSE, and CD34 are expressed in stromal tissue elements but are not expressed in cancer cells. CD45 is detected exclusively in infiltrating leukocytes. (B) HE-stained tissue section.

Figure 5

Immunostaining of cultured cell line PC14 with 11 different antibodies against EMA, AE1/3, MNF116, vimentin, desmin, α-SMA, CD45, NSE, S-100, CD34, and HMB35. (A) PC14 expresses EMA, AE1/3, MNF116, and vimentin and does not express non-epithelial markers such as CD45. Immunoreactivity was visualized with Fast Red for alkaline phosphatase. Original magnification ×200. (B) Gross appearance of an immunostained slide glass with cultured cells.