What Is the Difference Between IHC and ICC?
The principle of immunohistochemistry (IHC) has been known since the 1930s, but the first IHC research results were not officially published until 1942. This is regarded as a milestone in immunofluorescence technology. Albert Coons from Harvard Medical School published an article in the Journal of Immunology, using FITC-labeled antibodies to identify pneumococcal antigens in infected tissues. Since then, tissue fixation methods, detection labels, and microscopes have continuously improved, making IHC an essential tool in diagnosis and research.
In pathology, using specific tumor markers, doctors diagnose whether a tumor is benign or malignant through IHC, determine the tumor’s stage and grade, and identify cell types and the source of metastasis to locate the primary tumor. Similarly, IHC can also be used in drug development to assess drug efficacy by detecting the upregulation or downregulation of disease targets.
Immunohistochemistry refers to the detection of antigens (such as proteins) in tissue sections based on the principle of the specific binding of antibodies to antigens. The root of “immuno” comes from the antibodies used in the process, while “histo” means tissue. Another similar technique is immunocytochemistry (ICC). These two terms are often mixed up; they seem similar but do have some differences.
For IHC, the tissue comes from patients or animals and is frozen or embedded in paraffin. These tissues are sliced into approximately 4μm thick sections, processed after mounting. This method allows researchers to observe the localization of cellular components while maintaining the original structure of surrounding tissues.
For ICC, most extracellular matrix and other matrix components are removed, leaving whole cells for staining. The sources for ICC can be cell suspensions from patients or animals (such as blood smears, swabs, etc.) or cell lines cultured in the laboratory.
Apart from biological sources, IHC and ICC also differ in the extent of sample processing. ICC requires permeabilization, either through a fixation process or a separate permeabilization step, so that antibodies can bind to intracellular targets. IHC may not require a separate permeabilization step, depending on the thickness of the sections and the fixation method. IHC sections embedded in paraffin must be further processed before antibody staining. Once the samples are prepared, the staining procedures for IHC and ICC are almost the same. Of course, optimizing according to the antibodies used is still necessary.
Immunohistochemical Drug Resistance Prognostic Markers for Tumor Cells
Malignant tumor immunohistochemical drug resistance prognostic markers, complete set of 4 items:P-gp, GSTπ, TOPOⅡ, Ki-67.
Breast cancer immunohistochemical drug resistance prognostic markers, complete set of 7 items:P-gp, GSTπ, TOPOⅡ, Ki-67, ER, PR, C-erbB-2.
1、P-glycoprotein (P-gp)–Drug pump action–Cell membrane/cytoplasm–The higher the positive rate, the stronger the resistance to the following drugs:Doxorubicin, Epirubicin, Mitoxantrone, Vincristine, Vinblastine, Taxotere.
2、Glutathione S-transferase (GST π)–Detoxification action–Cytoplasm–The higher the positive rate, the stronger the resistance to the following drugs:Doxorubicin, Cisplatin, Nitrogen mustard, Cyclophosphamide, Tumor necrosis factor.
3、Topoisomerase II (TOPOⅡ)–Target action–Nucleus–The higher the positive rate, the more effective against the following drugs:Anthracycline antibiotics and podophyllotoxin, such as VP-16, Teniposide, Doxorubicin, Epirubicin, and VM26. Those with high positive rates are especially effective against VP-16.
4、Estrogen Receptor (ER)–Sex hormone action–Nucleus–The higher the positive rate, the more effective the tumor is against endocrine therapy, and the better the prognosis.
5、Progesterone Receptor (PR)–Sex hormone action–Nucleus–The higher the positive rate, the more effective the tumor is against endocrine therapy, and the better the prognosis.
6、C-erbB-2–Oncogene product–Cytoplasm–The higher the positive rate, the higher the malignancy of the tumor.ER, PR positive and C-erbB-2 also positive patients have poor treatment outcomes with Tamoxifen.
7、Ki-67–Cell proliferation marker–Nucleus–The higher the positive rate, the faster the tumor proliferation, the higher the malignancy.
Ki-67 is a marker of cell proliferation, expressed during the cell cycle G1, S, G2, M phases, and absent in G0 phase. It is closely related to the degree of tumor differentiation, infiltration, metastasis, and prognosis.
8、PCNA (Proliferating Cell Nuclear Antigen).
9、CEA Most adenocarcinomas express CEA.
10、Rb (Retinoblastoma) gene is a tumor suppressor gene that regulates the cell cycle.
11、P53 In immunohistochemistry, it is always mutated, and the higher the positive rate, the worse the prognosis.The wild type has a very short half-life.
12、Nm23 is a metastasis-suppressing gene, and its positive expression is negatively correlated with tumor metastasis.It has been widely applied in the detection of various malignant tumors such as breast cancer, non-small cell lung cancer, gastric cancer, colorectal cancer, liver cancer, and laryngeal cancer. Almost all studies have shown that patients with high nm23 protein expression have a relatively low rate of lymph node metastasis and a longer survival period.
13、E-CaE Cadherin, a transmembrane glycoprotein mediating intercellular adhesion, its loss of function causes destruction of connections between cells, mainly used in studies of tumor invasion and metastasis.
14、PS2 Estrogen-regulated protein, its expression is related to ER expression, can be used as an indicator for endocrine therapy and prognosis.
15、CK18 Low molecular weight keratin, mainly marks various simple epithelium including glandular epithelium, while stratified squamous epithelium is often negative, mainly used for adenocarcinoma diagnosis.
16、CK19 Distributed in simple epithelium and mesothelium, often used for adenocarcinoma diagnosis, hepatocytes do not express, while bile duct shows positive reaction.
17、Heppar 1 Hepatocyte antigen, positive in normal hepatocytes and well-differentiated hepatocellular carcinoma, weakly positive or negative in poorly differentiated hepatocellular carcinoma.
18、CK20 Used for diagnosing gastrointestinal adenocarcinomas, ovarian mucinous tumors, and skin Merkel cell carcinoma.Squamous carcinoma, breast cancer, lung cancer, endometrial and non-mucinous ovarian tumors are often negative.
19、CK7 Ovarian, lung, and breast epithelium are often positive, while colon, prostate, and gastrointestinal epithelium are negative.
20、Villin Villin, in normal tissues, is usually expressed only in cells with brush borders, such as gastrointestinal epithelial cells, pancreatic and bile duct epithelial cells, and renal parenchyma epithelial cells (especially proximal tubules).
Villin has a high expression rate in gastrointestinal cancer, pancreatic cancer, gallbladder cancer, and bile duct cancer tissues. If a tumor with a significant glandular structure shows no villin expression, the possibility of this tumor being of gastrointestinal, pancreatic, gallbladder, or bile duct origin is extremely low.
Breast cancer often becomes a disease that needs to be ruled out in female patients with unknown primary site metastatic cancer. If significant villin immunohistochemical positive staining is observed in metastatic cancer tissues, this tumor is very unlikely to be of breast origin.
Other tumors that usually show negative villin immunohistochemical staining include: serous ovarian cancer, transitional cell carcinoma of the urethra, and prostate cancer. Mesothelioma also often shows negative villin expression; therefore, in some cases, villin can also be used as an antibody to differentiate between mesothelioma and adenocarcinoma.
However, some non-gastrointestinal tumors may express villin, such as endometrioid adenocarcinoma, mucinous ovarian cancer, renal cell carcinoma, and some lung cancers. Some experts have reported villin expression in certain cases of cervical endometrial adenocarcinoma.
Diagnosis of liver cancer: Villin immunohistochemical staining can display capillary bile duct structures, thus it may also be very useful in expressing tubular structures in some liver cancers. Polyclonal CEA is the first reagent used for this purpose, and CD10 (CALLA) is also very useful in expressing this structure in liver cancer. The expression of polyclonal CEA, villin, and CD10 (CALLA) in liver cancer cases does not conflict with each other, so if liver cancer is suspected, it is recommended to use these three antibodies together to assist in diagnosing difficult cases.
Villin is also very helpful in the study of neuroendocrine tumors. It is well known that carcinoids and pancreatic islet cell tumors have similar morphological characteristics, and it is almost impossible to distinguish these two tumors morphologically. Villin is particularly useful in this case, as literature reports that 85% of gastrointestinal carcinoid cases express villin, while no positive expression has been reported in islet cell tumors. Villin expression in carcinoids is usually positive on the cell membrane.
Moreover, there is some evidence that villin expression rates are higher in small cell carcinomas of the stomach and lower gastrointestinal tract than in small cell carcinomas in other sites, such as the lung, esophagus, bladder, or prostate. According to literature, about 40% of lung carcinoid cases are villin positive, and there is also villin expression in other neuroendocrine tumors, such as medullary thyroid carcinoma and a few Merkel cell carcinomas.
21、MRP1 Multidrug resistance-associated protein 1, affects chemotherapy sensitivity, and is related to prognosis.
22、MDR Multidrug resistance gene
23、TS Thymidylate synthase, an important target of 5-FU; if it is highly expressed, with positive reaction ++ or above, it indicates that tumor cells are resistant to 5-FU.
24、Syn Synaptophysin, a neuro tissue marker
25、S-100 A neuro tissue marker, present in nervous tissue, pituitary, carotid body, adrenal medulla, salivary glands, and some mesenchymal tissues, often used for diagnosing neurofibromas, malignant melanomas, liposarcomas, and chondrosarcomas.
26、NSE Mainly used for diagnosing neuroendocrine tumors
27、Chr Chromogranin, highly concentrated in adrenal medulla, used to differentiate between adrenal medulla and cortex, for diagnosing neuroendocrine tumors.
28、CKH High molecular weight keratin, mainly marks squamous cell tumors
29、CKL Low molecular weight keratin, mainly marks simple epithelium, glandular epithelium
30、EMA Epithelial membrane antigen, glycoprotein, widely distributed in various epithelia and their tumors
31、Vim Vimentin, a mesenchymal tissue marker
32、P504 The sensitivity of detecting prostate cancer with methyl-CoA racemase is 97%, and specificity is 100%.
33、AMACR The advantage is that it is cancer-specific, only present in cancer tissues.Rubin stated that AMACR can also be used as a diagnostic marker for other cancers. After examining various cancer cells, it was found that colorectal cancer, ovarian cancer, breast cancer, bladder cancer, lung cancer, lymphoma, and melanoma all overexpress AMACR, with the highest expression in colorectal and prostate cancers.
34、CD117 Gastrointestinal stromal tumor
35、CD10 As a common acute lymphoblastic leukemia antigen, mainly expressed in immature lymphocytes, it has application value in the diagnosis of hematological diseases such as Burkitt lymphoma and chronic myeloid leukemia. In recent years, this antigen has been found to express in certain tumors outside the hematological system, such as endometrial stromal sarcoma and malignant melanoma. The antibody has certain reference value in diagnosing and differentiating renal cell carcinoma.
36、CD15 A cell adhesion molecule, due to its good marking effect on Reed-Sternberg cells in Hodgkin lymphoma (HD), is considered an important marker of HD.In addition to the differential diagnosis of HD, studies on CD15 expression in tumors such as gastric cancer, colorectal cancer, thyroid cancer, and breast cancer have found that CD15 expression increases significantly with the degree of cancer cell differentiation, lymph node metastasis, and clinical staging.
Immunoelectron microscopy shows that CD15 antigen is mainly distributed in the boundary membrane, endoplasmic reticulum, Golgi apparatus, and near the nuclear membrane of colon cancer cells; CD15 may affect and participate in tumor formation and metastasis through changes in the conformation of the bound substrate.
37、SMA Smooth muscle actin, marks smooth muscle
38、CD56 A neural cell adhesion molecule, mainly distributed in most neuroectoderm-derived cells, often used for diagnosing astrocytomas, neuroblastomas, neuroendocrine tumors, and is also an important marker for NK cell tumors, marking small cell lung cancer.
39、Des Desmin, widely distributed in smooth muscle, cardiac muscle, skeletal muscle cells, and myoepithelial cells, highly differentiated and expressed, low differentiation and low expression.
40、MSA Muscle-specific actin, widely distributed in almost all muscle-type cells.
41、CD68 Present in macrophages of bone marrow and various nervous tissues, used for diagnosing granulocyte leukemia, various monocyte-derived tumors, including malignant fibrous histiocytoma (first choice).
42、CD34 Expressed in early lymphoid hematopoietic stem cells, progenitor cells, endothelial cells, embryonic fibroblasts, and some neural tissue cells, mainly used to mark vascular endothelial cells, diagnosis of vascular tumors, GIST 80-90%.
43、CD31 Also marks vascular endothelium.
44、CD44 A widely distributed transmembrane glycoprotein molecule, divided into CD44s and CD44v.CD44s mainly serves as a receptor for hyaluronic acid, and its binding with hyaluronic acid affects tumor growth and metastasis. CD44v is mainly expressed in metastatic tumor cells.
Li Daoming et al. used the immunohistochemical LSAB method to detect CD44v expression in 42 cases of esophageal squamous cell carcinoma, finding that the positive expression rate in the lymph node metastasis group was 76.19% (16/21), while the non-metastasis group had a positive rate of 42.86% (9/12), showing significant differences between the two groups.
Cancer cells around the cancer nest, interstitial infiltrating cancer cells, mitotic cancer cells, and cancer cells in emboli, as well as cancer cells infiltrating the vascular wall, all show strong positive expression. Zhang Chengwu et al. detected CD44v6 expression in 20 cases of normal gastric mucosa, 43 cases of atypical hyperplasia, and 85 cases of gastric cancer, finding no expression in normal gastric mucosa, while the positive rates in atypical hyperplasia and gastric cancer tissues were 30.2% and 74.1%, respectively. Its expression intensity is closely related to the depth of gastric cancer infiltration, lymph node metastasis, tumor growth pattern, venous and lymphatic invasion, and distant metastasis. The above results indicate that high expression of CD44v constitutes the aggressiveness and ease of metastasis of tumor cells.
45、NESTIN Rich in neural stem cells
46、Ost Osteocalcin, secreted by osteoblasts.
47、AAT Anti-trypsin, fibrous tissue-derived tumors
48、ACT Anti-chymotrypsin
49、GFAP Glial fibrillary acidic protein, a neural tissue marker, often used for diagnosing astrocytomas
50、Tg Thyroglobulin, positive in thyroid cancer.
51、CT Calcitonin, positive in medullary thyroid carcinoma.
52、PH Parathyroid hormone, positive in parathyroid tumors
53、N-myc Enhanced expression in small cell lung cancer and neuroblastoma leads to lack of response to chemotherapy and rapid progression;
54、bcl-2 The mechanism of drug resistance is anti-apoptotic action; those with high expression are resistant to most anticancer drugs/radiation therapy.
55、TGA72 Tumor-associated antigen 72, expressed in various malignant epithelial tumors, especially breast cancer, ovarian cancer, and colorectal cancer.Normal epithelial cells, sarcomas, and hematopoietic system tumors are usually TGA72 negative. TGA72 antibodies are used more in breast cancer research; their high expression is usually associated with larger tumor size, poor differentiation of metastatic tumor cells, and high proliferative activity.
56、GA733 Tumor-associated antigen, encoding epithelial glycoprotein 40, is an epithelial cell adhesion molecule (EP-CAM), playing an important role in the growth and differentiation of epithelial cells.Many tumors can express GA733, especially breast cancer, colorectal cancer, and lung cancer.
Kubuschok et al. used GA733 to detect hidden micro-metastases in lymph nodes removed from non-small cell lung cancer surgery, finding that the detection of hidden foci is an independent prognostic factor for overall survival. The GA733 expression form in colorectal cancer is related to tumor prognosis; expression on the cell membrane and cytoplasm is worse than that on the basement membrane side.
57、TTF-1 Thyroid transcription factor-1, expressed in thyroid gland epithelial cells and lung epithelial cells.In lung tumor studies, it has been found that most small cell lung cancers, primary and metastatic lung adenocarcinomas, a small portion of undifferentiated large cell lung cancers, and most atypical neuroendocrine tumors show positive results for TTF-1, while lung squamous cell carcinoma and most typical carcinoids are negative for TTF-1. Thus, it is believed that TTF-1 can be used to differentiate between lung adenocarcinoma and squamous cell carcinoma and assist in distinguishing it from metastatic adenocarcinoma of the lung.
TTF-1 Expression in Thyroid and Its Tumors
TTF-1 is mainly expressed in thyroid follicular cells and the principal cells of the parathyroid gland. TTF-1 is the basic substance for the differentiation of the thyroid and the secretion of thyroglobulin; it can promote the transport of thyroid peroxidase, iodine/sodium, and is related to the activity of serum TSH, with active TSH-R enhancing TTF-1 expression.
TTF-1 expression differs between benign and malignant thyroid tissues. Normal thyroid and benign adenomas show more expression, while expression is less in papillary and follicular thyroid cancers, and absent in undifferentiated cancers.
TTF-1 expression intensity in malignant thyroid lesions increases with age, and the longer the duration of the tumor, the higher the recurrence rate.
TTF-1 Expression in Lung Cancer
75% of lung non-small cell lung cancers (NSCLCs) show positive expression, with adenocarcinomas (ACs) significantly higher than squamous cell carcinomas (SCCs), and over 90% of primary small cell lung cancers (SCLCs) express positively. The intensity of TTF-1 positive expression in NSCLCs is negatively correlated with patient prognosis, serving as an independent prognostic indicator.
Typical carcinoids of the lung (TCS) are all negative, indicating that small cell lung cancer and non-small cell lung cancer may have a theory of a common origin different from TCS.
Source: Medical Student Academic (MSA)
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