What Are the Differences Between IHC and ICC?
In the 1930s, the principle of Immunohistochemistry (IHC) was already known, but it wasn’t until 1942 that the first IHC research results were officially published, marking 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 been continuously improved, making immunohistochemistry an essential tool in diagnosis and research.
In the pathology department, using specific tumor markers, doctors diagnose whether tumors are benign or malignant through IHC, determine the stage and grade of tumors, and identify cell types and the source of metastasis to locate the primary tumor. Similarly, IHC can also be used in drug development by detecting the upregulation or downregulation of disease targets to assess drug efficacy.
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 operation, while histo means tissue. Another similar technique is Immunocytochemistry (ICC).
These two terms are often confused; they seem similar, but there are some differences.
For IHC, the tissue comes from patients or animals and is frozen or embedded in paraffin. These tissues are made into sections about 4μm thick, processed after mounting. This method allows researchers to observe the localization of cellular components while maintaining the original structure of the surrounding tissues.
For ICC, most extracellular matrix and other matrix components are removed, leaving whole cells to stain. The source of ICC can be cell suspensions from patients or animals (such as blood smears, swabs, etc.), or cell lines cultured in the laboratory.
In addition to biological sources, IHC and ICC also differ in the degree of sample processing. ICC requires permeabilization, either through the 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 operations for IHC and ICC are almost indistinguishable. Of course, optimization based on the antibodies used is still necessary for staining.
Immunohistochemical Drug Resistance Prognostic Markers in Tumor Cells
Malignant tumor immunohistochemical drug resistance prognostic markers, a full set of 4 items: P-gp, GSTπ, TOPOⅡ, Ki-67.
Breast cancer immunohistochemical drug resistance prognostic markers, a full 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, Taxol.
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: Anthracyclines and Vinca alkaloids, such as VP-16, Teniposide, Daunorubicin, Epirubicin, Doxorubicin, VM26. A high positive rate is especially effective against VP-16.
4. Estrogen Receptor (ER) — Hormonal action — Nucleus — The higher the positive rate, the more effective the tumor is to endocrine therapy and the better the prognosis.
5. Progesterone Receptor (PR) — Hormonal action — Nucleus — The higher the positive rate, the more effective the tumor is to 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. If ER and PR are positive and C-erbB-2 is also positive, treatment with Tamoxifen is ineffective.
7. Ki-67 — Cell proliferation marker — Nucleus — The higher the positive rate, the faster the tumor proliferation and the higher the malignancy. Ki-67 is a marker for cell proliferation, expressed during the cell cycle phases G1, S, G2, and M, and absent in G0 phase, closely related to the degree of differentiation, infiltration, metastasis, and prognosis of many tumors.
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 generally 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 suppressor gene, and its positive expression is negatively correlated with tumor metastasis. It has been widely used in the detection of various malignant tumors such as breast cancer, non-small cell lung cancer, gastric cancer, colon cancer, liver cancer, and laryngeal cancer. Almost all studies indicate that patients with high expression of nm23 protein have a relatively low lymph node metastasis rate and a relatively longer survival time.
13. E-CaE Cadherin, a transmembrane glycoprotein mediating intercellular adhesion, whose loss of function leads to disruption of connections between cells, mainly used in studies of tumor invasion and metastasis.
14. PS2 Estrogen Regulatory Protein, whose expression is related to ER expression, can serve as one of the indicators for endocrine therapy and prognosis assessment.
15. CK18 Low Molecular Weight Keratin, mainly marks various simple epithelia 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 it while bile duct shows positive reaction.
17. Heppar 1 Hepatocyte Antigen, positive in normal hepatocytes and well-differentiated hepatocellular carcinoma, while poorly differentiated hepatocellular carcinoma often shows weak positivity or negativity.
18. CK20 used for the diagnosis of gastrointestinal adenocarcinoma, 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 Often positive in ovarian, lung, and breast epithelium, negative in colon, prostate, and gastrointestinal epithelium.
20. Villin Brush Border Protein, in normal tissues, villin is usually only expressed in cells with a brush border, such as gastrointestinal epithelial cells, pancreatic and bile duct epithelial cells, and renal parenchyma epithelial cells (especially proximal convoluted tubules).
Villin has a high expression rate in gastrointestinal cancer, pancreatic cancer, gallbladder cancer, and bile duct cancer. If tumors with obvious glandular structures do not express villin, it is very unlikely that the tumor originates from the gastrointestinal tract, pancreas, gallbladder, or bile duct.
Breast cancer often becomes a disease that needs to be differentiated and excluded in female patients with unknown primary metastatic cancer. Because obvious villin immunohistochemical positive staining is observed in metastatic cancer tissues, this tumor is extremely unlikely to be of breast origin.
Other tumors that usually show negative expression for villin immunohistochemical staining include: such as ovarian serous carcinoma, transitional cell carcinoma of the urethra, and prostate cancer. Mesothelioma also often shows negative expression for villin, so in some cases, villin can also be used as an antibody to differentiate mesothelioma from adenocarcinoma.
However, there are some non-gastrointestinal tumors that can express villin, such as endometrial adenocarcinoma, ovarian mucinous carcinoma, renal cell carcinoma, and a small number of lung cancers. Some experts have reported villin expression in some cases of cervical endometrial adenocarcinoma.
Diagnosis of liver cancer: villin immunohistochemical staining can show capillary bile duct structures, so it may also be very useful in expressing tubular structures in some liver cancers. Polyclonal CEA was 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 the diagnosis of difficult cases.
Villin is also very helpful in the study of neuroendocrine tumors. It is well known that carcinoid tumors and pancreatic islet cell tumors have similar morphological characteristics, making it almost impossible to distinguish between these two tumors morphologically. Villin is particularly useful in this case, as literature reports indicate that 85% of gastrointestinal carcinoid cases express villin, while no positive expression has been reported in pancreatic islet cell tumors. The expression of villin in carcinoid tumors is usually positive at the cell membrane.
Additionally, there is some evidence that villin expression in small cell carcinomas of the stomach and lower digestive tract is higher than in small cell carcinomas at other sites, such as lung, esophagus, bladder, or prostate. Literature reports indicate that 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 Protein 1, affects chemotherapy sensitivity and is related to prognosis.
22. MDR Multidrug Resistance Gene
23. TS Thymidylate Synthase, is an important target of 5-FU; if its high expression is positive ++ or above, it indicates that tumor cells are resistant to 5-FU.
24. Syn Synaptophysin Neural tissue marker
25. S-100 Neural tissue marker, present in nerve tissue, pituitary, carotid body, adrenal medulla, salivary glands, and some mesenchymal tissues, often used for the diagnosis of neurofibromas, malignant melanoma, liposarcoma, and chondrosarcoma.
26. NSE Mainly used for the diagnosis of neuroendocrine tumors
27. Chr Chromogranin, highly present in adrenal medulla, differentiates between adrenal medulla and cortex, used for neuroendocrine tumor diagnosis.
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, mesenchymal tissue marker
32. P504 Detection of P504 (P504S) has a sensitivity of 97% and specificity of 100% for diagnosing prostate cancer.
33. AMACR Its advantage is that it is cancer-specific and only exists in cancerous 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, has application value in the diagnosis of hematologic diseases such as Burkitt lymphoma, chronic myeloid leukemia, etc. In recent years, it has been found that this antigen is expressed in certain tumors outside the hematopoietic system, such as endometrial stromal sarcoma, malignant melanoma, etc. The antibody has certain reference value in diagnosing and differentiating renal cell carcinoma.
36. CD15 is a cell adhesion molecule that has good labeling effect on Reed-Sternberg cells in Hodgkin lymphoma (HD) and is considered an important marker for HD. In addition to the differential diagnosis of HD, studies on the expression of CD15 in gastric cancer, colorectal cancer, thyroid cancer, breast cancer, etc. found that CD15 expression significantly increases with the decrease in differentiation degree of cancer cells, increase in lymph node metastasis, and clinical staging. It is believed that CD15 expression is a good indicator for assessing tumor progression, predicting lymph node metastasis, and prognosis.
Immunoelectron microscopy shows that CD15 antigen is mainly distributed in the plasma membrane, endoplasmic reticulum, Golgi apparatus, and near the nuclear membrane of colorectal cancer cells. CD15 may influence and participate in the formation and metastasis of tumors by altering 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 ectoderm-derived cells, commonly used in the diagnosis of 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 highly expressed, low differentiation and low expression.
40. MSA Muscle-specific Actin, widely distributed in almost all muscle-type cells.
41. CD68 Present in macrophages in bone marrow and various neural tissues, used for diagnosing granulocyte leukemia and various monocytic tumors, including malignant fibrous histiocytoma (first choice).
42. CD34 expression in early lymphoid hematopoietic stem cells, progenitor cells, endothelial cells, embryonic fibroblasts, and certain neural tissue cells, mainly used to mark vascular endothelial cells, vascular tumors diagnosis, GIST 80-90%.
43. CD31 Also marks vascular endothelium.
44. CD44 A widely distributed transmembrane glycoprotein molecule, divided into CD44s and CD44v. CD44s mainly acts as a receptor for hyaluronic acid, influencing tumor growth and metastasis after binding with hyaluronic acid. CD44v is mainly expressed in metastatic tumor cells.
Li Daoming et al. used immunohistochemistry LSAB method to detect the expression of CD44v 4/5 in 42 cases of esophageal squamous carcinoma, finding that the positive expression rate in the lymph node metastasis group was 76.19% (16/21), while the positive rate in the non-metastasis group was 42.86% (9/12), showing a significant difference between the two groups.
The cancer cells around the cancer nest, interstitial infiltrating cancer cells, mitotic cancer cells, and cancer cells in thrombi and infiltrating vascular walls all showed strong positive expression. Zhang Chengwu et al. detected the expression of CD44v6 in 20 cases of normal gastric mucosal epithelium, 43 cases of atypical hyperplasia, and 85 cases of gastric cancer, finding that normal gastric mucosa showed no expression, while atypical hyperplasia and gastric cancer tissues had positive rates of 30.2% and 74.1%, respectively, with expression intensity closely related to the depth of gastric cancer infiltration, lymph node metastasis, tumor growth pattern, and invasion of veins and lymphatics as well as distant metastasis. The above results indicate that high expression of CD44v constitutes the aggressiveness and metastatic potential of tumor cells.
45. NESTIN Extremely abundant in neural stem cells
46. Ost Osteocalcin, secreted by osteoblasts.
47. AAT Anti-trypsin Fibroblast-derived tumors
48. ACT Anti-chymotrypsin
49. GFAP Glial Fibrillary Acidic Protein Neural tissue marker, mainly used for diagnosing astrocytomas
50. Tg Thyroglobulin, positive in thyroid cancer Tg.
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 lacks response to chemotherapy and progresses rapidly;
54. bcl-2 The resistance mechanism is anti-apoptotic action, and those with high expression are resistant to most anticancer drugs/radiation therapy.
– THE END –