A 65-year-old man with a 40-pack-year smoking history presents with hemoptysis and a persistent cough. Chest CT shows a 3.5 cm centrally located mass in the right main bronchus. Positron emission tomography confirms a malignant nodule. Bronchoscopy with transbronchial biopsy is performed and a specimen sample of the nodule is sent for frozen section analysis. The tissue sample is most likely to show which of the following tumor types?

Metastasis of colorectal cancer

During an experiment, the immunophenotypes of different cells in a sample are determined. The cells are labeled with fluorescent antibodies specific to surface proteins, and a laser is then focused on the samples. The intensity of fluorescence created by the laser beam is then plotted on a scatter plot. The result shows most of the cells in the sample to be positive for CD8 surface protein. Which of the following cell types is most likely represented in this sample?

Activated regulatory T lymphocytes

A 43-year-old woman presents to your clinic for the evaluation of an abnormal skin lesion on her forearm. The patient is worried because her mother passed away from melanoma. You believe that the lesion warrants biopsy for further evaluation for possible melanoma. Your patient is concerned about her risk for malignant disease. What is the most important prognostic factor of melanoma?

Depth of invasion of atypical cells

Level of irregularity of the borders

An investigator is processing a blood sample from a human subject. A reagent is added to the sample and the solution is heated to break the hydrogen bonds between complementary base pairs. This solution is then cooled to allow artificial DNA primers in the solution to attach to the separated strands of the sample DNA molecules. An enzyme derived from the thermophilic bacterium Thermus aquaticus is added and the solution is reheated. These steps are repeated multiple times until the aim of the test is achieved. The investigator most likely used which of the following laboratory procedures on the test sample?

Fluorescence in-situ hybridization

A group of scientists is studying the mechanism by which the human papillomavirus (HPV) vaccine confers immunity. They observe that during inoculation of test subjects, certain viral proteins are taken up by the organism's antigen-presenting cells (APCs) and presented on major histocompatibility complex (MHC) class I molecules. Which of the following is the correct term for the process that the scientists are observing in this inoculation?

Endogenous antigen presentation

A 72-year-old man goes to his primary care provider for a checkup after some blood work showed lymphocytosis 3 months ago. He says he has been feeling a bit more tired lately but doesn’t complain of any other symptoms. Past medical history is significant for hypertension and hyperlipidemia. He takes lisinopril, hydrochlorothiazide, and atorvastatin. Additionally, his right hip was replaced three years ago due to osteoarthritis. Family history is noncontributory. He drinks socially and does not smoke. Today, he has a heart rate of 95/min, respiratory rate of 17/min, blood pressure of 135/85 mm Hg, and temperature of 36.8°C (98.2°F). On physical exam, he looks well. His heartbeat has a regular rate and rhythm and lungs that are clear to auscultation bilaterally. Additionally, he has mild lymphadenopathy of his cervical lymph nodes. A complete blood count with differential shows the following: Leukocyte count 5,000/mm3 Red blood cell count 3.1 million/mm3 Hemoglobin 11.0 g/dL MCV 95 um3 MCH 29 pg/cell Platelet count 150,000/mm3 Neutrophils 40% Lymphocytes 40% Monocytes 5% A specimen is sent for flow cytometry that shows a population that is CD 5, 19, 20, 23 positive. Which of the following is the most likely diagnosis?

Immune thrombocytopenic purpura

Immunohistochemistry for USMLE Step 3: High-Yield Pathology Lessons

IHC Principles - Staining the Unseen

A technique to visualize specific antigens (proteins) in tissue sections by binding them to highly specific antibodies.
The core principle is an antigen-antibody (Ag-Ab) reaction.
- An enzyme (e.g., peroxidase) is linked to the antibody.
- When a substrate (chromogen) is added, the enzyme creates a colored precipitate at the antigen's site.
This allows for the precise localization and identification of cellular components.

Direct vs. Indirect Immunohistochemistry Methods

⭐ The most common chromogen used is Diaminobenzidine (DAB), which produces a characteristic brown stain, indicating the presence of the target antigen.

IHC Technique - A Staining Story

Antigen Retrieval: Unmasks antigen epitopes hidden by formalin fixation. Methods: Heat-Induced (HIER) or Proteolytic-Induced (PIER).
Blocking: Prevents non-specific background staining from endogenous enzymes or proteins.
Primary Antibody: Monoclonal or polyclonal antibody that binds specifically to the target antigen.
Secondary Antibody: Binds to the primary antibody. Conjugated to an enzyme (e.g., Horseradish Peroxidase - HRP).
Chromogen & Counterstain: Substrate (e.g., DAB) forms a colored precipitate at the antigen site. Hematoxylin counterstain provides context.

⭐ The detection system often uses the high-affinity bond between biotin and avidin/streptavidin to amplify the signal, which dramatically increases the sensitivity of the assay.

Key Markers - The Cellular Lineup

Marker	Cell / Tumor Type	Notes
Cytokeratin	Epithelial cells (Carcinomas)	Distinguishes carcinoma from sarcoma.
Vimentin	Mesenchymal cells (Sarcomas)	Connective tissue, muscle, endothelium.
Desmin	Muscle (Myosarcomas)	Rhabdomyosarcoma, Leiomyosarcoma.
GFAP	Neuroglia (Astrocytoma, Glioblastoma)	Glial Fibrillary Acidic Protein.
Neurofilament	Neurons (Neuroblastoma)	Differentiates from other small round blue cell tumors.
S-100	Neural crest cells	Melanoma, Schwannoma, Langerhans cell histiocytosis.
CD45	All Leukocytes (Lymphoma, Leukemia)	Also known as Leukocyte Common Antigen (LCA).
CD20, CD19	B-cells (B-cell lymphomas)	CD20 is a target for Rituximab.
CD3, CD4, CD8	T-cells (T-cell lymphomas)	Coreceptors for T-cell recognition.

Clinical Use - Decoding the Colors

Diagnosis & Differentiation
- Distinguishes broad tumor types: Cytokeratin (Carcinoma), Vimentin (Sarcoma), LCA (Lymphoma).
- Identifies cell lineage in poorly differentiated neoplasms.
- Pinpoints origin of metastatic tumors (e.g., PSA for prostate, TTF-1 for lung).
Prognosis
- Gauges tumor aggressiveness.
- Ki-67: Measures proliferation rate (↑ Ki-67 = poorer prognosis).
- HER2/neu overexpression indicates aggressive disease.
Predictive Markers (Therapy Guidance)
- Guides targeted treatment selection.
- ER/PR status in breast cancer predicts response to hormone therapy.
- HER2 positivity predicts response to Trastuzumab.

⭐ IHC is crucial for evaluating a "Carcinoma of Unknown Primary" (CUP). A tumor positive for TTF-1 and Napsin-A strongly suggests a lung adenocarcinoma primary, guiding focused treatment.

TTF-1 IHC in lung adenocarcinoma and other tumors

High‑Yield Points - ⚡ Biggest Takeaways

IHC is a cornerstone technique using antibodies to detect specific antigens (proteins) in tissue, primarily for tumor diagnosis and classification.

Cytokeratin is the classic marker for carcinomas (epithelial tumors), while vimentin is characteristic of sarcomas (mesenchymal tumors).

S-100 is a key marker for melanoma, schwannomas, and other neural crest-derived tumors.

CD markers (e.g., CD20 for B-cells, CD3 for T-cells) are essential for subtyping lymphomas and leukemias.

IHC identifies prognostic and predictive markers, such as ER/PR and HER2 in breast cancer, to guide targeted therapy.

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Immunohistochemistry