Immunohistochemistry

Overview

Immunohistochemistry is a lab technique that uses antibodies to visualize specific proteins in thin slices of tissue. Patients often see the term immunohistochemistry and wonder what immunohistochemistry meaning really is — after all, it’s not your everyday blood test. This test is commonly ordered alongside biopsies when doctors need to nail down what type of cells are in a tumor, inflammation site, or any tissue of interest. It reflects how proteins are distributed and expressed within tissues, linking directly to physiology, cell markers, and tissue architecture. Inevitably, folks may feel confused or anxious when they get results mentioning staining intensities, “positive” versus “negative,” or subcellular patterns, since it’s not always black-and-white.

Purpose and Clinical Use

Why is immunohistochemistry ordered? Simply put, immunohistochemistry results help provide clues about what kind of cells are present, whether they’re normal, reactive, or malignant, and sometimes the underlying mechanism driving disease. It’s a key tool for tumor subtyping — think distinguishing breast cancer hormone receptors (ER/PR) or HER2 status, or classifying lymphomas by CD markers. Beyond oncology, immunohistochemistry interpretation can reveal infectious organisms (like viral antigens in a biopsy), track autoimmune processes, or assess tissue-specific differentiation in developmental disorders. Clinicians rely on immunohistochemistry not to make a diagnosis in isolation, but to support other findings, guide further tests, and monitor treatment response. In short, it’s all about adding molecular context to the microscopic picture — valuable screening, diagnostic support, monitoring, and risk assessment data rolled into one.

Test Components and Their Physiological Role

The core of immunohistochemistry (IHC) involves several key reagents and steps that reflect normal physiological processes. Understanding each component helps demystify immunohistochemistry interpretation.

• Antigens (Target Proteins): These are the proteins or peptides in cells that we want to detect—anything from hormone receptors (like estrogen or progesterone) to cell cycle regulators (like Ki-67) to lineage markers (such as cytokeratins for epithelial cells or CD3 for T lymphocytes). These antigens exist normally in tissues, often within specific subcellular compartments (nucleus, cytoplasm, membrane).
• Primary Antibody: Raised in animals (mouse, rabbit, goat), the primary antibody binds directly to the target antigen. It mimics the body’s own immune system recognition; in physiology, this is similar to how B cells produce antibodies against foreign proteins, but here it’s repurposed in vitro.
• Secondary Antibody: This binds to the primary antibody and is conjugated to an enzyme (like horseradish peroxidase) or fluorophore. It amplifies the signal, akin to the body’s complement cascade amplifying immune responses.
• Detection System: The enzyme linked to the secondary antibody converts a colorless substrate into a colored product (chromogen), marking where the antigen lives. This enzymatic reaction is reminiscent of physiological enzyme-substrate specificity (e.g., digestive enzymes). Fluorescent detection works similarly but uses light excitation.
• Chromogen or Fluorophore: The visible dye or fluorescent tag highlights antigen locations. Light microscopy or fluorescence microscopy then captures the pattern — somewhat parallel to how our immune cells “light up” inflamed sites, but studied on slides instead of living tissue.
• Counterstain: Hematoxylin or DAPI, for instance, stains nuclei and provides tissue context—very much like staining cells in histology to reveal general architecture.
• Controls: Positive and negative control tissues ensure the immunohistochemistry test worked properly, similar to how labs run control samples in blood work to validate assay performance.

Together, these elements recreate fundamental immunological principles on a tissue slide, enabling visualization of protein localization, density, and distribution. Each step must be carefully optimized—temperature, pH, antigen retrieval conditions, and antibody dilutions all play roles in detecting true physiological expression rather than artefacts.

Physiological Changes Reflected by the Test

Immunohistochemistry shines because it directly reflects shifts in cellular protein expression linked to physiology and pathology. When a cell upregulates a receptor, enzyme, or structural protein, immunohistochemistry staining intensity often increases. For example, heightened HER2 membrane staining in breast cancer isn’t just a lab finding—it signals a growth factor receptor that’s overexpressed, driving unchecked cell proliferation. On the flip side, decreased staining of hormone receptors like ER may indicate loss of normal differentiation in tumors.

Inflammation can be tracked too: immunohistochemistry for CD68 highlights macrophages in injured tissues, reflecting innate immune activation. A surge in CD3 or CD20 staining points to T- or B-cell infiltration during autoimmune flares. Likewise, Ki-67 labeling indexes the fraction of cells in active cell cycle phases, so a high Ki-67 rate suggests rapid cell turnover—useful in grading tumors or assessing regenerative processes after injury.

Importantly, not all changes equivocate to disease. Reactive hyperplasia in lymph nodes or transient upregulation of repair proteins following injury can produce robust staining patterns that normalize later. Thus, immunohistochemistry results need interpretation within the broader clinical and histological context, acknowledging that protein expression levels can be both adaptive and pathological.

Preparation for the Test

Preparing for immunohistochemistry hinges on proper collection and processing of the tissue specimen. Unlike blood tests, there’s no patient fasting or hydration protocol; instead, focus is on preserving tissue integrity and antigenicity:

• Biopsy or Surgical Resection: Collect tissue promptly after removal. Delay increases post-mortem autolysis and degrades antigens—think of it like letting fruit rot; proteins break down over time.
• Fixation: Immerse specimens in neutral buffered formalin (10%) for between 6 and 48 hours, depending on tissue thickness. Under-fixation can cause patchy staining (“underfix”), while over-fixation masks epitopes.
• Embedding: Dehydrate in graded alcohols, clear in xylene (or substitutes), and embed in paraffin. Any deviation in dehydration times or solvent concentrations may lead to incomplete paraffin infiltration, causing tissue tears or loss during sectioning.
• Sectioning: Cut 3–5 µm sections on a microtome. Too thick, and antibody penetration is poor; too thin, and tissue architecture may be lost—like over-slicing bread.
• Dewaxing and Rehydration: Remove paraffin in xylene and rehydrate through graded alcohols to water. This step is critical; incomplete dewaxing leads to uneven antibody binding.
• Antigen Retrieval: Heat-induced epitope retrieval (HIER) or enzymatic methods unmask antigen sites. pH and temperature must be tailored to each antibody. Skipping retrieval often yields false negatives.
• Blocking: Incubate sections with serum or protein-blocking solution to reduce non-specific binding—think of it as putting on sunscreen to prevent sunburn (non-specific staining).

All these prep steps greatly influence immunohistochemistry results; clinical labs maintain strict protocols to minimize variability and ensure reliable staining patterns.

How the Testing Process Works

Once tissue sections are prepped, the actual immunohistochemistry testing process takes place in a few hours to a day, depending on automation:

• Apply primary antibody and incubate (30–60 minutes at room temperature or as per antibody datasheet).
• Wash off unbound antibody with buffer (phosphate-buffered saline or TBS).
• Add secondary antibody conjugated to enzyme or fluorophore; incubate similarly.
• Wash again, then add chromogen (like DAB) or fluorescent detection reagents.
• Counterstain nuclei, dehydrate, mount with coverslip, and examine under the microscope.

Most patients experience no discomfort beyond the biopsy. Normal short-term reactions are related to the biopsy procedure (mild bruising or soreness). The staining itself is purely lab-based; you won’t feel a thing while your doctor reviews the slide under a microscope.

Reference Ranges, Units, and Common Reporting Standards

Immunohistochemistry reporting doesn’t use mg/dL or mmol/L. Instead, it relies on qualitative and semiquantitative systems. You might see:

• Percentage of Positive Cells: e.g., 10%, 50%, 90% of tumor cells staining for a marker.
• Intensity Scores: Commonly 0 (none), 1+ (weak), 2+ (moderate), 3+ (strong).
• H-Score or IRS: Combines intensity and percent to give a numerical value (0–300), though exact scales vary by lab.
• Pattern Descriptions: Nuclear, cytoplasmic, or membranous staining; focal versus diffuse distribution.

Labs typically list a “reference range” or “cutoff” for what’s considered clinically significant—for example, ER positive if ≥1% nuclear staining. These cutoffs are derived from clinical studies and validated controls. They can differ by institution, antibody clone, and platform, so clinicians always refer to the specific immunohistochemistry results sheet rather than generic charts.

How Test Results Are Interpreted

Interpreting immunohistochemistry results involves integrating staining patterns, intensity, and proportion of positive cells with clinical data. A single “positive” result for a marker like HER2 or PD-L1 can guide targeted therapy decisions, but is seldom a standalone verdict. Pathologists look for:

• Staining Localization: Membranous staining for HER2, nuclear for hormone receptors, cytoplasmic for some enzymes—each pattern suggests different physiology.
• Signal Intensity and Percentage: Higher intensity and wider distribution often correlate with more active or overexpressed targets, but semi-quantitative scoring accounts for borderline cases.
• Comparison to Controls: Internal (non-neoplastic stromal cells) and external control tissues verify assay performance.
• Correlations Over Time: In some diseases, tracking immunohistochemistry markers serially (e.g., Ki-67 in treatment response) helps assess trend rather than one-off value.

Context is king: results are always framed by histological architecture, patient history, and other lab or imaging findings. A pathologist’s report will typically state interpretation remarks such as “strong, diffuse positivity supporting diagnosis of X,” rather than simply “positive.”

Factors That Can Affect Results

Many variables can sway immunohistochemistry outcomes—some biological, others technical:

• Tissue Fixation: Improper formalin concentration, fixation time, or delayed fixation leads to epitope masking or degradation.
• Antibody Specificity and Clone: Different clones may recognize distinct epitopes; cross-reactivity can produce false positives.
• Antigen Retrieval Conditions: pH, buffer composition, temperature, and duration all matter; suboptimal retrieval yields weak or patchy staining.
• Section Thickness: Varying from the recommended 3–5 µm can change background staining or signal strength.
• Batch-to-Batch Reagents: Variability in antibody lots or chromogen solutions can alter staining consistency.
• Operator Technique: Manual staining relies on pipetting accuracy and timing; automated stainers reduce but don’t eliminate operator influence.
• Sample Handling: Freeze-thaw cycles or prolonged storage (paraffin blocks older than several years) can diminish antigenicity.
• Biological Factors: Tumor heterogeneity, necrosis, or poor cellularity may yield misleading results. Inflammatory cells might express markers that complicate interpretation.
• Pre-Analytical Variables: Patient medication, prior therapies (like chemoradiation), or ischemia time prior to fixation can change protein expression.
• Technical Controls: Failure to run positive/negative controls or skipping isotype controls can introduce uncertainty.

In a practical setting, pathologists routinely review control slides, repeat staining if necessary, and correlate with clinical history to minimize misinterpretation.

Risks and Limitations

Immunohistochemistry is generally low-risk for patients since it analyzes tissue already removed by biopsy or surgery. But there are noteworthy limitations:

• False Positives/Negatives: Non-specific antibody binding or epitope masking can mislead. For instance, endogenous peroxidase in some cells might react with chromogen unless blocked.
• Semi-Quantitative Nature: Scoring intensity and percentage involves subjectivity—even slight inter-observer variation can impact clinical decisions.
• Limited Multiplexing: Traditional chromogenic IHC usually detects one or two markers per slide; assessing multiple targets demands serial sections or multiplex platforms.
• Cannot Diagnose Alone: Immunohistochemistry supports, but doesn’t replace, morphological and clinical correlation.
• Pre-Analytical Variability: Tissue handling and processing differences across labs can affect comparability of results from different centers.

Despite these caveats, immunohistochemistry remains indispensable in pathology, provided results are interpreted within methodological constraints.

Common Patient Mistakes

Patients sometimes misunderstand immunohistochemistry, leading to common errors:

• Expecting immediate results: Processing and staining takes at least a day or two.
• Confusing it with blood tests: It requires tissue, not blood, so hydration or fasting won’t change results.
• Assuming quantitative precision: IHC is semi-quantitative, not absolute like glucose measurements.
• Altering medications without advice: Drugs affecting protein expression (e.g., steroids) can change staining patterns—always check with your clinician.
• Over-interpretation: Believing a single positive marker equals a definitive diagnosis rather than part of a broader evaluation.

Myths and Facts

There’s quite a few myths floating around immunohistochemistry. Let’s clear some up:

• Myth: IHC results alone can confirm cancer type. Fact: It’s a piece of the puzzle, used with morphology, genetics, and clinical data for accurate diagnosis.
• Myth: Stronger staining always means a worse prognosis. Fact: Sometimes high expression of certain markers predicts better response to targeted therapies (e.g., HER2-positive breast cancer).
• Myth: Any lab can run IHC the same way. Fact: Protocols, antibody clones, and platforms differ across labs, affecting immunohistochemistry interpretation—your doctor relies on the specific lab’s reference standards.
• Myth: You must fast or exercise control before a biopsy. Fact: There’s no fasting or exercise prep for the test—only proper tissue collection matters.

Understanding these myths helps patients ask better questions and appreciate why immunohistochemistry results can vary between institutions.

Conclusion

Immunohistochemistry is a powerful technique that reveals protein expression patterns in tissue samples, reflecting underlying physiological and pathological processes. It combines immunology and histology—using antibodies, detection enzymes, chromogens, and precise lab protocols—to show where and how intensely proteins are present. Though semi-quantitative and subject to pre-analytical and technical variability, immunohistochemistry results guide diagnosis, prognosis, and therapy selection in oncology and beyond. By grasping what immunohistochemistry includes and how results are interpreted, patients and clinicians can collaborate more confidently, asking informed questions and understanding reports that mention staining intensity, percent positivity, and control comparisons.

Frequently Asked Questions

• Q1: What is immunohistochemistry?
  A: Immunohistochemistry is a lab technique that uses specific antibodies to visualize the presence and location of proteins in thin tissue sections, helping identify cell types and disease markers.
• Q2: How does immunohistochemistry work?
  A: The process involves applying a primary antibody to bind target antigens, using a secondary antibody with an enzyme or fluorophore to amplify the signal, and adding a chromogen or fluorescent dye to detect the binding under microscopy.
• Q3: What does immunohistochemistry mean in cancer diagnosis?
  A: In cancer, immunohistochemistry meaning relates to determining tumor subtype, receptor status (e.g., ER, PR, HER2), or proliferation index (Ki-67), which guides prognosis and therapy decisions.
• Q4: Do I need special preparation for immunohistochemistry?
  A: No special fasting or hydration is needed. Preparation focuses on proper tissue collection, timely fixation, and processing to preserve antigens.
• Q5: What sample types are used?
  A: Formalin-fixed, paraffin-embedded tissue sections are standard. Frozen sections can also be used for certain antigens but require different protocols.
• Q6: How long does the test take?
  A: From sample receipt to final slide, the immunohistochemistry test typically takes 24–48 hours, depending on workflow and whether manual or automated stainers are used.
• Q7: What are common reporting units?
  A: Reports often use percent positive cells, intensity scores (0 to 3+), and composite scores (H-score), rather than mg/dL or mmol/L.
• Q8: Can medications affect results?
  A: Yes, drugs like steroids or targeted therapies may change protein expression in tissues. Always discuss recent treatments with your pathologist or clinician.
• Q9: Are false positives possible?
  A: False positives can occur due to non-specific antibody binding or inadequate blocking of endogenous enzymes. Controls help detect such issues.
• Q10: Is immunohistochemistry interpretation subjective?
  A: Semi-quantitative scoring involves some subjectivity, but standardized protocols and inter-lab quality controls aim to minimize variability.
• Q11: What’s antigen retrieval?
  A: Antigen retrieval uses heat or enzymes to unmask epitopes hidden by formalin fixation, ensuring antibodies can bind effectively.
• Q12: How do pathologists confirm results?
  A: They review positive and negative controls, internal tissue references, and may repeat staining or run alternate antibody clones if results are ambiguous.
• Q13: Can IHC diagnose all diseases?
  A: No, immunohistochemistry supports diagnosis and characterization of many conditions but must be combined with morphology, clinical data, and other tests for definitive conclusions.
• Q14: Why might two labs give different IHC scores?
  A: Differences in antibody clones, staining platforms, scoring criteria, and tissue processing can lead to inter-lab variability in immunohistochemistry results.
• Q15: When should I discuss results with my doctor?
  A: Always review your immunohistochemistry interpretation with your healthcare provider to understand implications for your diagnosis, prognosis, and treatment options.