9.4 Infectious IHC and Microorganisms

9.4 Infectious IHC and Microorganisms

Quick Answer: Infectious IHC labels organisms or virus-infected cells; it does not replace the H&E or silver/GMS morphology. HSV, CMV, and adenovirus are read in infected-cell nuclei (CMV also cytoplasmic). H. pylori sits in gastric mucus. Spirochete IHC is more directed than Warthin-Starry, which stains other bacteria and elastin. Fungus IHC can cross-react; GMS still shows wall shape. Pigment is not an organism. Use known-positive control tissues.

This OpenExamPrep section is independent teaching on microorganism staining patterns covering published QIHC tissue topics (microorganisms as a staining-pattern skill). It is not an ASCP publication and does not claim Board, CDC, or manufacturer approval. Do not treat named antibodies as the only valid clones.

Infectious IHC is still a staining-pattern problem

Section 9.1 rules still apply. You need the H&E (or a Giemsa, silver, or GMS) to know where organisms should be, which cells look cytopathic, and which brown granules were already pigment. Infectious antibodies are often polyclonal or broadly reactive. Background in mucus, necrosis, and granulomas is common. A true positive is structured: viral inclusions in the right cell, helical bacteria in surface mucus, yeast forms with walls, not a smear of DAB on debris.

Internal controls are different from cytokeratin. Residual normal epithelium is not supposed to be HSV-positive. The control for an infectious antibody is a known infected tissue (or a commercially prepared organism control) plus a negative reagent control. If the patient tissue has no organisms and the positive control is blank, the run failed. If the patient tissue is dirty and the control is clean, you are looking at background or pigment.

Herpes simplex virus (HSV)

HSV IHC is used on ulcers of skin, mucosa, esophagus, and similar sites when viral cytopathic effect is subtle or when rapid confirmation is needed.

• Where: infected squamous or other epithelial cells, especially at the ulcer edge.
• Compartment: nuclear labeling is required for a confident viral read; cytoplasmic labeling often accompanies it in packed infected cells.
• Morphology partner: multinucleation, molding, chromatin margination, and Cowdry-type inclusions on H&E. IHC can highlight infected cells that are not obvious on a crushed biopsy.
• Pitfalls: staining of debris in the ulcer bed; edge artifact; calling neutrophils positive. HSV-1/HSV-2 cocktails are common; subtyping is a clinical add-on, not required to recognize that viral antigen is present.

If residual uninfected epithelium is strongly dirty while nuclei of cytopathic cells are negative, believe morphology and troubleshoot the antibody, not the reverse.

Cytomegalovirus (CMV)

CMV IHC is common on GI biopsies (transplant, immunocompromised bleeding), lung, and placenta.

• Where: enlarged endothelial cells, fibroblasts, and stromal cells more often than surface epithelium; infected cells can be sparse.
• Compartment: nuclear and cytoplasmic. Classic owl-eye nuclear inclusions and granular cytoplasmic inclusions should match the IHC.
• Pitfalls: missing rare infected cells on a hasty screen; calling pigment-laden macrophages positive; using a control that never contained CMV.

Sparse true CMV is still a positive if the labeled cell is cytomegalic in the right compartment. Diffuse blush of every nucleus in a colonic biopsy is not CMV.

Adenovirus

Adenovirus IHC is used in hemorrhagic cystitis, transplant colitis, and necrotizing pneumonia, especially in immunocompromised patients.

• Where: epithelial nuclei (smudge cells) in lung, GI, or bladder, depending on site.
• Compartment: nuclear. Smudged chromatin on H&E is the morphologic partner.
• Pitfalls: confusing smudge cells with HSV or CMV. HSV prefers squamous mucosa with multinucleation; CMV prefers giant endothelial/stromal cells with nuclear and cytoplasmic inclusions; adenovirus prefers smudged epithelial nuclei. IHC is how you confirm when H&E overlap exists.

Helicobacter pylori

H. pylori IHC labels curved organisms in the surface mucus and pits of gastric (and sometimes duodenal) biopsies.

• Where: extracellular, in mucus, hugging foveolar epithelium—not inside macrophage pigment.
• Why IHC: it can highlight sparse organisms that H&E or Giemsa miss, including coccoid forms after therapy, as a typical use. Do not quote unpublished sensitivity percentages; say IHC is used because organisms can be rare.
• Pitfalls: mucus debris, other bacteria in the mouth/swallowed flora, and overstaining of mucus as a brown wash. True organisms have bacterial shape. Chronic gastritis on H&E without organisms is not an IHC-positive result.

Related helicobacters (for example H. heilmannii-type organisms) may or may not label depending on the antibody. If the H&E shows long tightly coiled organisms and the IHC is negative, report the morphology rather than forcing the reagent.

Spirochetes versus Warthin-Starry

Warthin-Starry is a silver impregnation stain. It can show slender spirochetes in tissue (syphilis, some GI spirochetosis contexts) but it also labels other bacteria and can blacken elastin and debris. It is sensitive to technique (pH, developer, overstain).

Treponema IHC (and similar spirochete antibodies) is typically more directed at the organism of interest than silver, provided the antibody’s intended target matches the clinical question (T. pallidum versus intestinal Brachyspira, which is a different organism). IHC still shows background in plasma-cell-rich dermis and in necrotic debris.

Do not call black elastic fibers in a vessel wall “spirochetes.” Do not skip a known-positive control because silver “always works.”

Fungi: IHC versus GMS (and PAS)

Grocott methenamine silver (GMS) and PAS are morphologic special stains of fungal wall carbohydrate. They answer shape: yeast versus hyphae, septate versus pauciseptate, width, branching angle, and whether the structures are in tissue or in a cavity. Pneumocystis on GMS is cup-shaped cysts in alveolar casts, not budding yeast.

Fungus IHC (Aspergillus, Candida, Mucorales, Cryptococcus, Pneumocystis, and similar reagents) can support a genus-level hypothesis on FFPE when culture is not available. Cross-reactivity among fungi is a known practical problem. An Aspergillus antibody that lights up Fusarium or a Candida antibody that dirties other yeasts will mislead if you ignore GMS morphology. Use IHC as an adjunct, not as a replacement for wall morphology and clinical microbiology.

Background pigment versus organisms

Brown on an infectious IHC slide is guilty until the H&E agrees it is an organism.

• Anthracotic carbon: chunky black, in pulmonary macrophages, already visible on H&E, GMS-negative as yeast.
• Hemosiderin: golden-brown, chunky, often after bleeding; Prussian blue positive on a separate slide.
• Melanin: fine brown in melanocytes or melanoma; bleach protocols exist where validated.
• Formalin-heme pigment: in bloody, poorly fixed tissue; not helical, not budding.
• DAB dirt: folds, necrosis, mucus wash.

A GMS-positive structure with a wall is a fungus until proven otherwise. A GMS-negative black chunk in a macrophage is pigment. Helicobacter does not live in alveolar macrophages. HSV does not label carbon.

Control tissues for infectious antibodies

Every infectious IHC run needs a positive organism control that actually contains the target, processed as similarly as the laboratory can manage (FFPE infected tissue or a commercial control).

Score the control first: organisms present, expected compartment, acceptable background. Then screen the patient slide at a magnification that can actually see bacteria and inclusions. Sparse CMV and sparse Helicobacter are easy to undercall if you only glance at 4×.

Worked example

A gastric biopsy for “rule out Helicobacter” shows a brown granular wash in mucus on IHC. The H&E shows chemical gastropathy without plasma-cell gastritis. The positive control has crisp curved organisms. The patient mucus wash has no bacillary shapes. Interpretation: background mucus staining, not H. pylori. Repeating the IHC will not create organisms the H&E never supported. Contrast that with a lung GMS on which cup-shaped cysts sit in alveolar casts and Pneumocystis IHC (if used) labels the same structures: morphology and IHC agree, and anthracotic pigment in nearby macrophages is ignored.

Infectious IHC is pattern recognition plus honesty about pigment. The antibody is only half of the stain.