Can tumor tissue with insufficient cellularity for immunohistochemistry be sent to an external laboratory for review?

Can Tumor Tissue with Insufficient Cellularity Be Sent to an External Laboratory for Review?

Yes, tumor tissue with insufficient cellularity for immunohistochemistry can and should be sent to an external reference laboratory for review, particularly when the specimen is inadequate at the originating institution but additional diagnostic testing is critical for patient management.

Key Principles for Handling Insufficient Specimens

Distinction Between Inadequate and Indeterminate Specimens

The first critical step is understanding what "insufficient cells" means. A sample is deemed inadequate when there is insufficient well-preserved lesional material for confident diagnosis by the pathologist 1. This differs from an adequate but indeterminate specimen, where sufficient material exists but definitive conclusions cannot be made due to the nature of the process itself 1.

When External Laboratory Review is Appropriate

Laboratories that require send-out of tests for treatment predictive biomarkers should process and send specimens to reference molecular laboratories in a timely manner 2. The benchmark recommendation is that 90% of specimens should be sent out within 3 working days 2.

Specimen Adequacy Assessment

Before sending tissue externally, pathologists must evaluate candidate specimens for biomarker testing to ensure specimen adequacy, taking into account:

• Tissue quality
• Tissue quantity
• Malignant tumor cell fraction

These specimen adequacy findings should be documented in the patient report 2.

Practical Solutions for Limited Tissue

Alternative Specimen Preparation Methods

When dealing with insufficient cellularity, several options exist 1, 3:

1. Touch imprint preparations (TPs) - Can be made from limited tissue and used for immunohistochemistry and FISH analysis
2. Cytospin preparations - Useful for concentrating samples with very low cellularity
3. Paraffin-embedded tissue sections - Can be used even with minimal material
4. Core biopsy sampling - If the originating institution is unwilling to release the entire block, a 4-mm core punch can be taken and sent to the reference laboratory, which can then be re-embedded and used for multiple analyses 4

Tissue Conservation Strategies

For specimens with limited tissue, laboratories should establish policies to ensure efficient allocation and utilization of tissue for molecular testing 2. This includes:

• Dividing core biopsies into multiple blocks with no more than 2 cores per block to minimize tissue depletion 5
• Prioritizing molecular analysis over immunohistochemistry when tissue is extremely limited 5
• Coordinating with cytopathologists to determine the best collection method based on clinical differential diagnosis 1

Quality Assurance Requirements

Laboratory Standards

External reference laboratories receiving insufficient specimens should:

• Be fully equipped and ISO 15189 accredited molecular pathology laboratories 6
• Have well-trained pathologists, clinical molecular biologists, and technicians 6
• Have expertise in the specific tumor type being evaluated 5

Validation Requirements

Immunohistochemistry performed on limited cellularity specimens requires laboratory validation specific to these preparations to avoid diagnostic pitfalls 7. This is particularly important because:

• Staining patterns may differ in scant material
• Controls must be appropriate for the specimen type
• Interpretation criteria may need adjustment

Communication and Documentation

Essential Information to Include

When sending specimens externally, ensure the following are documented 8, 2:

1. Pre-analytical variables:

   • Time tissue was removed from patient
   • Time tissue was placed in fixative
   • Duration of fixation
   • Fixative type
   • Cold ischemia time

2. Specimen characteristics:

   • Estimated tumor cellularity percentage
   • Areas of necrosis or artifact
   • Specific regions of interest marked on slides

3. Clinical context:

   • Differential diagnosis
   • Specific biomarkers needed
   • Urgency of results

Turnaround Time Expectations

Colorectal carcinoma molecular biomarker results should be made available as promptly as feasible, with a benchmark of 90% of reports available within 10 working days from date of receipt in the molecular diagnostics laboratory 2. Similar timelines should apply to other tumor types.

Common Pitfalls to Avoid

1. Not consulting with the reference laboratory before sending - Coordinate specimen handling requirements in advance 1

2. Sending unstained slides cut more than 6 weeks before analysis - This is not recommended as antigenicity may be compromised 8

3. Failing to mark tumor areas - A pathologist should review and delineate regions of tumor cells before external review, as it can be difficult to differentiate normal from malignant cells using only DAPI counterstain 9

4. Not preserving tissue for future testing - When possible, collect fresh/frozen tissue and obtain informed consent for tumor banking so tissue is available for later analyses 10

Special Considerations for Specific Tumor Types

For certain diagnoses where molecular analysis is critical (e.g., GIST, sarcomas, colorectal cancer), molecular analysis should be performed in centralized laboratories enrolled in external quality assurance programs with expertise in genomic analysis 10. This is particularly important when:

• Treatment decisions depend on specific mutations
• Rare tumor types require specialized expertise
• Multiple ancillary tests are needed from limited material

The bottom line: Insufficient cellularity at the originating institution should not prevent diagnostic workup. External reference laboratories with appropriate expertise and validated methods can often extract diagnostic information from limited specimens that would otherwise be deemed inadequate.