DIF

Dual-Stain Immunofluorescence

Multiplex Immunofluorescence for Cytopathology

Breakthrough multiplex immunofluorescence staining technology designed specifically for cytological samples – enabling precise, reproducible, and AI-optimized in situ immunodetection on a single slide.

Why DIF for Cytopathology?

Many cytological specimens – serous effusions, liquid-based cytology (LBC), and fine-needle aspiration (FNA) – are naturally available as cell suspensions, making them ideal for direct immunofluorescence staining. DIF requires only a small number of target cells, achieving multiplex immunodetection at the paucicellular to single-cell level.

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Paucicellular-Compatible

Detects 6-8 markers on a single slide without requiring cell block preparation.

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Morphology-Preserving

One slide supports both morphological evaluation (Pap/H and E) and multiplex in situ immunomarkers simultaneously.

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AI-Ready Platform

Digital IF signals integrate seamlessly with AI colocalization and quantitative analysis for superior diagnostic accuracy.

Five Application Frontiers

1. p16/Ki-67 Dual Staining in Cervical Cytology

Co-expression of p16 and Ki-67 within the same cell indicates cell cycle dysregulation independent of cytomorphology. DIF-based dual staining overcomes limitations of traditional IHC – no cell overlapping issues, clear interpretation criteria, and high inter-observer consistency.

Clinical validation (599 LBC cases):
High-grade detection rate: 97.1%
Overall agreement with LBC: 98.66%
Sensitivity: 95.87%
Specificity: 95.35%

Cervical cytology cell clusters – the ideal substrate for DIF dual staining

2. Multiplex Immunodetection on Cytology Slides

DIF enables simultaneous detection of 2-6 markers on a single conventional or liquid-based cytology slide, supporting diagnosis, tumor classification, and origin determination. Ideal for paucicellular samples where cell block preparation is not feasible.

Clinical examples:
Pleural effusion: TTF-1 (green) + Napsin A (red) dual positivity confirms lung adenocarcinoma
Urine cytology: HK2 + pan-CK + CD45 multiplex panel for target cell identification

3. Immune Microenvironment Analysis

DIF powered by multiplex immunofluorescence (mIHC/IF) enables quantitative analysis of up to 6-8 markers on a single cytology slide. Standardized identification of immune cell types while preserving spatial context through marker colocalization.

Applications:
Tumor immune microenvironment (TIME) profiling from serous effusions
Peripheral blood immune analysis – T cell, NK cell, macrophage subtyping
Rectal cancer-associated serous effusion immune profiling

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4. Flow Cytometry-Based Applications

Serous effusions are naturally occurring cell suspensions – excellent quality samples for flow cytometric immunophenotyping (FCM). DIF supports FCM-based diagnosis for lymphoid and hematopoietic malignancies, including lymphoma subtype classification (CLL-SLL, DLBCL, FL, LPL-WM, MCL, MZL), MRD detection, and CSF immunophenotyping for CNS lymphoma.

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5. CTC Detection via Immunofluorescence

Circulating tumor cells (CTCs) provide non-invasive access to tumor cell samples for liquid biopsy. DIF utilizes the most widely used multiplex IF platform for CTC identification, using the EpCAM+/CK+/CD45-/DAPI+ marker panel for detection and longitudinal monitoring.

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Spotlight · flagship application

Understanding p16/Ki-67 dual staining

Co-expression of p16 and Ki-67 in a single cell reveals cell-cycle dysregulation independent of morphology — an objective, biomarker-based triage recommended by the 2023 Chinese Expert Consensus on p16/Ki-67 dual staining.

p16 (CDKN2A)
Cell-cycle dysregulation marker
  • Over-expressed when HPV E7 disrupts the cell cycle
  • Stains both cytoplasm and nucleus
  • Flags HPV-transformed cells
Ki-67 (MKI67)
Proliferation marker
  • Expressed in all active phases (G1, S, G2, M)
  • Stains the nucleus only
  • Flags actively dividing cells
Co-expression = risk
Same-cell dual positivity
  • Normally p16 and Ki-67 are mutually exclusive
  • Both in the same cell = cell-cycle dysregulation
  • A dual-positive cell strongly indicates a precancerous lesion

Why fluorescence

Immunofluorescence vs. IHC dual staining

Separate fluorescence channels remove the colour overlap that limits chromogenic IHC, making same-cell co-localization unambiguous and AI-ready.

Feature IHC dual staining KFBIO IF dual staining
Staining method Chromogenic (DAB + Fast Red) Fluorescence (FITC + Cy3 + DAPI)
Colour overlap Yes — hard to confirm co-localization No — separate fluorescence channels
Interpretation Subjective, high inter-observer variability Objective, AI-assisted co-localization
Cell clusters Overlapping cells hard to read Channel separation resolves overlap
Sensitivity Moderate High — detects single dual-positive cells
Reproducibility Low — variable staining quality High — standardized protocol
AI integration Limited Full AI pipeline for detection & analysis
Pap-stain compatibility Not compatible Compatible — IF after LBC staining

One slide, two modalities

Staining & imaging workflow

IF is performed on the LBC-stained slide, so Pap morphology is preserved and no duplicate slide or cell block is required.

1
LBC preparation
Liquid-based cytology (LBC) slide prepared
2
Pap stain & scan
Pap staining + brightfield whole-slide scan
3
IF dual staining
p16 (FITC) + Ki-67 (Cy3) + DAPI on the same slide
4
Fluorescence scan
Multi-channel fluorescence whole-slide scan
5
AI analysis
Automated cell detection & co-localization
6
Diagnostic report
Integrated morphology + IF result report

Fluorescence channel configuration

DAPIBlue
Nuclear counterstain — marks all nucleated cells
p16 / FITCGreen
Cell-cycle dysregulation — cytoplasmic & nuclear
Ki-67 / Cy3Red
Proliferation marker — nuclear staining
MergeDual +
p16 + Ki-67 in the same cell = DUAL POSITIVE

AI-Assisted Dual-Modal Diagnostic Workflow

Fusing bright-field and fluorescence features effectively reduces missed diagnoses – one slide, two modalities, one AI platform.

TSA-based fluorescent labeling – high sensitivity, no chromogenic overlap
Single-slide workflow – multiplex detection on existing Pap/H and E slides

AI colocalization analysis – eliminates false positives from cell overlapping
Quantitative in situ analysis – preserves true cellular characteristics

No cell block required – direct IF staining on cell suspensions
End-to-end digital workflow – integrates with KFBIO PIS and AI platforms

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Clinical Validation Performance

Prospective clinical validation from 599 cytology cases – DIF p16/Ki-67 dual staining delivers superior diagnostic performance over conventional LBC alone.

97.1%

High-Grade Detection Rate

98.66%

Agreement with LBC

95.87%

Sensitivity

95.35%

Specificity

599-case validation, by cytology category

Per-category performance of DIF p16/Ki-67 dual staining across the full prospective series.

Cytology category Cases Dual positive Negative Contam. Prep success Positive rate
NILM 217 10 207 0 100.0% 4.6%
ASC-US 77 23 52 2 97.4% 30.7%
ASC-H 68 64 3 1 98.5% 95.5%
LSIL 131 43 86 3 98.5% 33.3%
HSIL 103 99 2 2 98.1% 98.0%
SCC 3 3 0 0 100.0% 100.0%
Total 599 242 350 8 98.8% —
Prospective series of 599 liquid-based cytology cases. Most false positives in the NILM group trace to cell overlapping, which AI cluster segmentation is tuned to de-prioritise.

Beyond basic screening

Clinical value in routine practice

LBC triage optimization
  • 110 of 217 low-grade LBC cases were IF-positive — clearer follow-up guidance
  • Negative IF safely reduces unnecessary colposcopy referrals
  • 3 over-called high-grade LBC cases corrected after IF review
Same-slide morphology + IF
  • IF runs on the LBC-stained slide — no separate slide needed
  • Pap morphology preserved and unaffected
  • Correlates morphology with biomarkers on the exact same cells
Paucicellular advantage
  • Needs only a few target cells
  • Ideal for LBC specimens that are natural cell suspensions
  • No cell-block preparation; single-cell detection sensitivity

AI Colocalization in KFBIO Platform

KFBIO digital pathology platform – left: bright-field Pap stain (ASC-US); right: fluorescence multiplex IF (DAPI/Ki-67/P16) with AI colocalization

The Optimal Multiplex Immunofluorescence Platform for Cytology

Powered by KFBIO’s end-to-end digital pathology ecosystem – from sample preparation to AI analysis.

One integrated solution

Scanner, software and reagents — end to end

KFBIO delivers the complete p16/Ki-67 in-situ LBC workflow: a brightfield-plus-fluorescence scanner, the AI co-localization platform and validated dual-staining reagents.

KF-PRX scanner
Brightfield + fluorescence WSI
  • Brightfield + up to 10 fluorescence channels
  • High-speed whole-slide scanning
  • 120-slide capacity (expandable to 480)
  • Outputs KFB / DCM / TIFF / SVS / QPTIFF
KFBIO AI software
Automated co-localization
  • Cell detection & classification across channels
  • p16/Ki-67 same-cell co-localization
  • Cell-cluster segmentation
  • Objective, quantitative reporting
p16/Ki-67 IF kit
Validated dual-staining reagents
  • Optimized for liquid-based cytology
  • Compatible with Pap-stained slides
  • p16-FITC + Ki-67-Cy3 + DAPI
  • Standardized protocol, 599+ cases validated