Recent Advances in Automation in Clinical Pathology

1. Total Laboratory Automation (TLA)

• Automated specimen input and bar-code / RFID tracking at every routing decision point
• Automated centrifuges (self-balancing, refrigerated or ambient)
• Decapper and recapper stations
• Level detection and specimen integrity checking (hemolysis, lipemia, icterus - often spectrophotometric at multiple wavelengths)
• Aliquot modules and automated diluters
• Refrigerated storage and retrieval units
• Autoverification middleware using rules-based logic to release results without technologist intervention
• Autoretrieval for reflex and repeat testing

2. Collaborative Robotics (Cobots) and Autonomous Mobile Robots (AMRs)

• Cobots (collaborative robots): Work safely alongside humans with sub-millimeter precision for fine-motor tasks such as colony picking, tube manipulation, and bench-level specimen handling - tasks that fixed conveyor lines cannot reach.
• Autonomous Mobile Robots (AMRs): Navigate hospital corridors autonomously to transport pathology carts, specimens, and medical supplies. Diligent Robotics' "Moxi" AMR has completed hundreds of thousands of supply deliveries in real hospital settings.
• Hybrid ecosystems: TLA "islands" are increasingly connected by cobotic workcells and AMRs, orchestrated by AI-enabled middleware, creating a modular rather than monolithic architecture.

3. Digital Pathology and Whole Slide Imaging (WSI)

• Multiple FDA-approved WSI scanners and image management systems (DICOM-based) are now operational
• New CPT codes for digital pathology facilitate billing and reimbursement
• Modern LIS platforms must handle DICOM WSI, integrate AI algorithms in an auditable/compliant manner, and support hybrid glass-plus-digital workflows

4. Artificial Intelligence in Pathology

a) Morphologic Diagnosis Automation

• Prostate cancer grading (Gleason scoring)
• Colorectal cancer detection
• Cervical cytology screening programs

b) Hematology - Automated Differential and Cell Classification

c) Foundation Models and Generative AI

d) AI for Oncology Biomarker Discovery

• Automated tumor detection and classification
• Identification of prognostic molecular biomarkers from H&E slides (without requiring IHC or molecular testing)
• Prediction of treatment response and patient outcomes
• Multi-omics integration for patient stratification

5. Laboratory Information Systems (LIS) - From Records to Action

• AI-first, cloud-native LIS: Moving from compliance-driven systems to AI-first architectures with cloud deployment and advanced interoperability
• Embedded AI agents: Clinisys CLS v2025.2 introduced an AI chat support agent (CLS CARE) with multilingual guidance built directly into the LIS environment
• TEFCA alignment and QHIN integration: Labs connecting to national interoperability networks for value-based care and public health reporting
• Orchestration layer: The LIS is now the human-plus-AI pathology orchestration hub, managing DICOM images, AI algorithm outputs, audit trails, and hybrid workflows
• Global LIS market estimated at ~$891 million in 2024, projected to reach ~$1.3 billion by 2032

6. Preanalytical Quality and Error Reduction

7. Microbiology and Molecular Diagnostics

• MALDI-TOF MS for rapid microbial identification is now standard
• Automated blood culture systems (e.g., BD BACTEC, bioMérieux BacT/ALERT) continuously monitor and flag positive cultures
• COPAN's WASP/WASPLab platforms automate plating, streaking, incubation, and imaging of culture plates - combined with AI for automated colony recognition and presumptive identification
• Next-generation sequencing (NGS) workflows are increasingly automated, with pool-to-pool testing and bioinformatic pipelines integrated into LIS platforms

Summary Table

• Mukherjee S et al. Diagnostics 2026 - Cobots, AMRs, TLA [PMID 41750668]
• Marra A et al. Ann Oncol 2025 - AI in oncology pathology [PMID 40307127]
• Zhang DY et al. Lab Invest 2024 - Digital pathology implementation [PMID 39053633]
• McGenity C et al. npj Digit Med 2024 - AI diagnostic accuracy meta-analysis [PMID 38704465]
• Tietz Textbook of Laboratory Medicine, 7th Ed., pp. 879-880 (TLA systems)
• Henry's Clinical Diagnosis and Management by Laboratory Methods (TLA chapter)

Recent Trends in the Diagnosis and Differential Diagnosis of Small Round Cell Tumours (SRCTs)

Overview

Classic Histology

Ewing sarcoma: sheets of uniform small round cells with scant clear cytoplasm. (Robbins & Kumar Basic Pathology)

1. The WHO 2020 Framework: Undifferentiated Small Round Cell Sarcomas (USRCS)

• Robbins & Kumar Basic Pathology, p. 786; Quick Compendium of Clinical Pathology, p. 411

2. Entity-by-Entity Features and Diagnostic Advances

2.1 Ewing Sarcoma

• Morphology: Sheets of small round cells, slightly larger than lymphocytes, scant clear glycogen-rich cytoplasm; Homer-Wright rosettes (pseudorosettes with a central fibrillary core) in ~10%
• Molecular: >90% carry EWSR1::FLI1 t(11;22)(q24;q12); rarer partners include ERG, FEV, ETV1, ETV4. EWSR1 is on 22q12, highly prone to rearrangement
• IHC panel: CD99 (diffuse strong membranous - hallmark but not specific), NKX2.2 (nuclear, more specific than CD99), FLI1 (nuclear), vimentin+; keratin/desmin/S100 negative
• FISH: EWSR1 break-apart probe (dual-colour) is the standard confirmatory test (see image below); note the separate red and green signals in rearranged nuclei
• Pitfall: EWSR1 FISH is positive in many non-Ewing tumours (DSRCT, clear cell sarcoma, myxoid liposarcoma); it is a "rule-in for EWSR1 rearrangement," not specific for Ewing

EWSR1 FISH break-apart probes. Left (a): normal nuclei with 2 red-green fusion signals. Right (b): abnormal nuclei with 1 fused + 1 red + 1 green signal, indicating EWSR1 rearrangement. (Quick Compendium of Clinical Pathology)

• Imaging (2026 update): Lytic "moth-eaten" bone destruction with aggressive periosteal reaction (onion-skin), large extraosseous soft tissue mass (Mori et al., RadioGraphics 2026, PMID 41886300)
• Prognosis: 75% 5-year survival with neoadjuvant chemotherapy + surgery ± radiation

2.2 CIC-Rearranged Sarcoma

• Demographics: Predominantly young adults; predominantly soft tissue (rarely bone)
• Morphology: Slightly larger cells than Ewing, more irregular nuclei, geographic necrosis, myxoid stroma; sometimes spindled cells
• Key IHC: WT1 (nuclear - useful), ETV4 (nuclear - highly sensitive), DUX4 (nuclear); CD99 is focal or absent (key distinguishing feature from Ewing)
• Molecular: CIC-DUX4 rearrangement (CIC on 19q13, DUX4 on 4q35 or 10q26) detected by FISH or RNA-seq fusion panels
• Prognosis: More aggressive than Ewing sarcoma; poor response to standard Ewing chemotherapy - an important clinical reason to distinguish it (Mori et al., RadioGraphics 2026)
• Imaging: Well-circumscribed lobulated soft tissue masses with extensive internal necrosis and haemorrhage, no calcification

2.3 Sarcoma with BCOR Genetic Alterations

• Two main subtypes:
  • BCOR-CCNB3: X-linked inversion (inv X(p11;p13)), predominantly adolescent boys, long bone metaphyses/pelvis
  • BCOR internal tandem duplication (ITD): Overlaps with infantile fibrosarcoma
• Morphology: Primitive round to spindle cells; myxoid stroma; occasional whorling
• Key IHC: BCOR (nuclear), CCNB3 (nuclear, specific for BCOR-CCNB3 subtype), CD99 (variable), TLE1 (nuclear)
• Imaging: Osteolytic or sclerotic lesion in long bones/pelvis, often with calcification in extraosseous component - helps distinguish from Ewing
• Prognosis: Potentially more favourable than Ewing; still benefits from Ewing-type chemotherapy regimens (Mori et al., 2026)

2.4 EWSR1/FUS::NFATc2 and EWSR1::PATZ1 Sarcomas (Newly Recognised)

• EWSR1 or FUS fuses with NFATc2 (transcription factor)
• Morphologically overlaps with Ewing but shows unique features including lobulated architecture and fibromyxoid stroma
• NFATc2 IHC (nuclear) serves as a surrogate marker
• Limited response to neoadjuvant chemotherapy compared with Ewing - critical for management
• Distinct methylation, transcriptomic, and epigenetic profile from Ewing sarcoma
• Definitive diagnosis requires NGS-based fusion detection

3. The Broader SRCT Differential

3.1 Desmoplastic Small Round Cell Tumour (DSRCT)

• Hallmark: Growth within strikingly desmoplastic stroma - the most distinctive morphologic clue
• Demographics: Adolescent/young adult males; intra-abdominal/peritoneal most common; rare sites include head/neck and GI tract
• Unique IHC profile: Polyphenotypic - expresses epithelial (EMA, cytokeratin), neural (NSE, synaptophysin), and mesenchymal (desmin - dot-like perinuclear) markers simultaneously. WT1 (C-terminal antibody, nuclear) is key
• Molecular: t(11;22)(p13;q12) fusing EWSR1 with WT1 - highly specific, detectable by RT-PCR or RNA-seq
• Recent work (2026, Brahmi et al., Curr Opin Oncol) highlights the role of androgen receptor (AR) expression in DSRCT - AR positivity in ~70% of cases, with implications for targeted therapy with AR antagonists. Multi-layered molecular profiling is informing novel treatment strategies in this ultra-rare tumour
• Prognosis: Poor; median survival ~2-4 years; hyperthermic intraperitoneal chemotherapy (HIPEC) under investigation

3.2 Neuroblastoma

• Age: Predominantly <5 years; arises from adrenal gland or sympathetic ganglia
• Morphology: Neuropil background (Homer-Wright rosettes); ranges from undifferentiated to differentiating
• IHC: Synaptophysin+, chromogranin+, NSE+, CD56+; PHOX2B (nuclear) is highly sensitive/specific
• Biochemical marker: Elevated urinary catecholamine metabolites (VMA, HVA) and MIBG avidity - unlike other SRCTs
• Molecular: MYCN amplification (poor prognosis), ALK mutations (targetable), segmental chromosomal aberrations
• Differential clue: The presence of neuropil is "highly distinctive" and essentially excludes other SRCTs; positive MIBG scan rules out all non-adrenergic SRCTs (Tietz Textbook)

3.3 Rhabdomyosarcoma (RMS)

• Subtypes: Embryonal (most common, children), Alveolar (PAX3/7-FOXO1 fusions, worse prognosis), Pleomorphic (adults), Spindle cell/Sclerosing (MYOD1 mutations)
• IHC: Desmin+, myogenin+ (nuclear, most specific), MyoD1+ (nuclear), MSA+; these myogenic markers are the diagnostic cornerstone
• Molecular: Alveolar RMS - t(2;13)(q35;q14) PAX3-FOXO1 or t(1;13)(p36;q14) PAX7-FOXO1; detection by FISH or RT-PCR is prognostically important (PAX3-FOXO1 = worse)
• Emerging markers: MYOD1 L122R mutation (spindle cell/sclerosing RMS) detectable by sequencing; relevant for targeted PI3K pathway inhibition

3.4 Wilms Tumour (Nephroblastoma)

• Classically presents in children <5 years as a renal mass; characterised by the classic triphasic pattern (blastema + epithelial + stromal)
• The blastemal component is the SRCT component
• IHC: WT1 (nuclear), cyclin D1, CD56; blastemal component is CD99+
• Molecular: WT1 mutations, WTX mutations, CTNNB1 mutations; anaplastic Wilms - TP53 mutations. Newer: SIX1/SIX2 mutations in blastemal-predominant Wilms

3.5 Lymphoma/Leukaemia

• Burkitt lymphoma: Extremely rapid proliferation; "starry sky" pattern; MYC rearrangement (8q24)
• Precursor B/T-ALL: Terminal deoxynucleotidyl transferase (TdT)+, CD34+, lineage-specific markers
• IHC differentiator: LCA (CD45) positivity effectively separates lymphoma from other SRCTs; TdT distinguishes precursor lesions
• Clinical clue: High WBC count, lymph node enlargement, bone marrow infiltration pattern (Tietz Textbook)

3.6 NUT Carcinoma (Midline Carcinoma)

• An aggressive carcinoma of midline structures (mediastinum, sinonasal, head/neck) with SRCT morphology; occasional "abrupt squamous differentiation"
• IHC: NUT IHC (nuclear, speckled pattern) - highly sensitive and specific; a single positive IHC is now considered diagnostic
• Molecular: BRD4-NUT t(15;19), BRD3-NUT, or NSD3-NUT fusions
• Bell et al. (Head Neck Pathol 2024, PMID 38315310) reviewed sinonasal SRCTs emphasising NUT IHC as a key "hack" in the IHC panel for poorly differentiated sinonasal SRCTs

3.7 Merkel Cell Carcinoma and Small Cell Carcinoma

• Merkel cell carcinoma: Neuroendocrine SRCT of skin; CK20+ (dot-like perinuclear), synaptophysin+, chromogranin+; associated with Merkel cell polyomavirus (MCPyV) - LT antigen IHC (CM2B4 antibody) positive in ~80%
• Small cell lung carcinoma: TTF-1+, CK7+, neuroendocrine markers+; rarely metastasises with SRCT morphology to sites that mimic primary SRCTs

4. Emerging and Recently Defined Entities

4.1 EWSR1::ATF1 and FET::CREB Fusion Tumours

4.2 SMARCA4-Deficient Undifferentiated Tumour

• Highly aggressive SRCT occurring in adults (especially thoracic)
• Loss of SMARCA4 (BRG1) protein by IHC - now a standard panel member in mediastinal/thoracic SRCTs
• WHO 2022 thoracic tumour classification recognises this as a distinct entity

4.3 Infantile Fibrosarcoma / Congenital Fibrosarcoma

• SRCT variant in neonates/infants; ETV6-NTRK3 fusion (targetable with TRK inhibitors - larotrectinib, entrectinib)
• NTRK IHC pan-Trk (EPR17341) serves as a screening tool before confirming by FISH/NGS

5. Diagnostic Approach: Tiered Algorithm

Step 1: Morphology + clinical context (age, site, serum markers)
         ↓
Step 2: First-line IHC panel
         • CD99, TdT, LCA, desmin/myogenin, synaptophysin/chromogranin, WT1,
           NKX2.2, S100, pan-keratin, NUT IHC, SMARCA4
         ↓
Step 3: Targeted molecular testing based on IHC
         • EWSR1 FISH (break-apart) → if positive, partner identification needed
         • BCOR IHC + CCNB3 IHC (for BCOR-CCNB3)
         • CIC FISH (break-apart) + ETV4 IHC
         ↓
Step 4: RNA-based NGS fusion panel (if Steps 1-3 inconclusive)
         → Detects all known fusion transcripts in a single assay
         → Identifies rare/novel fusions (EWSR1::NFATc2, EWSR1::PATZ1, etc.)
         ↓
Step 5: Methylation array profiling (in research/reference centres)
         → Can resolve diagnostically challenging cases
         → WHO 5th edition recognises "essential" vs. "desirable" diagnostic criteria
            to allow diagnosis in centres without full molecular capabilities

6. Role of Next-Generation Sequencing (NGS)

• Detects known and novel gene fusions simultaneously in one assay
• Required for definitive classification of EWSR1/FUS::NFATc2 and EWSR1::PATZ1 sarcomas
• Cytology specimens (FNA smears, cell blocks) yield high-quality nucleic acid and are now validated for NGS in round cell sarcomas (Gajdzis & Klijanienko, Acta Cytol 2026, PMID 41615867)
• DNA methylation profiling complements fusion detection for borderline/unclassifiable cases
• The 2025 reviews emphasise that accurate subclassification of EWSR1/FUS fusion variants carries direct therapeutic implications - different chemotherapy sensitivity and prognosis (Jennings et al., 2025)

7. Summary IHC Differential Table

Key Recent References

• Dehner CA et al. "Updates on WHO classification for small round cell tumors: Ewing sarcoma vs. everything else." Hum Pathol 2024 - PMID 38280658
• Mori K et al. "Undifferentiated Small Round Cell Sarcomas: Radiologic-Pathologic Correlation for the Updated WHO 5th Ed." RadioGraphics 2026 - PMID 41886300
• Davis JL & Cheesman E. "Emerging round cell sarcomas in children." Virchows Arch 2025 - PMID 39576278
• Jennings LJ et al. "EWSR1/FUS::NFATC2 Sarcomas: Clinicopathologic review." Arch Pathol Lab Med 2025 - PMID 41167264
• Marcelis L & Sciot R. "Undifferentiated small round cell sarcomas of bone and soft tissue." Ann Pathol 2025 - PMID 39510958
• Makise N & Yoshida A. "Undifferentiated Small Round Cell Sarcomas of Bone." Surg Pathol Clin 2025 - PMID 40716917
• Gajdzis P & Klijanienko J. "Cytological, Immunocytochemical, and Molecular Findings of Extraskeletal Round Cell Sarcomas in Paediatric Patients." Acta Cytol 2026 - PMID 41615867
• Bell D. "Top IHC/ISH Hacks for Sinonasal Small Round Cell Tumors." Head Neck Pathol 2024 - PMID 38315310
• Robbins & Kumar Basic Pathology, p. 786 (Ewing sarcoma morphology)
• Quick Compendium of Clinical Pathology 5th Ed., p. 411 (EWSR1 family)
• Tietz Textbook of Laboratory Medicine 7th Ed. (neuroblastoma differential)