HALO® Module

Serial Section Analysis Add-on

The Serial Registration Analysis add-on includes options for both serial section analysis and serial stain fusion analysis.

Serial section analysis allows users to create a tissue classifier based off a reference slide (e.g., H&E or Pan-CK) and then apply the resulting classification mask to serial section(s) stained for additional IHC markers (e.g., HER2, ER, PR, Ki-67, CD8, CD4).  In the example above, a tissue biopsy section is stained with hematoxylin and eosin. A classifier has been created segmenting tumor from stroma (Tumor in red, stroma in green, glass in yellow). The serial section add-on takes the Classifier created from the H&E image and allows the user to apply the resulting classification mask to IHC-stained serial sections (e.g., ER, HER2) as regions of interest. The tumor regions, based off of the H&E classifier, are analyzed on the ER and HER2 slides using the Cytonuclear Module and Membrane Module, respectively.  Serial registration analysis is compatible with both brightfield and fluorescent images and any of our HALO modules can be used for the final analysis step.

Where serial section analysis involves different tissue sections that have been stained with different markers, serial stain fusion facilitates analysis of single slides that have been stained, stripped and restained (as described in Romain Remark et al. Science Immunology, 2016).  As demonstrated below, the brightfield images are deconvolved into their composite stains (nuclear counterstain and biomarker stain) and converted to a pseudo-fluorescent images in Step 1. In Step 2, the images are registered using a customizable set of configuration settings. Serial Stain Registration uses an Image Consistency Phase, a Similarity-Only Phase, and a High-Resolution Registration to precisely align single cells. Additionally, a Single Channel Registration option allows the user to align the images using one consistent fluorescent channel, typically DAPI, to improve the registration. Then, the registered serial stain images can be fused into a single composite fluorescent image that can be analyzed using Highplex FL or (any of our other FL modules).

File formats supported by the HALO image analysis platform:

  • Non-proprietary (JPG, TIF, OME.TIFF)
  • Nikon (ND2)
  • 3D Histech (MRXS)
  • Akoya (QPTIFF, component TIFF)
  • Olympus / Evident (VSI)
  • Hamamatsu (NDPI, NDPIS)
  • Aperio (SVS, AFI)
  • Zeiss (CZI)
  • Leica (SCN, LIF)
  • Ventana (BIF)
  • Philips (iSyntax, i2Syntax)
  • DICOM (DCM*)
    *whole-slide images

Sequential Same Slide Multiplex IF and H&E Staining for Combined Phenotypic and Morphological Characterization of FFPE Tissue Sections

Read this eBook for an introduction to a sequential staining and image analysis workflow that leverages a terminal H&E stain for the delineation of tissue architectural regions in subsequent HALO image analysis.

Introduction of a Robust Workflow for the Whole-Slide Acquisition and Co-Registration of Multiplex Immunofluorescence Tissue Images for Analysis of a 9-Color, 8-Marker Immunophenotyping Assay

Download our eBook to learn about a HALO workflow for co-registration and cellular analysis of immunofluorescence images from cyclically stained and imaged slides.

Publication Spotlight

The table below includes publications that cite the Serial Section Analysis Add-on. 

Your publication not on the list?  Drop us an email to let us know about it!

TitleAuthorsYearJournalApplicationHALO Modules
A Cdh3-β-catenin-laminin signaling axis in a subset of breast tumor leader cells control leader cell polarization and directional collective migrationHwang P, Mathur J, Cao Y, Almeida J, Ye J, Morikis V, Cornish D, Clarke M, Stewart S, Pathak A, Longmore G2023Developmental CellOncologyHighplex FL, RegistrationHALO
Standardized pathology screening of mature Tertiary Lymphoid Structures in cancersVanhersecke L, Bougouin A, Crombe A, Brunet M, Sofeu C, Parrens M, Pierron H, Bonhomme B, Lembege N, Rey C, Velasco V, Soubeyran I, Begueret H, Bessede A, Bellera C, Scoazec J-Y, Italiano A, Fridman C, Fridman W, Le Loarer F2023Laboratory InvestigationOtherHighplex FL, RegistrationHALO
Fucosylation of HLA-DRB1 regulates CD4+ T cell-mediated anti-melanoma immunity and enhances immunotherapy efficacyLester D, Burton C, Gardner A, Innamarato P, Kodumudi K, Liu Q, Adhikari E, Ming Q, Williamson D, Frederick D, Sharova T, White M, Markowitz J, Cao B, Nguyen J, Johnson J, Beatty M, Mockabee-Macias A, Mercurio M, Watson G, Chen P-L, McCarthy S, MoranSegura C, Messina J, Thomas K, Darville L, Izumi V, Koomen J, Pilon-Thomas S, Ruffell B, Luca V, Haltiwanger R, Wang X, Wargo J, Boland G, Lau E2023Nature CancerImmuno-oncologyHighplex FL, RegistrationHALO
CXCL9 and CXCL13 coordinately shape the immune-activated microenvironment of endometrial cancer via tertiary lymphoid structure formationKodama M, Nagase Y, Aimono E, Nakamura K, Takamatsu R, Abe K, Yoshimura T, Chiyoda T, Yamagami W, Aoki D, Nishihara H2023Research SquareImmuno-oncologyRegistrationHALO
African-Ancestry Associated Gene Expression Signatures and Pathways in Triple Negative Breast Cancer, a Comparison across Women of African Descent
Martini R, Delpe P, Chu T, Arora K, Lord B, Verma A, Chen Y, Gebregzabher, Oppong J, Adjei E, Jibril A, Awuah B, Bekele M, Abebe E, Kyei I, Aitpillah F, Adinku M, Ankomah K, Osei-Bonsu E, Chitale D, Bensenhaver J, Nathanson S, Jackson L, Jiagge E, Petersen L, Proctor E, Gyan K, Gibbs L, Monojlovic Z, Kittles R, White J, Yates C, Manne U, Gardner K, Mongan N, Cheng E, Ginter P, Hoda S, Elemento O, Robine N, Sboner A, Carpten J, Newman L, Davis M
Multiplex IHC, Serial Section
Multiplex Immunohistochemical Phenotyping of T Cells in Primary Prostate Cancer Ozbek B, Ertunc O, Erickson A, Vidal I, Gomes-Alexandre C, Guner G, Hicks J, Jones T, Taube J, Sfanos K, Yegnasubramanian S, DeMarzo A
2022The Prostate
Classifier, Cytonuclear, Highplex FL, Serial Section, Registration, Figure Maker, TMA
Assessment of associations between clinical and immune microenvironmental factors and tumor mutation burden in resected nonsmall cell lung cancer by applying machine learning to whole-slide imagesOno A, Terada Y, Kawata T, Serizawa M, Isaka M, Kawabata T, Imai T, Mori K, Muramatsu K, Hayashi I, Kenmotsu H, Oshima K, Urakami K, Nagashima T, Kusuhara M, Akiyama Y, Sugino T, Ohde Y, Yamaguchi K, Takahashi T T2020Cancer MedicineClassifier, Multiplex IHC, Serial Section
Neoadjuvant immunotherapy leads to pathological responses in MMR-proficient and MMR-deficient early-stage colon cancersChalabi M, Fanchi LF, Dijkstra KK, Van den Berg JG, Aalbers AG, Sikorska K, Lopez-Yurda M, Grootscholten C, Beets GL, Snaebjornsson P, Maas M, Mertz J, Veninga V, Bounova G, Broeks A, Beets-Tan RG, de Wijkerslooth TR, van Lent AU, Marsman HA, Nuijten E, Kok NF, Kuiper M, Verbeek WH, Kok M, Van Leerdam ME, Schumacher TN, Voest EE, Haanen JB2020Nature MedicineImmuno-oncologyMultiplex IHC, Serial Section
Multidimensional, quantitative assessment of PD-1/PD-L1 expression in patients with Merkel cell carcinoma and association with response to pembrolizumab Giraldo NA, Nguyen P, Engle EL, Kaunitz GJ, Cottrell TR, Berry S, Green B, Soni A, Cuda JD, Stein JE, Sunshine JC, Succaria F, Xu H, Ogurtsova A, Danilova L, Church CD, Miller NJ, Fling S, Lundgren L, Ramchurren N, Yearley JH, Lipson EJ, Cheever M, Anders RA, Nghiem PT, Topalian SL, Taube JM2018Journal for ImmunoTherapy of CancerOncology, Immuno-oncologySpatial Analysis, Serial Section

Related HALO Modules

Multiplex IHC

Quantify expression of up to five brightfield stains in any cellular compartment - membrane, nucleus or cytoplasm.

Learn More
Tissue Classifier Add-On

Separate multiple tissue classes across a tissue using a learn-by-example approach. Can be used in conjunction with all other modules (fluorescent and brightfield) to select specific tissue classes for further analysis.

Learn More
Spatial Analysis

Plot cells and objects from one or more images and perform nearest neighbor analysis, proximity analysis, and tumor infiltration analysis.

Learn More

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