Laser Capture Microdissection System Research Articles

Abstract 3768: Optimization of digital pathology through laser capture microdissection with a 405 nm laser

Abstract Tumor cellular heterogeneity is a complex problem in cancer molecular diagnostics and personalized therapy. The tumor is a product of the different types and interactions of host and immune cells. Investigators have created a variety of methods to procure separate subpopulations of the tumor microenvironment for individual analysis. One of the most successful methods of this is laser capture microdissection (LCM). This method captures specific subpopulations of cells under direct microscopic visualization. This LCM technology has successfully been used for over 20 years, revealing a variety of insights into cancer pathogenesis and mechanisms of therapeutic response employed in numerous clinical trials. Nevertheless, there are two drawbacks to the current systems. For UV cutting (365 nm), the tissue is significantly damaged by the UV light energy. Infrared (808 nm) laser systems do not damage the tissue however, this method has low power and poor resolution. Our first hypothesis to be tested is that a new class of near UV 405 nm laser will have small capture areas approaching single cells while simultaneously preventing cellular damage. The second weakness of LCM is the requirement for an open-faced tissue section without a coverslip. Moreover, when visualizing the tissue of interest, the identification of regions of interest is obscured due to the refractive index mismatch between the laser microdissection cap and the tissue surface. We hypothesize that using a volatile organic compound with a refractive index similar to glass would elucidate the tissue region of interest to allow proper visualization. We successfully developed and tested a near 405 nm wavelength LCM system that utilizes a “liquid” coverslip to visualize the sample of interest with higher clarity, resolution, and contrast for immunohistochemistry, immuno-fluorescence, blood smear slides, and conventional hematoxylin-eosin tissue staining. Overall, these critical improvements LCM technology now allow for single-cell tissue capture, system automation, tissue visualization, and opens the door for artificial intelligence-assisted spatial profiling. Citation Format: Thomas Philipson, Marissa Howard, Kevin Johnson, Amanda Still, Furkat Yunusov, Emanuel Petricoin, Virginia Espina, Noel Gonzalez, Alan Carpino, Lance Liotta. Optimization of digital pathology through laser capture microdissection with a 405 nm laser [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3768.

A Thermoplastic Transfer Film Spin-Coated for Near-Field Probe Laser-Capture Microdissection System

To improve laser-capture microdissection (LCM) resolution, we designed a near-field probe LCM system that pushed microdissection resolution from 7.5 μm to 400 nm. Here, we report a transfer film for our constructed system, replacing commercial transfer films used in conventional LCM systems. A low-melting-point ethylene vinyl acetate (EVA) copolymer was chosen as the main material. Adequate concentrations of EVA and near-infrared (NIR) absorbing dyes of an appropriate thickness to prepare the transfer film were investigated. The flatness of the film surface and the relationship between different film thicknesses and melt dot sizes were studied using atomic force microscopy and scanning electron microscopy, respectively. The results show that the flatness is approximately 4.3 nm, and the melt dot size is proportional to the transfer film’s thickness. The proper conditions for preparing the film were 17.5% (w/v) of EVA in the toluene solution with 0.001 M of NIR absorbing dyes. Using a 30 μm thick transfer film, 200 nm diameter particles on a 20 nm thick monolayer of gold particles could be captured.

Laser Capture Microdissection-Based RNA Microsequencing Reveals Optic Nerve Crush-Related Early mRNA Alterations in Retinal Ganglion Cell Layer.

PurposeTo establish a method of laser capture microdissection (LCM) and RNA microsequencing for exploring optic nerve crush (ONC)–related early mRNA alterations in retinal ganglion cell (RGC) layer.MethodsAn LCM protocol was developed using retinal tissue sections to obtain high-quality RNA for microsequencing. Cells in the RGC layer were collected by laser pressure catapulting (LPC) using a PALM Zeiss UV LCM system. The effect of section thickness and slide type on tissue capture success and RNA yield and the integrity after LCM were evaluated. The optimal LCM protocol was used to explore ONC-related early mRNA alterations in the RGC layer. Candidate genes were validated by real-time polymerase chain reaction of the RGC layer tissue dissected by “cut and LPC” using the same LCM system.ResultsWe successfully established an optimal LCM protocol using 30-µm–thick retinal tissue sections mounted on glass slides and laser pressure catapulting (LPC) to collect cells in the RGC layer and to obtain high-quality RNA for microsequencing. On the basis of our protocol, we identified 8744 differentially expressed genes that were involved in ONC-related early mRNA alterations in the RGC layer. Candidate genes included Atf3, Lgals3, LOC102551701, Plaur, Tmem140, and Maml1.ConclusionsThe LCM-based single-cell RNA sequencing allowed a new sight into the early mRNA changes of RGCs highlighting new molecules associated to ONC.Translational RelevanceThis technique will be helpful for more accurate transcriptome analysis of clinical pathological samples of ophthalmology and provide important reference for the discovery of new pathological diagnosis indicators and drug development targets.

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones.

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-µm-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum.

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (≥8) as needed for transcriptional profiling experiments.

Laser Capture Microdissection of Murine Embryonic Neural Crest Cells.

The purpose of this chapter is to provide a step-by-step protocol to enable performance of laser capture microdissection (LCM) on tissue sections from mammalian embryos or postnatal organism stages in order to collect pure populations of neural crest cells from which sufficient amounts of nucleic acids and/or protein can be obtained for quantitative analysis. The methods (1) define a strategy to genetically and indelibly label mammalian neural crest-derived cells with a fluorescent marker, thus enabling their isolation throughout the pre- and postnatal life span of the organism, and (2) describe subsequent isolation by LCM of the labeled neural crest cells, or their derivatives, from embryonic/postnatal tissue cryosections. Details are provided for using the Arcturus PixCell®IIe Laser Capture Microdissection System (Arcturus) and CapSure LCM Caps (Thermo Fisher Scientific), to which the selected cells adhere upon laser-mediated capture. The protocol outlined herein can be applied in any situation wherein limited cellular samples are available for isolation by LCM. Nucleic acids or proteins can be extracted from LCM-isolated cells and processed for high-density gene expression profiling analyses (microarrays or RNA sequencing), Real-Time PCR (q-PCR) for specific candidate gene expression, investigation of DNA methylation, as well as for varied protein analyses.

Laser Capture Microdissection in Traumatic Brain Injury Research: Obtaining Hippocampal Subregions and Pools of Injured Neurons for Genomic Analyses.

The methods presented here are based on our laboratory's 15years of experience using laser capture microdissection to obtain samples for the study of gene expression after traumatic brain injury (TBI) using a well-established rat model of experimental TBI. Here, we describe how to use the ArcturusXT laser capture microdissection system to capture swaths of specific regions of the rat hippocampus as well as specific neuronal populations defined by Fluoro-Jade C staining. Staining with Fluoro-Jade C identifies a neuron that is in the process of degeneration. We have optimized our protocols for Fluoro-Jade C tissue staining and laser capture microdissection to maintain RNA integrity which is essential for a variety of downstream applications, such as microarray, PCR array, and quantitative real-time PCR analyses.

Laser capture microdissection tailored to type 1 diabetes mellitus research.

RNA isolation from pancreatic islets poses unique challenges. Here, we present a reproducible means of obtaining high-quality RNA from juvenile rodent islets in sufficient quantities for use in ex vivo expression studies. Tissue was extracted from female non-obese diabetic (NOD) toll-like receptor 3 (TLR3)(+/+) and (TLR3)(-/-) mice in the pre-diabetic stage. Samples were frozen in liquid nitrogen, sectioned, fixed in a highly alcoholic solution, and stained with an alcoholic cresyl violet (CV) solution. Rehydration of the fixed sections was minimized. Islets were identified visually and isolated with the Leica LMD6000 laser capture microdissection (LCM) system to yield samples highly enriched in islet RNA. Real time qPCR was performed on the islet cDNA using probes for CXC chemokine ligand 10 (CXCL10), an inflammatory marker that plays a critical role in the pathogenesis of type 1 diabetes mellitus (TIDM). This method represents an improvement over currently described LCM techniques for rodent pancreatic islets and makes feasible expression studies using small amounts of starting tissue without the need for RNA pre-amplification. This has immediate implications for ongoing TIDM studies using the NOD mouse.

Anti-inflammatory effect of topical administration of tofacitinib on corneal inflammation

We evaluated an anti-inflammatory effect of topical administration of tofacitinib, janus kinase (JAK) blocker, on corneal inflammation. Topical instillation of either tofacitinib or PBS was applied after wounding BALB/c mice corneas with alkali burn. Topical instillation was performed until day 14 after injury and injured eye was analyzed. The vascularized area in the alkali burned cornea was significantly reduced in the tofacitinib group compared with that in the PBS group. The immunoreactivity of Gr-1, F4/80, IFN-γ, and phosphorylated STAT(signal transducer and activator of transcription)1 in corneal stroma was diminished significantly in the tofacitinib group. Using laser capture microdissection system and quantitative PCR array analysis, the expression levels of CXCL9, CXCL5, CCL7, CCL2, MMP(matrix metalloproteinase)-9, and STAT1 in corneal stroma were down-regulated in the tofacitinib group. In in vitro study, human fibroblast pretreated by IFN-γ showed phosphorylation of STAT1, and this phosphorylation was down-regulated by adding tofacitinib to the culture medium. These results indicate the topical application of JAK inhibitor causes down-regulation of JAK- or IFN-γ-related molecules. Therefore, we deduce that application of JAK inhibitor for topical instillation may contribute to the treatment of corneal inflammation.

Potential Application of Biological Products for the Treatment of Ocular Surface Inflammation.

Various biological products have been introduced for the treatment of autoimmune diseases. The injection of tocilizumab [anti-interleukin (IL)-6R antibody] and a tumor necrosis factor receptor fusion protein (TNFR-Fc) has been approved for the treatment of rheumatoid arthritis. We investigated the effect of the anti-IL-6R antibody and TNFR-Fc on corneal inflammation. Topical instillation of the anti-IL-6R antibody (MR16-1, 2 μg/μL; anti-IL-6R group) or TNFR1-Fc (100 μg/mL; TNFR1 group) was performed after corneal wounds were induced in BALB/c mice by alkali burns. The injured eye was analyzed on day 14 or 28 after injury, and topical instillation was performed until day 14 or day 28. Corneal stromal sections were made using a laser capture microdissection system, and total RNA from the specimens was subjected to quantitative polymerase chain reaction array analysis. Topical instillation of phosphate-buffered saline (PBS) served as a control. The vascularized area was significantly reduced in the anti-IL-6R (day 14) and TNFR1 groups (day 28) compared with that in the PBS group. In the anti-IL-6R group, the expression levels of matrix metalloproteinase-13, monocyte chemotactic protein-1, and C-C motif ligand-22 were downregulated compared with those in the PBS group. In the TNFR1 group, expression of mitogen-activated protein kinase 8 was downregulated. These results indicate the possible application of biological products for topical instillation for the treatment of corneal inflammation.

Abstract 555: A workflow for enrichment and whole genome amplification (WGA) of circulating tumor cells (CTCs) for next generation sequencing

Abstract Metastasis is the leading cause of cancer deaths. Circulating tumor cells (CTCs) are thought to be responsible for the seeding of metastases. These tumor cells are heterogeneous with variation occurring at the single-cell level. Using a flexible micro spring array (FMSA) device for label-free mechanical enrichment of CTCs from whole blood samples, we established a workflow from isolation followed by whole genome amplification (WGA) and finally sequencing of circulating tumor cells. Briefly, circulating tumor cells were collected from the FMSA using the Zeiss Laser Capture Microdissection System. Isolated cells underwent whole genome amplification using a commercial WGA kit. The quality of the amplified genomic DNA was analyzed using the TaqMan® Real-Time PCR Assays. Specific loci were then amplified with the Ion AmpliSeq™ Cancer Hotspot Panel v2 and sequenced using the Ion PGM™ System. These studies were performed to understand the genomic heterogeneity observed in circulating tumor cells. Citation Format: Merisa Nisic, Sijie Hao, Ramdane Harouaka, Si-Yang Zheng. A workflow for enrichment and whole genome amplification (WGA) of circulating tumor cells (CTCs) for next generation sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 555. doi:10.1158/1538-7445.AM2015-555

Laser capture microdissection for gene expression analysis.

Laser capture microdissection (LCM) is an excellent and perhaps the only platform to isolate homogeneous cell populations from specific microscopic regions of heterogeneous tissue section, under direct microscopic visualization. The basic operations of the LCM system are based on (a) microscopic visualization of phenotypically identified cells of interest, (b) selective adherence of cells to a melting thermolabile film/membrane using a low-energy infrared laser (IR system) or photovolatization of cells within a selected region (UV system), (c) capturing or catapulting of structurally intact cells from a stained tissue section. RNA/DNA or protein can be extracted from the cell or tissue fragments for downstream applications to quantitatively study gene expression. This method can be applied to many downstream analyses including but not limited to quantitative real-time polymerase chain reaction (PCR), microarray, DNA genotyping, RNA transcript profiling, generation of cDNA library, mass spectrometry analysis, and proteomic discovery.The application of LCM is described here to specifically and reliably obtain a homogeneous cell population in order to extract RNA to study microRNA expression by quantitative real-time PCR.

Great insight created by tiny holes; celebrating 40 years of brain micropunch technique.

Great insight created by tiny holes; celebrating 40 years of brain micropunch technique.

The application of laser microdissection in molecular detection and identification of aspergillus fumigatus from murine model of acute invasive pulmonary aspergillosis.

Invasive aspergillosis (IA) is a major concern in patients with severe immune deficiency. As antifungal susceptibility varies in different fungal pathogens, accurate and timely identification of species is becoming imperative for guidance of therapy and reducing high mortality rates in patients with IA. But, in fact, the diagnosis is challenging and new validated techniques are required for the detection and identification of clinically relevant isolates. The laser capture microdissection (LCM) system enables analysis of cytologically and/or phenotypically defined cell types from heterogeneous tissue and has been used in diagnosis and fungal species identification in pulmonary aspergillosis of white storks. To establish the experimental foundation for clinical application of the system, we microdissected and collected Blankophor-stained single hyphal strands from tissue cryosections of murine model of invasive pulmonary aspergillosis (IPA) with A. fumigatus by LCM, subsequently processed for DNA extraction, PCR sequencing, and species molecular identification. The sensitivity of LCM-PCR sequencing was 89 % (89/100), and the specificity was 100 %. Moreover, the positive predictive value and negative predictive value were 100 and 78.43 %, respectively. The result approved that the LCM-based methods had the potential for accurately diagnosis and rapidly identification fungal pathogens of IPA.

Laser‐Capture Microdissection

Laser‐Capture Microdissection

Laser microdissection and atmospheric pressure chemical ionization mass spectrometry coupled for multimodal imaging

Improvement in spatial resolution of atmospheric pressure molecular chemical imaging is required to resolve distinct surface features in the low micrometer and sub-micrometer scale. Laser capture microdissection systems have the capability to focus laser light to a few micrometers. This type of system, when employed for laser ablation (LA) mass spectrometry (MS)-based chemical imaging, has the potential to achieve high spatial resolution with multimodal optical and chemical imaging capability. A commercially available laser capture microdissection system was coupled to a modified ion source of a mass spectrometer. This design allowed for sampling of laser-ablated material via a transfer tube directly into the ionization region. Ionization of the ablated material was accomplished using atmospheric pressure chemical ionization (APCI). Rhodamine 6G dye of red permanent marker ink in a laser etched pattern as well as cholesterol and phosphatidylcholine in a cerebellum mouse brain thin tissue section were identified and imaged from the mass spectral data. Employing a spot diameter of 8 µm using the 10× microscope cutting objective and lateral oversampling resulted in a pixel size of about 3.7 µm in the same dimension. Distinguishing between features approximately 13 µm apart in a cerebellum mouse brain thin tissue section was demonstrated in a multimodal fashion co-registering optical and mass spectral images. A LA/APCI-MS system was developed that comprised a commercially available laser microdissection instrument for transmission geometry LA and a modestly modified ion source for secondary ionization of the ablated material. The set-up was successfully applied for multimodal imaging using the ability to co-register bright field, fluorescence and mass spectral chemical images on one platform.

Laser capture microdissection: Should an ultraviolet or infrared laser be used?

Laser capture microdissection (LCM) is a well-established cell separation technique. It combines microscopy with laser beam technology and allows targeting of specific cells or tissue regions that need to be separated from others. Consequently, this biological material can be used for genome or transcriptome analyses. Appropriate methods of sample preparation, however, are crucial for the success of downstream molecular analysis. The aim of this study was to objectively compare the two main LCM systems, one based on an ultraviolet (UV) laser and the other based on an infrared (IR) laser, on different criteria ranging from user-friendliness to sample quality. The comparison was performed on two types of samples: peripheral blood mononuclear cells and blastocysts. The UV laser LCM system had several advantages over the IR laser LCM system. Not only does the UV system allow faster and more precise sample collection, but also the obtained samples—even single cell samples—can be used for DNA extraction and downstream polymerase chain reaction (PCR) applications. RNA-based applications are more challenging for both LCM systems. Although sufficient RNA can be extracted from as few as 10cells for reverse transcription quantitative PCR (RT–qPCR) analysis, the low RNA quality should be taken into account when designing the RT–qPCR assays.

Aberrant Stratifin Overexpression Is Regulated by Tumor-Associated CpG Demethylation in Lung Adenocarcinoma

We previously have shown the aberrant overexpression of stratifin (SFN, 14-3-3 ς) in lung adenocarcinoma. Although SFN is known to facilitate tumor cell proliferation, the mechanism that underlies its aberrant expression has remained unclear. SFN, the downstream target of p53, often has been reported to be hypermethylated and subsequently silenced in certain cancers; however, its hypomethylation-linked reactivation has not yet been validated. In this study, we investigated the DNA methylation status of the SFN promoter region using 8 lung cancer cell lines and 32 specimens of adenocarcinoma tissue. Real-time methylation-specific PCR analysis showed that although both normal lung tissue and adenocarcinoma in situ bore a completely methylated SFN promoter, the promoter region in almost all invasive adenocarcinomas was at least partially methylated. The expression of SFN and its level of methylation were correlated strongly. Furthermore, statistical analysis revealed that the level of methylation became reduced with progression of the pathologic stage, although no clear relationship between methylation level and p53 abnormality was found. These results suggest that methylation-related silencing of SFN occurs in both normal lung tissues and adenocarcinoma in situ, and that demethylation of the SFN promoter participates in the aberrant expression of SFN in invasive adenocarcinoma cells, independently of p53 alteration. This novel finding might be informative for clarifying the mechanism that underlies the progression of early lung adenocarcinoma.

Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection

BackgroundLaser-capture microdissection (LCM) that enables the isolation of specific cell populations from complex tissues under morphological control is increasingly used for subsequent gene expression studies in cell biology by methods such as real-time quantitative PCR (qPCR), microarrays and most recently by RNA-sequencing. Challenges are i) to select precisely and efficiently cells of interest and ii) to maintain RNA integrity. The mammary gland which is a complex and heterogeneous tissue, consists of multiple cell types, changing in relative proportion during its development and thus hampering gene expression profiling comparison on whole tissue between physiological stages. During lactation, mammary epithelial cells (MEC) are predominant. However several other cell types, including myoepithelial (MMC) and immune cells are present, making it difficult to precisely determine the specificity of gene expression to the cell type of origin. In this work, an optimized reliable procedure for producing RNA from alveolar epithelial cells isolated from frozen histological sections of lactating goat, sheep and cow mammary glands using an infrared-laser based Arcturus Veritas LCM (Applied Biosystems®) system has been developed. The following steps of the microdissection workflow: cryosectioning, staining, dehydration and harvesting of microdissected cells have been carefully considered and designed to ensure cell capture efficiency without compromising RNA integrity.ResultsThe best results were obtained when staining 8 μm-thick sections with Cresyl violet® (Ambion, Applied Biosystems®) and capturing microdissected cells during less than 2 hours before RNA extraction. In addition, particular attention was paid to animal preparation before biopsies or slaughtering (milking) and freezing of tissue blocks which were embedded in a cryoprotective compound before being immersed in isopentane. The amount of RNA thus obtained from ca.150 to 250 acini (300,000 to 600,000 μm2) ranges between 5 to 10 ng. RNA integrity number (RIN) was ca. 8.0 and selectivity of this LCM protocol was demonstrated through qPCR analyses for several alveolar cell specific genes, including LALBA (α-lactalbumin) and CSN1S2 (αs2-casein), as well as Krt14 (cytokeratin 14), CD3e and CD68 which are specific markers of MMC, lymphocytes and macrophages, respectively.ConclusionsRNAs isolated from MEC in this manner were of very good quality for subsequent linear amplification, thus making it possible to establish a referential gene expression profile of the healthy MEC, a useful platform for tumor biomarker discovery.

Low prevalence of HPV in Brazilian children with retinoblastoma

Retinoblastoma is the most frequent intra-ocular malignant tumor of the childhood, occurring in 1 of 18,000-30,000 live births. Little is known about the causes of sporadic retinoblastoma and only a few authors have investigated the etiologic role of human papillomavirus (HPV), with controversial results. Formalin-fixed, paraffin-embedded tissue blocks containing retinoblastoma were retrieved from the archives of the Department of Pathology at Hospital A C Camargo, São Paulo, Brazil. All patients were treated with enucleation (21 children had both eyes enucleated). Retinoblastoma and, when possible, normal retina of each specimen, were micro-dissected under direct light microscopic visualization by using a PixCell II Laser Capture Micro-dissection System. The DNA quality was evaluated by polymerase chain reaction (PCR) amplification of 110 base pairs fragment of the human β-globin gene using primers PCO3+/PCO4+. All globin positive specimens were analyzed by PCR for the presence of HPV DNA using consensus primers GP5+/GP6+. A total of 154 specimens were evaluated. Forty-four patients also had normal retinal specimens available for analysis of DNA HPV. The DNA HPV prevalence among all tumor specimens was 4.6% (95% CI 2.0; 8.8) (7 positive specimens/153 adequate specimens). Among normal retinal specimens, the DNA HPV prevalence was 9.1% (95% CI 2.9; 20.5) (4 positive specimens/44 specimens). There was no statistically significant difference between these rates (P = 0.318). Excluding any experimental failure, our results indicate a low prevalence of HPV DNA in retinoblastomas. We were therefore unable to conclude about the association between these oncogenic viruses and this rare pediatric neoplasm.