Comparison of conventional and liquid-based cytology using The Paris System for Reporting Urinary Cytology.

BACKGROUND: This retrospective study was conducted to compare the conventional cytospin method and ThinPrep liquid-based urinary cytology in diagnosing bladder cancer using The Paris System (TPS) of classification. METHODS: We retrieved files for 2020, at the Cytopathology Department of Laiko Hospital, of urinary cases diagnosed according to TPS. Cytospin and ThinPrep slides were separately reviewed and new diagnoses were rendered, then compared with the original diagnosis and histology when available. Risk of high-grade malignancy (ROHM) for each TPS category was assessed, along with accuracy parameters of each method and their combination. RESULTS: The study material comprised 100 cases of void urinary cytology classified as 20 high-grade urothelial carcinoma (HGUC = TPS5) cases, 20 of suspicion for HGUC (SHGUC = TPS4), 25 of atypical urothelial cells (AUC = TPS3), and 35 of negative for HGUC (NHGUC = TPS2). A single inadequate (TPS1) case and 4 of low-grade urothelial neoplasm (TPS6) were excluded as small in number. The ROHM was 95% for HGUC, 55% for SHGUC, 28% for AUC and 5.7% for NHGUC. Agreement with the original diagnosis was 86% for cytospin and 82% for ThinPrep. No significant differences were observed among the two techniques or their combination regarding sensitivity and specificity, with a mild advantage for cytospin. Interobserver reproducibility and repeatability were high. CONCLUSION: No significant differences were found concerning sensitivity and specificity between cytospin and ThinPrep when applying TPS criteria. TPS is a reliable classification scheme for either conventional/cytospin or liquid-based cytology, or their combination.

Stability of canine and feline cerebrospinal fluid samples regarding total cell count and cell populations stored in "TransFix®/EDTA CSF sample storage tubes".

BACKGROUND: Because of fast leucocyte degeneration in cerebrospinal fluid (CSF) laboratory examinations of CSF samples should be performed approximately within 30 min after withdrawal. This study examines the storage of canine and feline CSF samples in "TransFix®/EDTA CSF Sample Storage Tubes" (Cytomark, Buckingham, UK) for preventing leucocytes from degeneration, so that routine and flow cytometry examinations are feasible up to 3 days after sampling. RESULTS: After storage in TransFix® tubes, leukocytes could not be adequately stained with Türk's solution and differentiating between erythrocytes and leukocytes was cumbersome. In addition, the cell morphology could not be sufficiently assessed on cytospin preparations because of shrunken leukocytes and indistinct cell nuclei. In contrast, by flow cytometry, a significantly higher cell count was measured over the entire study period in the samples stored in TransFix® tubes compared to the untreated samples. The antibodies (AB) against CD3, CD4 and CD21, against CD11b and against CD45 showed a good binding strength and thus enabled a good differentiation of cell populations. However, after storage in the TransFix® tubes, monocytes were no longer detectable using an AB against CD14. CONCLUSION: Based on these results, "TransFix®/EDTA CSF Sample Storage Tubes" can be used for extended storage prior to flow cytometric analysis of lymphocytes and granulocytes in CSF samples but not for detecting monocytes. However, standard examinations, such as microscopic cell counting and morphological cell assessment should be performed on fresh CSF samples.

Management of cytologic material, preanalytic procedures and biobanking in lymph node cytopathology.

The range of pathologies that lymph node (LN) fine needle cytology (FNC) may encounter is extremely wide and ancillary techniques, in addition to traditional smears, are generally required to reach reliable cytologic diagnoses. Storing part of the cytologic material may be useful or necessary for molecular testing. The main difficulties concern the generally small size of the sample and the different methods of acquisition of LN-FNC. Therefore, the preanalytic phase is extremely important for LN-FNC. This article outlines the management of LN-FNC material, vials, technical devices (e.g.: additional smears, cytospin slides, LBC slides, cards, resins, etc.) and main ancillary techniques to assess their optimal application, taking into account the different diagnostic needs and cell storage.

JC and BK virus DNA detection in archival slides of urine cytospin from renal transplant patients.

BACKGROUND: Although identifying cytological viral inclusions (decoy cells) in the urine is relatively easy, distinguishing between Polyomaviruses BKV and JCV is not possible. Few studies have been published regarding JCV detection in kidney transplant recipients. OBJECTIVE: To evaluate the incidence of BKV and JCV DNA in archival slides of urine cytospin material from renal transplant patients. METHODS: A total of 44 urine specimens were evaluated cytologically for the presence of viral inclusions (decoy cells) and by nested polymerase chain reaction to differentiate between JCV and BKV in DNA isolated from archival slides of urine cytospin material. RESULTS: Of the 44 urine specimen donors, 9 (20.5%) patients had at least 1 sample with alterations suggestive of or compatible with viral infection (decoy cells), and 3 had urine samples with cellular atypias/neoplasias. Additionally, 24/44 (54.5%) patients had PCR-positive DNA for Polyomavirus in at least 1 sample, including 11/44 who were positive for BKV (25%) and 16/44 who were positive for JCV (36.36%), with 3 (6.8%) patients showing viral coinfection. Regarding transplantation time, only JCV was statistically significant (P = .019) for periods longer than 10 years. CONCLUSIONS: The results highlight the potential use of archival slides of urine cytospin material to differentiate BKV and JCV and demonstrate the importance of improved JCV detection for later kidney transplant recipients.

Cytological preparations for molecular analysis: A review of technical procedures, advantages and limitations for referring samples for testing.

Minimally invasive procedures such as endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) must yield not only good quality and quantity of material for morphological assessment, but also an adequate sample for analysis of molecular markers to guide patients to appropriate targeted therapies. In this context, cytopathologists worldwide should be familiar with minimum requirements for refereeing cytological samples for testing. The present manuscript is a review with comprehensive description of the content of the workshop entitled Cytological preparations for molecular analysis: pre-analytical issues for EBUS TBNA, presented at the 40th European Congress of Cytopathology in Liverpool, UK. The present review emphasises the advantages and limitations of different types of cytology substrates used for molecular analysis such as archival smears, liquid-based preparations, archival cytospin preparations and FTA (Flinders Technology Associates) cards, as well as their technical requirements/features. These various types of cytological specimens can be successfully used for an extensive array of molecular studies, but the quality and quantity of extracted nucleic acids rely directly on adequate pre-analytical assessment of those samples. In this setting, cytopathologists must not only be familiar with the different types of specimens and associated technical procedures, but also correctly handle the material provided by minimally invasive procedures, ensuring that there is sufficient amount of material for a precise diagnosis and correct management of the patient through personalised care.

Detection of central venous catheter-related bloodstream infections in haematooncological patients.

BACKGROUND: Catheter-related bloodstream infections (CRBSIs) are currently detected in patients with clinically suspicion. The aim of our study was to evaluate whether CRBSIs could be anticipated and detected in a subclinical stage by peptide nucleic acid fluorescence in situ hybridization (PNA FISH) using universal hybridization probes or acridine orange leucocyte cytospin (AOLC) tests in haematooncological patients with central venous catheters (CVCs) in situ. MATERIALS AND METHODS: Peptide nucleic acid fluorescence in situ hybridization and AOLC tests using blood samples from one CVC lumen/port chamber in haematooncological patients were continuously performed. These results were compared to those obtained from routinely performed CRBSI diagnostic tests. RESULTS: One hundred and eighty-two patients with 342 catheter periods were investigated. Seventeen CRBSI cases were detected in 6466 CVC days by routine measures resulting in a CRBSI rate of 2.6/1000 catheter days. Two of 17 showed positive PNA FISH tests, and five positive AOLC test results before the diagnosis were established with routine measures. The screening revealed further seven patients with positive universal PNA FISH tests and 10 positive AOLC tests without symptoms indicative for infection and were therefore considered not to have CRBSI. CONCLUSIONS: Sampling of only one CVC lumen/port chamber screening for CRBSI in haematooncological patients seems not to be a useful tool for anticipative diagnosis of CRBSI. Reasons for false-negative results might include origin of CRBSIs from the other CVC lumina not sampled for screening, and false-positive results might origin from catheter colonization without subsequent spread of micro-organisms into the peripheral bloodstream.

A Comparison of Different Sample Processing Protocols for MALDI Imaging Mass Spectrometry Analysis of Formalin-Fixed Multiple Myeloma Cells.

Sample processing of formalin-fixed specimens constitutes a major challenge in molecular profiling efforts. Pre-analytical factors such as fixative temperature, dehydration, and embedding media affect downstream analysis, generating data dependent on technical processing rather than disease state. In this study, we investigated two different sample processing methods, including the use of the cytospin sample preparation and automated sample processing apparatuses for proteomic analysis of multiple myeloma (MM) cell lines using imaging mass spectrometry (IMS). In addition, two sample-embedding instruments using different reagents and processing times were considered. Three MM cell lines fixed in 4% paraformaldehyde were either directly centrifuged onto glass slides using cytospin preparation techniques or processed to create paraffin-embedded specimens with an automatic tissue processor, and further cut onto glass slides for IMS analysis. The number of peaks obtained from paraffin-embedded samples was comparable between the two different sample processing instruments. Interestingly, spectra profiles showed enhanced ion yield in cytospin compared to paraffin-embedded samples along with high reproducibility compared to the sample replicate.

Cytospin performance when using Paris system for reporting urinary cytology.

Objectives: The Paris System (TPS) for Reporting Urine Cytology has significantly improved the approach to evaluating urine cytology. TPS criteria were defined mainly according to ThinPrep and SurePath preparations, as they are widely utilized. The objective of this study is to validate urine cytology interpretation according to the TPS classification using cytospin technique in relation to the gold slandered histology. Material and Methods: This retrospective study examined and analyzed 316 urine specimens from King Abdulaziz University Hospital between 2015 and 2020. Cytospin technique is performed for all cases. Slides were recategorized using TPS criteria, then compared with the original histology diagnosis. Results: According to the TPS, 108 cases were classified as 101 AUC (32%), 95 NEG (30%), 59 HGUC (18.7%), 31 SHGUC (9.8%), and 30 (9.5%) others. The computed sensitivity of cytospin in urine cytology was 94.7%, with 73.9% specificity, a positive predictive value of 85.6%, a negative predictive value of 89.5%, and overall accuracy of 86.8%. Conclusion: Urine cytology testing is considered to be a non-invasive and sensitive method to screen for urothelial carcinoma. TPS defined standards are reliable on cytospin prepared slides for reporting urine cytology.

Cytospin-A Regulates Colorectal Cancer Cell Division and Migration by Modulating Stability of Microtubules and Actin Filaments.

Proteins that interact with cytoskeletal elements play important roles in cell division and are potentially important targets for therapy in cancer. Cytospin-A (CYTSA), a protein known to interact with actin and microtubules, has been previously described to be important in various developmental disorders, including oblique facial clefting. We hypothesized that CYTSA plays an important role in colorectal cancer (CRC) cell division. The effects of CYTSA depletion on CRC cell proliferation were analyzed using cell growth assays, microscopic analyses of live and fixed cells, and time-lapse imaging. CYTSA depletion led to inhibition of cell proliferation, significant increases in CRC cell death, and accumulation of doublet cells during and following cell division. Depletion of CYTSA also resulted in strong inhibition of CRC cell migration and invasion. Mechanistically, CYTSA depletion resulted in significant decreases in the stability of microtubules and altered polymerization of actin filaments in CRC cells. Finally, bioinformatic analyses were performed to determine the correlation between CYTSA expression and survival of patients with CRC. Interestingly, a strong correlation between high CYTSA expression and poor survival was observed in the TCGA adenocarcinoma data set but not in an independent data set. Since inhibiting CYTSA significantly reduces CRC cell proliferation, migration, and invasion, targeting CYTSA may be a potential novel therapeutic option for patients with metastatic CRC.

Refining M1 stage in medulloblastoma: criteria for cerebrospinal fluid cytology and implications for improved risk stratification from the HIT-2000 trial.

BACKGROUND: Medulloblastoma is the most common malignant paediatric brain tumour, and cerebrospinal fluid (CSF) dissemination (M1 stage) is a high-risk prognostic factor. Criteria for CSF evaluation and for differentiating M0 from M1 stage are not clearly defined, and the prognostic significance of M1 stage in this context is unknown. PATIENTS AND METHODS: CSF investigations from 405 patients with medulloblastoma of the prospective multicenter trial HIT-2000 (HIirnTumor-2000) were reviewed. Data from 213 patients aged ≥4 years were related to 5-year progression-free (5y-PFS) and overall survival. RESULTS: Patients with cytological tumour dissemination only (M1 stage only) aged ≥4 years (n = 18) and patients with radiologically detected metastases (M2/3, n = 85) showed a worse 5y-PFS than M0 patients (n = 110) without signs of metastatic disease (5y-PFS 61.1% and 59.6% vs 80.7%; p < 0.02 and p < 0.01, log rank). Patients with positive samples drawn early after surgery who turned negative within 14 days postoperatively (n = 9) and patients with atypical cells (n = 6) showed a 5y-PFS similar to M0 patients. No tumour cells were detected in samples containing <10 nucleated cells. Analysis of cytological criteria showed a better predictive value for tumour cell clusters than ≥2 individual tumour cells. CONCLUSION: Based on our results, we suggest that CSF medulloblastoma staging should be performed 14 days postoperatively by lumbar puncture, and specimens should contain at least 10 nucleated cells. Cytological tumour dissemination alone (M1 stage only) appears a high-risk prognostic factor associated with an outcome comparable to M2/M3 stage. Tumour cell clusters seem to have a greater impact on prognosis than single tumour cells. This should be validated further.

Isolation of Microvesicles from Human Circulating Neutrophils.

Neutrophil-derived microvesicles (NDMVs) are liberated by neutrophils upon cell activation by molecules. Once activated, neutrophils are primarily involved in acute inflammation; however, the microvesicles they produce are largely anti-inflammatory. NDMVs have been shown to protect cartilage during inflammatory arthritis. They exert these effects by inhibiting or affecting the function of target cells, including macrophages. NDMVs have the potential to act as disease-modifying agents, especially for inflammatory diseases. This protocol describes a method using differential centrifugation to separate neutrophils from whole human blood. Subsequently, neutrophils are identified by cytospin and Wright's staining, and then the NDMVs are isolated using differential centrifugation.

Functionally Active Eosinophil Purification from Peripheral Blood.

The choice of isolation technique for human peripheral blood eosinophils contributes to the understanding of clinically relevant data derived from in vitro research. Since the 1990s, eosinophils have been conventionally isolated via density gradient centrifugation followed by negative immunomagnetic selection using anti-CD16 antibody-coated magnetic beads. Due to recent advancements in molecular techniques, "newer" methods have been made commercially available that drastically reduce user handling and processing time while maintaining high population purity. Here, we describe an isolation procedure using one of these methods, the human MACSxpress® Whole Blood Isolation Kit, as well as outline protocols for differential staining and flow cytometry analysis to evaluate the purity and activation state of isolated cells. In addition, we highlight an in vitro degranulation assay that may be used to verify the intact functionality of the isolated eosinophils.

Optimization of Sample Preparation Using Glass Slides for Spectral Pathology.

The clinical translation of Fourier transform infrared (FT-IR) microspectroscopy in pathology will require bringing this technique as close as possible to standard practice in pathology departments. An important step is sample preparation for both FT-IR microspectroscopy and pathology. This should entail minimal disruption of standard clinical practice while achieving good quality FT-IR spectral data. In fact, the recently described possibility of obtaining FT-IR spectra of cells placed on glass substrates brings FT-IR microspectroscopy closer to a clinical application. We have now furthered this work in order to identify two different types of lung cancer cells placed on glass coverslips. Two types of sample preparation which are widely used in pathology, cytospin and smear, have been used. Samples were fixed with either methanol, used in pathology, or formalin (4% paraformaldehyde) used widely in spectroscopy. Fixation with methanol (alcohol-based fixative) removed lipids from cells causing a decrease in intensity of the peaks at 2850 cm-1 and 2920 cm-1. Nevertheless, we show for the first time that using either type of sample preparation and fixation on thin glass coverslips allowed to differentiate between two different types of lung cancer cells using either the lipid region or the fingerprint region ranging from 1800 cm-1 to 1350 cm-1. We believe that formalin-fixed cytospin samples would be preferred to study cells on thin coverslips using FT-IR microspectroscopy. This work presents a clear indication for future advances in clinical assessment of samples within pathology units to gain a deeper understanding of cells/tissues under investigation.

Immunocytochemistry practices in European cytopathology laboratories-Review of European Federation of Cytology Societies (EFCS) online survey results with best practice recommendations.

BACKGROUND: Variability in preanalytical and analytical steps for immunocytochemistry (ICC) on cytology samples is poorly defined. The objective of this study was to evaluate current practices for ICC on cytology samples in European laboratories. METHODS: A link to an online survey with 19 questions about ICC practices was distributed to cytology laboratories through national representatives in the European Federation of Cytology Societies. RESULTS: In total, 245 laboratories responded to the survey by January 30, 2019. Cell blocks, cytospins, liquid-based cytology (LBC) preparations, and smears alone or in combination with other preparations were used for ICC in 38%, 22%, 21%, and 19% of laboratories, respectively. In general, various combinations of preparations were used for ICC in greater than one-half of laboratories (147 of 245; 60%), whereas only 1 specific type of cytology preparation was used in the remaining 98 of 245 laboratories (40%) laboratories. The majority of laboratories (217 of 226; 96%) performed ICC on automated platforms using protocols that were the same as those used for formalin-fixed, paraffin-embedded samples (238 of 527 laboratories; 45%), either optimized (138 of 527 laboratories; 26%) or optimized and validated (151 of 527 laboratories; 29%) for cytology preparations. Positive control slides, negative control slides, and external quality control were used in 174 of 223 (78%), 112 of 223 (50%), and 111 of 120 (50%) laboratories, respectively. Greater than 1000 ICC tests were performed yearly in 34% of laboratories (65 of 191; average, 1477 tests; median, 500 tests). CONCLUSIONS: ICC is extensively performed in European laboratories using variously prepared cytology preparations on automated platforms, mostly without quality-assurance measures.

Adequacy criteria for voided urine cytology using cytospin preparations.

BACKGROUND: Previous studies have suggested that urine cytology adequacy may be related to both specimen volume and cellularity. METHODS: The authors reviewed cytospin preparations from 314 urine specimens (162 voided and 152 instrumented specimens) found to have high-grade urothelial carcinoma on biopsy. RESULTS: The sensitivity of instrumented urine cytology was significantly higher than that of voided cytology (82% vs 25%; P < .001). The cellularity (at least 30 urothelial cells/10 high-power fields [HPF]) of instrumented urine specimens also was significantly higher than that of voided specimens (57% vs 9%; P < .001). The sensitivity of voided urine cytology with a cellularity of 20 to 39 cells per 10 HPF was significantly higher than that of cases with <20 cells per 10 HPF (77% vs 19%; P < .001). The sensitivity decreased with higher cellularity for both types of specimens. The sensitivity of voided cases with a volume of at least 30 mL was higher than that of cases with a volume <30 mL, but this was not statistically significant (31% vs 17%; P = .07). CONCLUSIONS: The sensitivity of voided urine cytology for high-grade urothelial carcinoma is significantly associated with urothelial cellularity but not specimen volume. Both the cellularity and sensitivity of voided urine specimens are less than that of instrumented specimens.

Application of Fluorescence In Situ Hybridization on Cerebrospinal Fluid Cytospins for the Detection of Residual Leukemic Cells in Patients With Childhood Acute Lymphoblastic Leukemia.

OBJECTIVES: Diagnosis of central nervous system involvement in acute lymphoblastic leukemia (ALL) requires morphologic expertise; therefore, we evaluated interphase fluorescence in situ hybridization (iFISH) of cerebrospinal fluid (CSF) cytospin preparations as a potential complementary test. METHODS: Twenty-three CSF cytospin specimens from 13 pediatric patients with ALL were included. iFISH probes detecting BCR-ABL1, ETV6-RUNX1, and KMT2A rearrangement and CDKN2A deletion, which were present at initial diagnosis, were used on follow-up CSF cytospin specimens and were compared with cytology. RESULTS: Seventeen (73.9%) follow-up specimens showed concordant results between iFISH and cytology. Two (8.7%) samples with discordant results were positive by iFISH but not by cytology; one (4.3%) was positive only by cytology. In the remaining three (13.0%) specimens, too few cells were available for cytology, whereas iFISH interpretation was possible. CONCLUSIONS: iFISH of CSF cytospin preparations improves malignant cell detection in pediatric ALL.

Utility of Cytospin and Cell block Technology in Evaluation of Body Fluids and Urine Samples: A Comparative Study.

BACKGROUND: Cytologic examination of body fluids commonly involves the use of direct or sediment smears, cytocentrifuge preparations, membrane filter preparations, or cell block sections. Cytospin and cell block techniques are extremely useful in improving cell yield of thin serous effusions and urine samples, and ensure high diagnostic efficacy. MATERIALS AND METHODS: We studied cytospin preparations and cell block sections prepared from 180 samples of body fluids and urine samples to compare the relative efficiency of cell retrieval, preservation of cell morphology, ease of application of special stains, and diagnostic efficacy. Samples were collected and processed to prepare cytospin smears and cell block sections. RESULTS: We observed that overall, cell yield and preservation of individual cell morphology were better in cytospin preparations as compared to cell blocks, while preservation of architectural pattern was better in cell block sections. The number of suspicious cases also decreased on cell block sections, with increased detection of malignancy. It was difficult to prepare cell blocks from urine samples due to low cellularity. CONCLUSIONS: Cytospin technology is a quick, efficient, and cost-effective method of increasing cell yield in hypocellular samples, with better preservation of cell morphology. Cell blocks are better prepared from high cellularity fluids; however, tissue architecture is better studied, with improved rate of diagnosis and decrease in ambiguous results. Numerous sections can be prepared from a small amount of material. Special stains and immunochemical stains can be easily applied to cell blocks. It also provides a source of archival material.

Histones Acetylation and Cancer Stem Cells (CSCs).

Chromatin decondensation is a key mechanism that guarantees gene transcription and repair of the genome, regulated mainly by the acetylation of histones. Emerging evidence has pointed out to histones as a new controlling mechanism of stem cell maintenance and fate. In this chapter, we will focus on the methods used to enrich tumor cell lines for cancer stem cells, and in the methods to identify the status of the histone acetylation in cancer cells and stem cells using immunofluorescence, invasion, and adhesion assays and identification of nuclear size.

High-Grade Urothelial Carcinoma on Urine Cytology Resembling Umbrella Cells.

CONTEXT: High-grade urothelial carcinoma (UC) cells have many appearances on urine cytology, but according to The Paris System, they can be easily distinguished from umbrella cells. OBJECTIVE: We aimed to define the incidence and appearance of high-grade UC cells that resemble umbrella cells in Cytospin preparations on urine cytology. RESULTS: Cytospin preparations from 331 cases with biopsy follow-up (230 benign/low-grade and 101 malignant [22 carcinoma in situ, 52 papillary, 19 invasive UC, 8 other] cases) were reviewed. A total of 18 cases with malignant cells resembling umbrella cells were identified (17.8% of the malignant cases) and were the only type of malignant cell in 3% of the cases. Two patterns were identified. Tumor cells were either identifiable by at least 20 abnormal cells which were large, had abundant cytoplasm but an elevated nuclear-to-cytoplasmic ratio, and markedly enlarged, round-to-elongated nucleoli, or else rare cells with abundant cytoplasm but obviously malignant nuclei. Cells without nucleoli or obviously malignant nuclei were not specific. CONCLUSIONS: Malignant cells resembling umbrella cells can be seen in up to 17% of urine cytology specimens.

Risk stratification of cervical disease using detection of human papillomavirus (HPV) E4 protein and cellular MCM protein in clinical liquid based cytology samples.

BACKGROUND: While human papillomavirus (HPV) DNA testing offers high sensitivity for the detection of significant cervical disease, its specificity is suboptimal given the high prevalence of transient HPV infections (CIN1 or less). Biomarkers to identify those suffering from low grade disease from those with high grade disease could save healthcare costs and reduce patient anxiety. OBJECTIVE: The objective of the present work was to develop and test an immunohistochemistry (IHC)-based dual viral and cellular biomarker strategy which was applicable to liquid based cytology (LBC) samples. STUDY DESIGN: We developed a novel IHC assay for detection of HPV E4 and cellular minichromosome maintenance (MCM) proteins in routinely taken cervical LBC samples using cytospin-prepared slides. The assay was applied to a prospective cohort of Scottish women referred to a colposcopy clinic due to preceding cytological abnormalities. The performance of the biomarkers for detection of clinically insignificant (CIN1 or less) versus significant disease was determined. RESULTS: A total of 81 women were recruited representing 64 cases of <=CIN1 and 28 of CIN2 + . Biomarker performance relative to histopathology outcomes showed high levels of MCM detection was significantly associated with CIN2+ (p = 0.03) while E4 was detected more frequently in <=CIN1 (p = 0.06). CONCLUSIONS: Combined detection of a host proliferation marker and a marker of viral gene expression could allow triage of cases of clinically insignificant disease prior to colposcopy. However, there was overlap between distributions of MCM levels in CIN2+ and <=CIN1 suggesting that additional biomarkers would be required for improved specificity. Combined with cytospin-prepared slides this approach could provide a means of risk stratification of disease in low resource settings.