Cytogenetic Slides Research Articles

Cell Culture and Slide Preparation for Cytogenetic Studies of Hematological Neoplasms.

Hematological neoplasms are heterogeneous diseases with various subtypes, each with its unique genomic features. Cell culture and slide preparation are essential steps to enrich and collect sufficient neoplastic cells for cytogenetic studies of the neoplasms. This chapter describes methods that are commonly used for culturing hematological neoplastic cells and preparing cytogenetic slides for clinical diagnosis and research of the neoplasms.

Fluorescence In Situ Hybridization of DNA Probes on Mitotic Chromosomes of the Mexican Axolotl.

Fluorescence in situ hybridization (FISH) is used extensively for visual localization of specific DNA fragments (and RNA fragments) in broad applications on chromosomes or nuclei at any stage of the cell cycle: metaphase, anaphase, or interphase. The cytogenetic slides that serve as a target for the labeled DNA probe might be prepared using any approach suitable for obtaining cells with appropriate morphology for imaging and analysis. In this chapter, we focus on the application of molecular cytogenetic methods such as DNA labeling, slide preparation, and in situ hybridization related to cells from Mexican axolotl.

Telomere aberrations, including telomere loss, doublets, and extreme shortening, are increased in patients with infertility

To test the hypothesis that telomere shortening and/or loss are risk factors for infertility. Retrospective analysis of the telomere status in patients with infertility using conventional cytogenetic data collected prospectively. Academic centers. Cytogenetic slides with cultured peripheral lymphocytes from 50 patients undergoing fertility treatment and 150 healthy donors, including 100 donors matched for age. Cytogenetic slides were used to detect chromosomal and telomere aberrations. Telomere length and telomere aberrations were analyzed after telomere and centromere staining. The mean telomere length of patients consulting for infertility was significantly less than that of healthy donors of similar age. Moreover, patients with infertility showed significantly more extreme telomere loss and telomere doublet formation than healthy controls. Telomere shortening and/or telomere aberrations were more pronounced in patients with structural chromosomal aberrations. Dicentric chromosomes were identified in 6/13 patients, with constitutional chromosomal aberrations leading to chromosomal instability that correlated with chromosomal end-to-end fusions. Our findings demonstrate the feasibility of analyzing telomere aberrations in addition to chromosomal aberrations, using cytogenetic slides. Telomere attrition and/or dysfunction represent the main common cytogenetic characteristic of patients with infertility, leading to potential implications for fertility assessment. Pending further studies, these techniques that correlate the outcome of assisted reproduction and telomere integrity status may represent a novel and useful diagnostic and/or prognostic tool for medical care in this field.

Качество цитогенетических исследований: проблемы и пути улучшения

Представлены результаты экспертной оценки качества цитогенетических исследований в лабораториях РФ в системе межлабораторных сличительных испытаний «ФСВОК» в 2018-2019 гг. Обсуждаются наиболее частые причины неудовлетворительных результатов экспертизы и возможные пути улучшения качества цитогенетических исследований. We report the results of quality assessment for preparation of cytogenetic slides and chromosomal analysis in the laboratories of Russian Federation in the system of the interlaboratory comparative examinations “FSVOK” in 2018-2019. Common causes of poor results of assessment and the ways for improvement of quality for cytogenetic investigations are discussed.

Retrospective cohort study and biobanking of patients treated for hemangioma in childhood – telomeres as biomarker of aging and radiation exposure

Purpose: Cohorts allowing joint epidemiological and biological analyses are essential for radiation risk assessment. The French Hemangioma Cohort (FHC), studied within the European project EpiRadBio, is one of the rare cohorts suitable for studying the effect of low dose radiation exposure (<100 mGy at organs), with a long-term follow-up. This highly homogeneous cohort consists of healthy individuals belonging to a normal population, except for the presence of skin hemangioma (age at exposure: between 6 months and 3 years of age). Published epidemiological studies have demonstrated that the risk of developing cancer is three times higher in the exposed individuals than in the general population. Here, we present the biobanking of samples (nucleated blood cells, cytogenetic slides of T and B lymphocytes) from the FHC and a primary feasibility study of biomarker analysis focusing on mean telomere length (MTL). Telomeres act as an internal clock, regulating the lifetime of the cell by their shortening during cell division. MTL is thus a biomarker of age. Many in vitro studies have linked MTL and radiosensitivity. The FHC will make it possible to discriminate between the effects of aging and radiation on this biomarker.Conclusion: The establishment of a biobank of essentially healthy individuals (369 in total), exposed 40–70 years before, during their early childhood, is a logistical challenge. Even among those who previously participated to a self-questionnaire based study, the response rate was only 30%. The first biomarker to be studied was the MTL to discriminate age effects from those of radiation exposure. MTL showed significant variation within age groups (4–11 kb) in both the exposed and non-exposed groups. MTL within the limited age window (i.e. 40–73 year) examined, showed age-dependent changes of 46 bp/year, consistent with the age-dependent decline of 41 bp/year previously reported. We observed no significant changes in MTL according to the average active bone marrow dose. However, we were able to demonstrate that exposure to radiation causes the loss of cells with, on average, shorter telomeres, by applying a model in which both the heterogeneity of the individual dose received at the bone marrow and the heterogeneity of the intercellular distribution of MTL were taken into account.

Establishing cytogenetic biodosimetry laboratory in Saudi Arabia and producing preliminary calibration curve of dicentric chromosomes as biomarker for medical dose estimation in response to radiation emergencies.

In cases of public or occupational radiation overexposure and eventual radiological accidents, it is important to provide dose assessment, medical triage, diagnoses and treatment to victims. Cytogenetic bio-dosimetry based on scoring of dicentric chromosomal aberrations assay (DCA) is the “gold standard” biotechnology technique for estimating medically relevant radiation doses. Under the auspices of the National Science, Technology and Innovation Plan in Saudi Arabia, we have set up a biodosimetry laboratory and produced a national standard dose–response calibration curve for DCA, pre-required to estimate the doses received. For this, the basic cytogenetic DCA technique needed to be established. Peripheral blood lymphocytes were collected from four healthy volunteers and irradiated with radiation doses between 0 and 5 Gy of 320 keV X-rays. Then, lymphocytes were PHA stimulated, Colcemid division arrested and stained cytogenetic slides were prepared. The Metafer4 system (MetaSystem) was used for automatic and manually assisted metaphase finding and scoring of dicentric chromosomes. Results were fit to the linear-quadratic dose–effect model according to the IAEA EPR-Biodosimetry-2011 report. The resulting manually assisted dose–response calibration curve (Y = 0.0017 + 0.026 × D + 0.081 × D2) was in the range of those described in other populations. Although the automated scoring over-and-under estimates DCA at low (<1 Gy) and high (>2 Gy) doses, respectively, it showed potential for use in triage mode to segregate between victims with potential risk to develop acute radiotoxicity syndromes. In conclusion, we have successfully established the first biodosimetry laboratory in the region and have produced a preliminary national dose–response calibration curve. The laboratory can now contribute to the national preparedness plan in response to eventual radiation emergencies in addition to providing information for decision makers and public health officials who assess the magnitude of public, medical, occupational and accidental radiation exposures.

FISHprep: A Novel Integrated Device for Metaphase FISH Sample Preparation

We present a novel integrated device for preparing metaphase chromosomes spread slides (FISHprep). The quality of cytogenetic analysis from patient samples greatly relies on the efficiency of sample pre-treatment and/or slide preparation. In cytogenetic slide preparation, cell cultures are routinely used to process samples (for culture, arrest and fixation of cells) and/or to expand limited amount of samples (in case of prenatal diagnostics). Arguably, this expansion and other sample pretreatments form the longest part of the entire diagnostic protocols spanning over 3–4 days. We present here a novel device with an integrated expansion chamber to culture, arrest and fix metaphase cells followed by a subsequent splashing protocol leading to ample metaphase chromosome spreads on a glass slide for metaphase FISH analysis. The device provides an easy, disposable, low cost, integrated solution with minimal handling for metaphase FISH slide preparation.

Oligonucleotide (ODN) fluorescence in situ hybridization (Oligo-FISH) and conventional FISH allow enumeration of 24 chromosomes in 6 successive hybridizations performed in a single day

OBJECTIVE: Enumerate 24 human chromosomes on a single cell in less than 24 h by FISH. DESIGN: Using conventional FISH experiments, no more than 3 hybridizations can be performed sequentially. Oligo-FISH probes are currently available in 4 visible fluors permitting enumeration of 4 chromosomes per hybridization. Because of low stringency conditions and short hybridization time (5 min), up to 6 successive FISH experiments can be performed on the same slide without signal loss. Performing 5 successive Oligo-FISH with a combination of 4 chromosomes (FISH 1: 3, 7, 12, 16; FISH 2: 2, 13-21, 18, 20; FISH 3: 6, 8, 9q12, 11; FISH 4: X, Yq12, 15, 17; FISH 5: 1q12, 4, 10, 14-22) and 1 hybridization with 4 chromosome speceific BAC probes (FISH 6: 5, 19, 21, 22), 24 chromosomes in the same cell were enumerated in less than 24 h. All cocktail probes were evaluated on peripheral blood and on discarded human blastomeres. MATERIALS AND METHODS: Cytogenetic Slides from 5 chromosomally normal males were prepared as previously described. Blastomere Slides from 20 discarded embryos were prepared as previously described. ODN sequence Design: ODN probes have been designed to detect 20 human chromosomes that harbor specific repeats. BAC probes were selected from the RP11 library (Empire Genomics, LLC, NY) to detect chromosomes 5, 19, 21 and 22. FISH was performed as previously described. Image Acquisition was performed using a Nikon microscope equipped with a CCD camera. Images were captured using NIS elements software. Analytical Specificity, Sensitivity and S/N were calculated as previously described according to the Standards and Guidelines for Clinical Genetic Laboratories of the American College of Medical Genetics. RESULTS: We successfully detected 24 chromosomes in 6 successive hybridizations in a single day. CONCLUSIONS: ODN probes' short length allows rapid hybridization and reduces cell damage, which are significant advantages for time critical procedures such as enumeration of chromosomes in interphase nuclei for PGD.

Non-Random Telomere Lengthening at Specific Chromosome Ends as a Novel Clonal Event in Chronic Myeloid Leukemia

It is widely accepted that chromosomal telomere dysfunction caused either by telomere shortening or lesions in the capping machinery is an important factor in carcinogenesis. However, in our recent study using quantitative FISH (Q-FISH), telomere restriction fragment (TRF) analysis and fiber FISH techniques on the measurement of telomere length in 32 cases of chronic myeloid leukemia (CML), we found that telomere lengthening at some specific chromosome ends is apparently a non-random event showing a clonal nature.Methods:TRF: genomic DNA was digested by frequently cutting restriction enzymes. After gel electrophoresis and Southern blotting, the blotted DNA was hybridized to a digoxigenin (DIG)-labeled probe specific for telomeric repeats. A DIG-specific antibody covalently coupled to alkaline phosphate followed by the chemiluminescence detection was used to detect telomere signals. The quantitative measurements of mean TRF length can be reached by scanning the signals on the film and analyzing them with the computer software.Q-FISH: chromosomes on cytogenetic slides were hybridized with a peptide nucleic acid (PNA) telomere probe (Panagene, Korea). The leukemia cells can be traced by the particular chromosome rearrangement presented in the metaphase cells, e.g. t(9;22). The signal intensity, which is proportional to telomere length, of each individual telomere was automatically measured with the software of the imaging system (ISIS 2 MetaSystems, Belmont, MA). The relative telomere length was calculated using the ratio of individual signal intensity to the standard deviation.fiber FISH: fiber FISH was performed with cohybridization of a specific subtelomere probe (with a known size and close to the telomere site of interest) and the telomere probe to the DNA/chromatin fibers preparation on the same slide.Results: The telomere lengthening at short arm of a X chromosome (Xp) was the most frequent event (seen in about 50% of CML cases we studied) in leukemia cells that can be identified by the chromosome 9 and 22 translocation [t(9;22)(q34;q11.2)]. The longest telomere length at Xp end for the CML cases reached 200 kb, which is about 20-fold longer than in normal cells. The other telomeres involved in the non-random lengthening include 18p (7/32), Yp (4/32), 4q (4/32), 5p (3/32), 7q (3/32), and 15p (3/32).Conclusion: While the relationship between the sequence organization for telomere shortening and their function is relatively clear, it has not been well stated if telomere lengthening at specific chromosome ends could be involved in cell proliferation, in maintenance of the genome stability and in evolution of the cancer. Our findings have shown the first evidence that the telomere lengthening at some specific chromosome ends is such a salient clonal event. Further investigation on picking up specific individual telomere lengthening in leukemia cells would greatly aid studies of chromosomal stability, telomerase activity, proliferative capacity and the evaluation of clinical status and thus one can use telomere length as an indicator to follow-up the cancer progression, to evaluate the treatment efficacy and to predict the prognosis in cancer.

Building Connecticut's clinical biodosimetry laboratory surge capacity to mitigate the health consequences of radiological and nuclear disasters: A collaborative approach between the state biodosimetry laboratory and Connecticut's medical infrastructure

Biodosimetry, based on the analysis of dicentric chromosomes in circulating mononuclear cells, is considered the “gold standard” for estimating radiation dose and is used to make informed decisions regarding the medical management of irradiated persons. This paper describes the development of biodosimetry laboratory surge capacity for the health consequences of radiological and nuclear disasters in Connecticut, including: (1) establishment of the Biodosimetry Laboratory for the timely assessment of radiation dosage in biodosimetry specimens; (2) identification of clinical laboratories qualified and willing to process biodosimetry specimens from a large number of victims; (3) training of clinical laboratorians in initial biodosimetry specimen processing; and (4) conducting a functional drill that evaluated the effectiveness of these elements. Descriptive information was obtained from: (1) personal observations; (2) a needs assessment of clinical laboratories in Connecticut; (3) records from a training program of clinical laboratorians in biodosimetry specimen processing that was developed and provided by the Yale New Haven Center for Emergency Preparedness and Disaster Response; and (4) records from a statewide functional drill in biodosimetry specimen processing that was developed and conducted by the State of Connecticut Biodosimetry Laboratory. A needs assessment of clinical laboratories in Connecticut identified 30 of 32 clinical laboratories qualified and willing to perform initial biodosimetry specimen processing. Currently, 79 clinical laboratorians in 19 of these qualified clinical laboratories have been trained in biodosimetry specimen processing. A functional exercise was conducted involving 37 of these trained clinical laboratorians in 18 qualified laboratories as well as the Biodosimetry Laboratory. The average turnaround time for biodosimetry specimen processing in this drill was 199 min. Exercise participants provided feedback which will be used to further optimize biodosimetry specimen processing protocols in Connecticut. Based on our findings, we conclude that clinical laboratory professionals are an important resource for assisting with the processing biodosimetry specimens that are used for triage of patients from accidental or terrorist-related mass-casualty radiological or nuclear catastrophies. The approach described in this paper to enroll and train clinical laboratorians in sample preparation for dicentric analysis forms the basis for the next step (namely, further training on harvesting cultured cells and preparing cytogenetic slides) in collaborative efforts between the State of Connecticut's Biodosimetry Laboratory and the state's medical infrastructure towards building laboratory surge capacity to estimate radiation dose in victims of a mass casualty event.

The optimization of sample treatment for spectral karyotyping with applications for human tumour cells

Spectral karyotyping (SKY) represents an important tool for the investigation of the complex chromosomal rearrangements (CCRs) in many human malignancies which may be difficult to characterize by conventional banding techniques. The main goal of our work was to optimize the most important steps in the preparation of molecular cytogenetic slides for a SKY protocol. This approach consisted of optimization of both the aging procedure and protease pretreatment of the slides, with special regard given to the preservation of chromosome structure and shape, as well as to the intensity of hybridization signals. The best results were obtained with a chemical aging procedure using SSC or ethanol in combination with trypsin pretreatment applied at a higher concentration for a shorter period of pretreatment. A resulting protocol for SKY also applicable to human solid tumour cells was subsequently proposed. The practical potential of the SKY technique was demonstrated on examples of two types of human embryonal tumours – neuroblastoma and Wilms’ tumour, in which some kinds of chromosomal aberrations were not detectable by means of classic cytogenetic methods.

Proliferation studies on chromosome preparations of bone marrow in hematological disease

The growth rate of neoplastic cells has been the subject of numerous scientific and diagnostic approaches. The study presented here analyses the relationship between mitotic activity in standardised cytogenetic bone marrow preparations from three haematological diseases and diagnostic and clinical parameters, most importantly the outcome. The disorders studied were: Acute lymphoblastic leukemia (ALL) (N=107), chronic myeloid leukemia (CML) (N=166) and aplastic anemia in childhood (AA) (N=39). A strict protocol of quantitative standardisation of cytogenetic slides was adhered to ensuring comparability both cross-sectionally and longitudinally. The samples were studied after short-term incubation without mitogenic in vitro stimuli. The most important findings include: i) ALL: Immunological subtypes can be differentiated according to their proliferation profile; there is a striking difference between childhood and adult ALL in proliferation activity; most importantly initial proliferation is much higher in patients who will relapse than in those with stable remission. ii) CML: Philadelphia-positive CML shows proliferation activities quite distinct from Philadelphia-negative CML; however there is only a small change in the proliferative activity from the chronic phase to the accelerated phase or blast crisis. iii) AA: Very low proliferation scores rise quickly to near normal levels during immunosuppressive therapy in most patients. Higher levels at diagnosis are associated with a faster and better response to therapy. In conclusion, assessment of the proliferative activity in cytogenetic preparations made from bone marrow samples of patients with haematological disease may add valuable information as to diagnostic sub-groups and clinical course and may contribute to therapeutic decisions.

The ploidy of in vitro matured bovine oocytes is related to the diameter

The aim of the present study was to investigate a possible relationship between bovine oocyte diameter and the ploidy after maturation in vitro. The cumulus-oocyte-complexes (COCs) were collected by slicing slaughterhouse ovaries and were matured in vitro in standard conditions. Oocytes were collected separately from each ovary and then processed in groups according to their origin. After maturation, the inside zona pellucida diameter of each cell was measured and cytogenetic slides were made. Four size categories were distinguished: <110, 110–115, 115–120 and >120 μm. Altogether, 600 oocytes derived from single ovaries of 50 donors were measured and cytogenetically analyzed. The diploid chromosome number (2MII) was found for 8.4% of oocytes (36/427) and for 44% of donors (22/50). The observed number of 2MII cells varied between 1 and 6 per donor. The size of secondary oocytes with unreduced chromosome numbers was significantly smaller ( P<0.01) than the haploid ones. We conclude that bigger oocytes underwent normal meiotic division, whereas their smaller counterparts tended to follow an abnormal path of maturation.

Small Marker Chromosome Identification in Metaphase and Interphase Using Centromeric Multiplex FISH (CM-FISH)

Multicolor karyotyping procedures, such as multiplex fluorescence in situ hybridization (M-FISH), spectral karyotyping, or color-changing karyotyping, can be used to detect chromosomal rearrangements and marker chromosomes in prenatal diagnosis, peripheral blood cultures, leukemia, and solid tumors, especially in cases where G-banding is not sufficient. A regular M-FISH analysis requires relatively large amounts of labeled DNA (microgram quantities), is not informative in interphase nuclei, hybridization can take up to 2 to 3 days, and unlabeled human chromosome-painting probes are not available commercially. Unique probes (plasmids, PAC), specific for centromeric or subtelomeric chromosomal regions, can replace the painting probes in M-FISH to address specific issues, such as the identification of marker chromosomes and aneuploidies. A set of plasmid probes carrying repetitive sequences specific for the α-satellite region of all human chromosomes were combined in a metaphase assay and an interphase assay, allowing identification of aneuploidies in one hybridization step, on a single cytogenetic slide. The fluorophore-dUTP and the labeled antibodies required to label and detect the DNA probes can be prepared in any laboratory. All DNA probes can be easily isolated and labeled using common molecular cytogenetic procedures. Because of the repetitive nature of the probes, hybridization time is short, usually less than 1 hour, and the analysis can be performed with nonspecialized image-processing software.

Improvements in cytogenetic slide preparation: Controlled chromosome spreading, chemical aging and gradual denaturing

Improvements in cytogenetic slide preparation: Controlled chromosome spreading, chemical aging and gradual denaturing

Improvements in cytogenetic slide preparation: Controlled chromosome spreading, chemical aging and gradual denaturing

Improvements in cytogenetic slide preparation: Controlled chromosome spreading, chemical aging and gradual denaturing

Improvements in cytogenetic slide preparation: Controlled chromosome spreading, chemical aging and gradual denaturing

Metaphase spreading is an essential technique for clinical and molecular cytogenetics. Results of classical banding techniques as well as complex fluorescent in situ hybridization (FISH) applications, such as comparative genomic hybridization (CGH) or multiplex FISH (M-FISH), are greatly influenced by the quality of chromosome spreading and pretreatment of the slide prior to hybridization. Materials and Methods Using hot steam and a metal plate with a temperature gradient across its surface, a reproducible protocol for slide preparation, aging, and hybridization was developed. This protocol yields good chromosome spreads from even the most difficult cell suspensions and is unaffected by the environmental conditions. Chromosome spreads were suitable for both banding and FISH techniques common to the cytogenetic laboratory. Chemical aging is a rapid slide pretreatment procedure for FISH applications, which allows freshly prepared cytogenetic slides to be used for in situ hybridization within 30 min, thus increasing analytical throughput and reducing benchwork. Furthermore, the gradually denaturing process described allows the use of fresh biologic material with optimal FISH results while protecting chromosomal integrity during denaturing. The slide preparation and slide pretreatment protocols can be performed in any laboratory, do not require specialized equipment, and provide robust results.

Detection of cryptic Y chromosome mosaicism by coamplification PCR with archived cytogenetic slides of suspected Turner syndrome.

Turner syndrome is one of the most common cytogenetic abnormalities. It is known that the Y chromosome or Y derived material is present in 6-9% of TS patient and it may develop a high risk of gonadoblastoma in 15-25%. So it is crucial to carry out cyto genetic analysis and Y-specific probe studies for all persons with gonadal dysgenesis to rule out mosaicism with Y-bearing cell line; eg 45,X/46,XY. In this study, 26 archival slides previously analyzed cytogenetically as 45,X, 45,X/46,X,i(X), 45,X/46,X,r(X), and 45,X/46,XX were examined. Coamplification PCR, having the advantage of providing rapid result and confirming PCR failure, was performed with the slide samples in the regions of dystrophin gene in Xp21and DYZ3 in the Y centromeric region. All of archived slides were positive for X-specific gene and one slide of 45,X was found to have the cryptic Y chromosome material. Our result suggests that the archived cytogenetic slides could be applied for the detection of Y chromosome rapidly and efficiently in TS patients.

Retroactive DNA analysis for sex determination and dystrophin gene by polymerase chain reaction with archived cytogenetic slides.

We describe a rapid and efficient diagnostic method for sex determination and the dystrophin gene by the polymerase chain reaction (PCR) using archived cytogenetic slides. Archived cytogenetic slides stored for about 4 years at room temperature were used. To confirm whether DNA analysis is possible using the archived cytogenetic slides, we extracted the DNA from the slides and amplified the Y centromeric region (DYZ3), the X centromeric region (DXZ1) and the exon 46 of the dystrophin gene. Of the 50 cases, 24 were peripheral bloods, 13 were amniotic fluid cells, 5 were chorionic villus samplings and 8 were cord bloods. The PCR related sex determination in 22 females and 28 males, showed 100% concordance with the results of chromosome analysis, and all cases showed positive band for the exon 46 of the dystrophin gene. Of the 50 cases of the archived cytogenetic slides, we were fortunate enough to obtain the fresh blood sample from one fetus whose karyotype showed 45,X[34]/46,X,+mar[145] to compare the results of the gDNA with that from archived cytogenetic slide. To confirm whether the marker chromosome was derived from Y chromosome, we studied the six loci (PABY, SRY, RPS4Y (SY16, 17), ZFY, DYS14) on the short arm, one locus (DYZ3) on the centromere and one locus (DYZ1) on the long arm. Of the 8 loci studies, all PCR related Y chromosome showed positive band from both gDNA obtained from cord blood and archived cytogenetic slides. We could conclude from the above results that the marker chromosome was derived from the Y chromosome. We believe our experiment is rapid and efficient for studies of over 10 independent loci from a single slide which has been kept in storage for up to 4 years and that archival Giemsa-stained cytogenetic slide repositories represent valuable DNA resources for clinical and forensic studies.

Point mutation analysis of archived cytogenetic slide DNA.

Archived Giemsa-stained cytogenetic slide repositories represent valuable DNA resources for medical, scientific, and forensic studies. Sequencing readily identified a Charcot-Marie-Tooth disease point mutation in a 209-bp PCR amplified product. With optimal PCR primers and amplification conditions, our protocol quickly and reliably isolated sufficient DNA for at least 12 independent PCR amplification reactions for forensic and medical applications from single slides up to 5 years old.