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DNA Ploidy Investigation Aided by Image Cytometry


Medical editor Håvard E. Danielsen Ph.D.
Professor
Oslo University Hospital

General

DNA ploidy investigation is defined here as the sequence of operations originating from paraffin embedded tumor blocks consisting of selection of tumor area, sectioning for preparation, preparation, Feulgen staining, measurement, editing, and classifying. 

DNA ploidy is a cytogenetic term describing the number of chromosome sets (n) or deviations from the normal number of chromosomes in a cell.  In cytometry, the expression is used either to describe the DNA content in a cell (c) or the total DNA distribution in a cell population.

Image cytometry is based on the Feulgen technique which is a widely used staining procedure within biology. The Schiff or Schiff-related reagent is used to bind to aldehydes created after the DNA is hydrolyzed with acid. This allows for staining of DNA in situ. The staining intensity is proportional to the DNA concentration and the amount of DNA in the nucleus is expressed by light absorbed by the Feulgen stain in the whole nucleus.

The Feulgen reaction is used to quantify the amount of DNA in a tumor nucleus. A digital camera captures images of individual nuclei in the specimen. The images are divided into image elements (picture elements - pixels). The gray tone value for each pixel represents the intensity of DNA specific staining. The value is saved in the computer which numerates between 0 (black) and 1023 (white).  The relative amount of DNA in each nucleus (DNA ploidy) is yielded by summing the optical density of all the pixels in the nucleus. DNA-ploidy measured in this way has shown to be a good prognostic and predictive method. Optical Density (OD) and Integrated Optical Density (IOD) are defined as:

During ploidy measurement, the IOD values of all of the nuclei in the specimen are registered and can be graphically developed into a histogram (x-axis: IOD value, y-axis: number of nuclei).

Clinical DNA ploidy investigations are routinely performed mainly on material from different gynecological cancers, prostate cancer, and oral cancer.

Goal

The goal of the investigation is to estimate a prognosis for the patient's cancer type. The treatment is customized depending on the ploidy result.


Equipment

Use protective gloves for sectioning, preparation, and staining. Preparation up to rinsing and Feulgen staining should occur under a fume hood.

Standard equipment for sectioning and staining of sections:

Preparation

  • Centrifuge tube with cap, 10 ml, 2 per specimen
  • Glass pipettes for xylene and plastic pipettes for other solutions 
  • Specimen glasses with lids, 10 ml, 1 per specimen
  • Centrifuge
  • Water bath with thermostat
  • Cytospin centrifuge with equipment
  • 60 µm nylon filter
  • Polysinä slide

Measuring and Editing

Measuring must occur in a dedicated room with dimmed lighting and minimal interruptions.

  • PC with minimum 512 Mb RAM, 1,2 Gb hard disk, 3 com-ports
  • Minimum 17"-screen with good resolution 
  • Software from Fairfield Imaging Ltd., GB
  • Digital camera (Hamamatsu)
  • Microscope (Zeiss Axioplan 2, with 40 x lens, stage with controller (Prior))
  • Focusing unit with controller (LVPZT – amplifier, PI) 

Chemicals 

  • Aluminum potassium sulfate (AlK(SO4)2•12H2O)
  • Basic Fuchsin
  • Glacial acetic acid
  • Eosin
  • Absolute ethanol (96%)
  • Eukitt®
  • Hematoxylin
  • Potassium disulfite (K2S2O5)
  • Activated charcoal
  • Sodium disulfite (Na2S2O5)
  • Sodium iodate (NaIO3)
  • PBS (phosphate buffer)
  • Protease (type XXIV, bacterial)
  • HCl
  • Xylene

Solutions

The following solutions are standard solutions used for different procedures. Prepare well before the start of the procedure.

Eosin

  • 1.25 g eosin is dissolved in 1 L of 80% ethanol
  • Add 2.5 ml glacial acetic acid
  • Mix

Schiff reagent

  • Dissolve 5 g of basic fuchsin into 150 ml of 1 M HCl.
  • Add sodium disulfite (5 g dissolved into 850 ml distilled water). The solution should be red.
  • Let it stand overnight in the absence of light.
  • Add 3 g active charcoal
  • Shake for 2 minutes.
  • Filter multiple times until the solution is clear. Use a mask and gloves. Prepare the solution in a fume hood.

5 M HCl

  • 292 ml distilled water
  • 208 ml concentrated HCl (37 %)

Hematoxylin (Mayers)

  • 100 aluminum potassium sulfate is dissolved in 2 L distilled water
  • Add 4 g hematoxylin
  • Add 0.4 g sodium iodate for culturing
  • The solution should be stirred until the next morning.
  • Filter 200 ml and add 4 drops of glacial acetic acid 
  • Empty solution into a staining dish
  • Filter the solution each time before use
  • Add more glacial acetic acid as more solution is added

The following solutions should be fresh and mixed before use.

Protease solution (0,05 %)

  • 10 mg protease dissolved in 20 ml PBS.

Sulfite solution

  • 1 gram sodium disulfite in 190 ml distilled water.
  • Add 10 ml 1 M HCl.

This is enough for one staining jar.

 

 

 


Procedure

Image cytometry, specimen collection, and registration

Tissue specimens for ploidy investigations are summoned from an external or the present hospital. The examinations are ordered by a doctor. A pathologist examines the HE sections from the actual operation biopsy and chooses one or more blocks which contain the suspicious tissue. The tumor areas are marked on the HE sections. The blocks and corresponding HE sections are retrieved. The documents for the specimens are created.

Section protocol

  • From each chosen block, 1-10 sections of 50 µm thickness are taken. The number of sections prepared depends on the size of the tumor area.
  • At the end, a thin section is prepared and marked HE2 and stained with HE. All of the slides should be labeled with at least two different identifications.

The HE2 section is delivered to the doctor for examination for tumor tissue. The Feulgen-stained monolayer is delivered in the absence of light (for example under aluminum foil) for measurement.

Monolayer preparation of 50 µm thick paraffin sections

Preparation should be performed under a fume hood. The xylene waste should be in a dedicated container. Observe the slide/specimen precisely and be careful with removal of all fluid.

  • Remove paraffin by adding 4 ml xylene to the specimen container.  Let it stand for 30 minutes. Remove xylene and repeat.
  • Remove the xylene by adding 4 ml absolute ethanol. Let stand it for 5 minutes. Remove the ethanol and repeat. Avoid cross-contamination!
  • Rehydration occurs by adding 4 ml of 96% ethanol to each specimen. Let it stand for 10 minutes. In 10 minute intervals, add 1 ml, 1 ml, and 2 ml of distilled water (concentration of ethanol after each addition will be 85%, 74%, and 50%).
  • Rinsing: Remove the ethanol and add 4 ml of PBS. Centrifuge for 5 minutes at 1000 rpm (150 G) and remove supernatant. Repeat. Use new pipette for each specimen. This applies for the rest of the procedure.
  • During enzyme treatment, the nuclei are liberated. Preheat the protease solution in a water bath (37ºC and 200 rpm). Incubation time is determined by trial and can vary for tissue type.
  • To stop the enzyme activity: place the specimens on ice and add 4 ml of cooled PBS. Let the specimens stay in ice until the spinning is finished. Observe the suspensions to obtain an impression of the density of cells while pipetting multiple times. Filter the suspension through a 60 µm nylon filter into a new tube. Centrifuge at 1000 rpm (150 G) for 10 minutes. Remove the supernatant. Add 4 ml PBS, resuspend and centrifuge again. Remove the supernatant, but let approximately 1 ml remain (assess based on pellet).
  • Spin: Pipette the right amount of nuclei suspension in the test chambers. This is based on experience. The fist specimen spin is assessed unstained under the microscope. With a 40x objective, there should be at least 30-40 nuclei in the field of vision. Centrifuge at 600 rpm (40 G) for 5 minutes. Slowing should occur gradually. Make at least 2 spins for each specimen. Finished monolayers are post-fixed with 4% buffered formalin overnight at minimum, but tolerate standing over a weekend.   
  • Stain the spins with the Feulgen stain.

Feulgen staining of monolayer

  • Post fixation: 4 % formalin over night. Rinse carefully in water (dip the slides in distilled water).
  • Hydrolysis: 5 M HCl for 60 minutes. The time may vary for different specimens, and a hydrolysis curve for different tissue types should be prepared to ensure correct hydrolysis time. Dispose of HCl in the proper container. Fill and empty the glass with distilled water x 3.
  • Sulfite rinse: Rinse 3 x 10 minutes in sulfite solution. Rinse one more time for 5 minutes in running water.
  • Dehydration: Place the slides into a metal slide holder and rinse in distilled water, 70 % ethanol, 96 % etanol, 2 x absolute ethanol. Rinse in xylene x 2.

Mount cover glass with Eukitt® from xylene.

Automatic measurement of monolayer

  • Find the best monolayer (dense with nuclei, but without many overlapping nuclei) and place in position in the microscope.
  • Start the measurement program, choose storage location, and enter detail of case.
  • Click Accept. A window appears with image field and menu – the program is in Capture-mode.
  • Check the monolayer position with the help of Image Position and autofocusing.
  • Click Blank Shade Image Position, check that the screen area is totally blank, set the value to 800 ±2 and click Capture.
  • Go to Automatic mode and set the maximum limit for number of fields to be scanned and total number of nuclei to be measured - remember to adjust the total amount if suspicious of a large number of non-tumor nuclei in the specimen.
  • Start Auto Capture and the measurement with automatically start until one of the following occurs:
    • The limit for the number of nuclei is reached.
    • The limit for the number of fields is reached.
    • The machine does not find more nuclei and automatically stops.
  • Record the number of fields and gallery distribution in the measurement form.
  • Click Exit and save the measurement.
  • Make necessary file transfers for later editing and back-up.

Editing of automatic measurement

  • Open "Histogram Draftsman" and the actual case for editing.
  • It is recommended that the galleries are sorted by area before assessment and editing. Browse through all galleries to get an impression of the quality of the specimen (for example poor segmenting, cytoplasm disturbances etc. are more the rule than the exception, in certain specimens).
  • The dynamic for editing is subjective, however here is a method:
    • First edit gallery 4, followed by gallery 3, 2 and lastly gallery 1.
    • Editing of each gallery occurs in the series:
      • Excluding (to gallery 6)
      • Moving (to other galleries)
      • Browse through each gallery at least twice, with and without masking lines and in different modes. Some properties are more visible in certain modes.
  • Goal for editing:
    • Gallery 1: tumor nuclei (main gallery)
    • Gallery 2: lymphocytes (reference gallery)
    • Gallery 3: large immune nuclei (partly reference gallery)
    • Gallery 4: fibroblasts partly reference gallery)
    • Gallery 5: nuclei with uncertain properties
    • Gallery 6: excluded nuclei
  • The following nuclei are excluded:
    • Sliced nuclei (mikrotom)
    • Nuclei, for other reasons, without intact membrane
    • Overlapping nuclei
    • Necrotic and apoptotic nuclei
    • Nuclei with foreign bodies
    • Heavily over-segmented or under-segmented nuclei

The edited file must be saved before closing "Histogram Draftsman".

Classification of DNA-histogram

Classification of DNA histogram is based on edited galleries in Histogram Draftsman. The classification is based on a visual assessment of all galleries (quality of editing and nuclei composition of the specimen).

 When editing has been verified, the histogram is retrieved. Assess the composition/placement of the different nuclei types and place the 2c peak (diploid G1 top).  Place "tags" for each potential G1 peak in the tumor nuclei area. These tags give information about the peaks':

  • Mean IOD-value (Integrated Optical Density).
  • CV (coefficient of variation)
  • DI (DNA-Index) 
  • percent share of total histogram
  • 5c ER (Exceeding Rate)
  • 9c ER

The histogram is classified into one of four possible categories:

  • Diploidy: There is a normal amount (23 chromosome pairs = duplicate set) of DNA in the cell nucleus. This is the normal situation for the resting phase of the cell cycle.
  • Tetraploidy: DNA-content in the cell nucleus is double. This is normal in liver cell nuclei and in the G2 phase before mitosis. 
  • Polyploidy: A further doubling of DNA content in the cell nucleus without cell division (eight copies of each chromosome). In general, polyploid is used to name nuclei where there are multiple copies of cell DNA.
  • Aneuploidy: The DNA content in the cell nuclei has not been equally represented under cell division. The chromosomes might be missing partly or completely, or whole or parts of chromosomes might be added.

Due to constant improvements in technical quality, classification criteria is steadily developing and international consensus conferences are held at regular intervals (1). The following should be regarded as the present criteria for DNA ploidy classification: 

  • DIPLOIDY  
    • Only one G0/G1-peak
    • G2-peak is under 10 % or less than SFF (S-phase fraction)
    • 5c ER is less than 1 %
  • TETRAPLOIDY
    • Peak at 4c, 8c and DI 1,9-2,1 (if DI is 1,8-1,9 or 2,1-2,2  the rule is used for ± 2xCV).
    • 4c is over 10 % (and larger than SFF) or visibly SFF/G2 for one 4c-peak over 1 %.
  • POLYPLOIDY
    • Peak at 8c, 16c and DI 3,8-4,2 (if 4c has high CV then DI-limit is 3,6-4,4).  
    • 8c is over 10 % or visibly SFF/G2 for one 8c-peak over 1 %.
  • ANEUPLOIDY    
    • Non-euploid G1-peak (euploid: 2c, 4c, 8c, etc.)
    • 5c/9c ER is larger than 1 % and does not represent euploid populations

REFERENCE

Haroske G, Baak JP, Danielsen H, Giroud F, Gschwendtner A, Oberholzer M, Reith A, Spieler P, Bocking A. Fourth updated ESACP consensus report on diagnostic DNA image cytometry. Anal Cell Pathol. 2001 23(2):89-95.


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