Setting Up and Management of an Ideal GI Pathology Laboratory with Emphasis on Hospital Information System

Abstract

The setup of a gastrointestinal (GI) pathology laboratory focuses on building the infrastructure, procurement, delivery of services through trained technical experts, efficient communication with the clinical team, and a thorough clinicopathological correlation. All these efforts should be intended towards satisfactory patient care and deciding therapeutic guidelines. Hence, recent focus has been laid on immunohistochemistry (IHC), not only as a diagnostic tool, but also  for deciding therapeutic guidelines (theranostics). Furthermore, integration of Hospital and Laboratory services and communicating important patient investigations to the treating physicians through Hospital information system (HIS) and Laboratory information system (LIS) has also gained prime importance. Overall, this helps in keeping patient care services updated with the evolving trends.

Appendices

Annexure I

Annexure II

1.1 Requirements for Histopathology Laboratory with Some Demo Specifications

Microslide Cabinet

• For keeping 75 × 25-mm slides in a horizontal or vertical arrangement.

• Wooden or stainless steel with lock and key.

• Slide trays should be arranged separately to avoid any disturbance to other trays while taking out or putting them in.

• Index card holder and handle to be provided on each tray.

• Compactor systems can be used for slide storage to save space.

Block Cabinet for Keeping Blocks in Numerical Arrangement

• Body and internal drawers made of stainless steel with lock and key.

• The transverse diameter of the compartments should be such that one block can fit in vertically, snugly, and easily in it.

• Index card holder and handle to be provided on each drawer and the front panel.

• Compactor systems can be used for block storage to save space.

Lab Rectangular Heating Plate

• Scratch-resistant cast iron/aluminum top Teflon-coated

• Usual dimensions of top approx. 70 × 18 cm

• With electronic temperature controller up to 100 °C

• Should operate on 220 V

Slide Rack

• To keep slides in a vertical position before and after mounting

• Made of anodized aluminum

• Should hold a minimum of 20 slides

Slide Carrying Tray

• Made of anodized aluminum for keeping 20 slides in the flat position.

• Slots should be equal and allow easy insertion and removal of slides.

Refrigerator 450 L

• Double door

• Temperature range 0–4 °C

• Frost-free, five-star electrical consumption

Microwave Oven:

• Power levels: 6

• Clock system option: +30s plus

• Reminder end signal

• Capacity size (cu. ft.): 0.8 cu.ft./20 L

• Power source: 230 V/50 Hz

• Power consumption: 1150 Watts

• Minimum 2-year warranty

Tissue Flotation Bath:

• Circular water bath with digitally controlled temperature regulation.

• The working temperature range of the tissue flotation bath +30 to +90 °C.

• The interior should be of seamless stainless steel with a black coating.

• The exterior should be epoxy powder-coated and should be resistant to corrosion and chemical damage.

• Accessory set including control thermometer 0–100 °C.

• The temperature uniformity should be ±0.2 °C at 37 °C.

• Chamber capacity should be: 2.5 L.

• Dimensions overall: 340-mm circumference, 90-mm height.

• Dimensions bath well: 210-mm circumference, 50-mm height.

• Power supply: 220 V/50/60 Hz/200 VA.

Specifications for Hot Air Oven:

• Stainless steel body

• At least three adjustable stainless steel shelves

• Timer: 5–120 min

• Graduated temperature control Knob: 50–250 °C ± 1 °C

• On/off switch with pilot lamp indicator

• Operable at 220 V AC

Stainless Steel Racks to Stain Slides with Suitable Heavy-Duty Glass Troughs

• Soda-lime glass dish with cover

• Stainless steel rack with hinged handle to fit inside the dish

• Allowing staining for 25 or 50 microscope glass slides

Paraffin dispenser

Paraffin dispenser is essential equipment for making paraffin-embedded block of tissues.

It should have the following configurations:

• Must have minimum 5-L high-capacity tank.

• Must have a digital display with the temperature set digital controller for the paraffin tank.

• The temperature range should be from ambient to 75 °C with +/− 1 °C increments.

• Must have a paraffin nozzle for liquid paraffin dispensing.

• The set temperature, as well as the actual temperature of the paraffin tank, must be displayed with a press of a button.

• Dimension:

• Width: 310 mm

• Depth: 350 mm

  • Height: 410 mm

  • Weight: 45 kg

Temperature can be set with 1 °C increments.

Specifications of Embedding Station:

• Imported compact, benchtop unit

• Approx. 70 cassette mold capacity

• Should have two separate modules, cold plate, and heat-embedding module

Heat-embedding module:

• Independent programmable temperature settings for paraffin reservoir, mold warmer, cassette bath, and work surface.

• Should have paraffin reservoir of the capacity of 3–5 L with temperature control up to 45–70 °C in 1 °C increment.

• Temperature control of mold warmer should be set up to 35–70 °C (95–158 °F) in 1 °C increment, cassette bath 100 cassettes 45–70 °C (113–158 °F) in 1 °C increment, and heated work surface 45–70 °C (113–158 °F) in 1 °C increment.

• Height-adjustable, rotary, and fold-back clip for activating the paraffin flow with the embedding mold, manually.

• Display showing temperature, time, filling level, and flow rate: alarm indicator when molten paraffin levels are low.

• Peltier cooled cold spot for making blocks, temperature maintained at −5 °C.

• Bright, even illumination of the work area by an adjustable halogen lamp.

• Adjustable magnifier facilitating specimen orientation when embedding.

• Stainless steel cold plate to hold at least 100 blocks.

• Constant temperature maintained at −15 °C.

Semiautomatic Rotary Microtome:

• Semi-motorized rotary microtome, independent control of section, and trimming thickness.

• Microtome cutting range from 0.5 to 100 μm.

• Trimming ranges from 1 to 50 μm.

• Specimen holder with the provision of rotating specimen in X-, Y-, and Z-axis.

• Section thickness should be on the LED display.

• Universal knife holder for a disposable blade with lateral knife holder adjustment.

• Total specimen advance at least 18 mm.

• Coarse specimen advance at least 150 microns per revolution.

• Vertical stroke more than 60 mm, horizontal stroke more than 25 mm.

• Microtome base plate with a ruler.

• The instrument should come with the following accessories: universal blade holder; low-profile and high-profile disposable blades – 50 packs each; section waste tray; storage tray at the top of the instrument; two large brushes and two small brushes; and dust cover.

• Operable at 230 V, 50 Hz.

Semiautomatic/Automated Tissue Processor:

• Carousel type with 12 stations.

• Ten reagent vessels and two paraffin baths for single basket operation.

• Reagent vessel tops and charcoal-enhanced ventilation help control processing vapors.

• Basket capacity of 120 cassettes.

• Ergonomic control panel with foil-protected keyboard and LCD.

• Immediate and delayed start processing modes.

• Up to ten different processing programs maintained.

• Programmable immersion time in each station (from 1 min to 99 h: 59 min).

• Provision of interrupting an automatic process for reloading or removing cassettes before the end of a run.

• Battery backup system in case of power failure.

• Audible alarms, error messages, and warning codes.

• Operable at 230 V, 50 Hz.

Cryostat Specifications

This instrument is required for cutting frozen sections for rapid reporting to guide intraoperative decision-making. Open top modular cryostat system for frozen sectioning in the hospital with the following features:

• Corrosion proof cryochamber with an illumination facility.

• Even stainless steel cooling chamber with a draining system for easy cleaning.

• A filtering system should be provided for wastes.

• Insulation of body to maintain constant temperatures and lower power consumption.

• Independent temperature setting for the specimen up to −50 °C and cryochamber/knife up to 35 °C.

• Additional independent quick specimen freezing device with Peltier cooling technology with temperature control reducible to up to −40 °C within few seconds.

• Microtome cutting ranges from 0.5 to 100 μm.

• Trimming ranges from 1 to 50 μm

Binocular Microscope:

• Good-quality microscopes and light sources

• Calibration should be performed and documented, yearly servicing.

• Infinity corrected optical system.

• Pre-centered lamp house with halogen 6 V 20 Watt with power supply of 220–240 V.

• Coaxial coarse/fine focusing with cross roller guide incorporated, 22-mm coarse/fine focusing range; coarse motion, 37.7 mm/rotation; fine motion is 0.2 mm/rotation with 2-micron scale increments.

• Condenser lens – swing out Abbe condenser with objective guide marking position and leaf-like aperture diaphragm NA 1.25.

• Plan achromatic antifungal lens; objective five nosepiece attachment with 4×, 10×, 20×, 40×, and 100× spring-loaded.

• Eyepieces – 10 × 22-mm-wide field binocular diopter adjustment for both eyes with eyepiece guard and cap. Lenses – plan achromatic approx. 30° incline, 360° rotation, or movable incline with IPD approx. 40–80 mm.

• Mechanical slide holder – double plate rectangular mechanical stage with travel area of 76 mm (x-axis) and 40 mm (y-axis), with removable slide holder.

• Accessory three bulbs.

Fluorescent Microscope with Camera Attachment:

• Should have a basic stand with LED illumination, four nosepieces, and HCS (harmonic component system) optics, with 20-mm field of view.

• Should have an automatic adjustment of illumination to the contrast methods, auto-off function, LED with a service life of 50,000 h, constant color temperature.

• Should have a bright field, phase contrast, integrated modulation contrast upgradeable to fluorescence.

• Should have a trinocular tube with beam splitting position vis/phot: 100/0, 50/50, and 0/100.

• Should have objective 4×, 10×, 20× FWD 3.7 mm and 40× FWD 2.0 mm and 10× eyepiece.

• Should have condenser suitable for all the above techniques.

• Should have fluorescence filters for rhodamine, FITC, and DAPI with 100-Watt Hg lamp for fluorescence.

• Should have fluorescence slider with three positions for filter cubes.

• Should be provided with high-resolution cooled digital camera (same make as that of the microscope) with 5 MP, 2/3″ CCD sensor, pixel size 3.4 μm × 3.4 μm, exposure time 1 ms to 600 s, fast live image (24 frames/s) with FireWire cable, interface to produce 1:1 image on PC.

• Should be provided with a branded PC with the latest configuration.

• Should be provided with UPS one KV offline with 30-min backup.

Slide Scanner:

• The scanning device must be based online scanning technology.

• Slide capacity of five or more, higher capacity preferred.

• The scanner must automatically self-calibrate for each slide without manual intervention.

• The scanner should have a resolution of 0.5 μm per pixel at 20× and 0.25 μm per pixel at 40×.

• Apochromat objectives with high numerical aperture are preferred.

• Typical scanning should less than 2 min, for 15 × 15 mm area in 20× magnification from the scan start till the slide is displayed.

• The scanner should have mechanisms in place to accommodate topological variations for a high probability of success at the first scanning.

• Barcode scanner/reader should be included.

• The digital image created by the scanner must allow the following functions:

  • Smooth tracking and zoom

  • Synchronization of multiple images

  • Grid overlay

  • Efficient serving of the image over the web (low latency, etc.)

  • Automatic color correction to standards using an ICC profile

• Computer appropriate for this device along with antivirus preloaded; high-resolution monitor for the display of images 24″ (1900 × 1200).

• Software required should be included with free updates for 5 years.

• 20 TB of storage space supplied along with rack.

• 5-KVA UPS with 1-h battery backup.

Annexure III

1.1 SOPs for Some Common Histochemical Stains

• 1. Periodic Acid Schiff:

Reagents:

Periodic acid solution:

• (a) Periodic acid crystals: 1 g

• (b) Distilled water: 100 mL

Schiff’s reagent:

• Basic fuchsin: 1 g

• Sodium metabisulphite (anhydrous) 2 g

• Concentrated HCl: 2 mL

• Charcoal 2 g

Boil 200 mL of distilled water and 1 g of basic fuchsin. Cool to 50 °C and add 2 g of sodium metabisulphite. Dissolve and cool to room temperature. Add 2 mL of HCl. Add activated charcoal (acting as adsorbent) and shake for 2 min. Filter and store in a dark bottle and store at 4 °C.

Procedure:

• Bring sections to water.

• Controlled oxidation in 1% periodic acid: 10 min.

• Rinse in distilled water.

• Schiff’s reagent: 20 min.

• Wash in running tap water for 5 min; section will appear pink.

• Counterstain in Harris hematoxylin for 5 min.

• Wash in running tap water.

• Dehydrate in ascending graded alcohol, clear in xylene, and mount in DPX.

End results:

• Mucin/glycogen: deep pink/magenta

• Basement membrane: deep pink/magenta

• Nucleus: blue

Safety precautions:

• Periodic acid is hygroscopic; keep the lid closed immediately after use.

• Basic fuchsin and potassium metabisulphite can be stored at room temperature.

• 2. Alcian Blue pH 2.5 (Acid Mucopolysaccharides)

Reagents:

Glacial acetic acid (3%):

• Acetic acid: 3 mL

• Distilled water: 100 mL

The solution is stable for 1 year.

Alcian blue solution:

• Glacial acetic acid (3%): 100 mL.

• Alcian blue 8GX: 1 g.

• Mix, adjust pH to 2.5, using acetic acid. Filter and add a crystal of thymol.

• Label with initial and date. The solution is stable for 2–6 months. Store in refrigerator.

Nuclear fast red (Kernechtrot) solution:

• Aluminum sulfate: 25 g.

• Distilled water: 500 mL.

• Nuclear Fast Red: 0.5 g.

• Dissolve the aluminum sulfate in water. Add the nuclear fast red; dissolve with aid of heat. Filter and add a crystal of thymol. The solution is stable for 1 year.

Procedure:

• Bring sections to water.

• Add 3% acetic acid for 3 min.

• Add Alcian blue solution at room temperature for 30 min.

• Wash in running tap water for 2 min, rinse in distilled water.

• Nuclear fast red for 5 min; rinse in tap water

• Dehydrate in ascending graded alcohol, clear in xylene, and mount in DPX.

End results:

• Acid mucins/mucosubstances: blue

• Nuclei (with nuclear fast red): reddish pink

• 3. Congo Red

Reagent Preparation:

Congo red solution:

• Congo red: 0.2 g.

• Ethanol (80%): 100 mL.

• Saturated sodium chloride.

• Sodium hydroxide.

• Dissolve 0.2 g of Congo red in 100 mL of 80% ethanol.

• Prepare a saturated solution of sodium chloride in 100 mL of 80% ethanol. Adjust pH to 10.5–11 with sodium hydroxide.

Procedure:

• Bring sections to water.

• Immerse sections in saturated sodium chloride in 80% ethanol: 1 h.

• Congo red solution: 1 h.

• Rinse in 70% alcohol.

• Counterstain in Harris hematoxylin.

• Dehydrate in ascending graded alcohol, clear in xylene, and mount in DPX.

End results:

• Amyloid: red (apple-green birefringence in polarized light)

• Nucleus: Blue

• 4. Masson’s Trichrome Stain

Equipment:

• Rinse glassware in deionized water

• Coplin jars, 60 °C oven or water bath, microwave

Reagent Preparation:

• Biebrich scarlet

• 1% acid fuchsin: 10 mL

• 1% Biebrich scarlet red: 90 mL

• Glacial acetic acid: 1 mL

Mix well; label with initial and date. The solution is stable for 6 months.

Caution:

Avoid contact and inhalation

Weigert’s Iron Hematoxylin

Stock solution A:

• Hematoxylin 5 g

• Alcohol (95%): 500 mL

Mix well; label with initial and date. The solution is stable for 1 year.

Stock solution B:

• Ferric chloride (29%): 20-mL distilled water: 475 mL

• HCl: 5 mL

Mix well; label with initial and date. The solution is stable for 1 year.

Weigert’s Hematoxylin (Working Solution):

• Solution A: 25 mL

• Solution B: 25 mL

• Mix well; the solution will remain stable for 3–4 days.

Phosphotungstic/phosphomolybdic acid solution:

• Phosphotungstic acid: 12.5 g

• Phosphomolybdic acid: 12.5 g

• Distilled water: 500 mL

Mix well; label with initial and date. The solution is stable for 6 months.

Caution:

Avoid contact and inhalation.

Aniline blue:

• Aniline blue: 2.5 g

• Distilled water: 100 mL

• Glacial acetic acid: 1 mL

Mix well; label with initial and date. The solution is stable for 6 months.

Caution:

Avoid contact and inhalation.

Acetic acid (1%):

• Glacial acetic acid: 10 mL

• Distilled water: 1000 mL

Mix well; label with initial and date. The solution is stable for 1 year.

Caution:

Avoid contact and inhalation.

Laboratory safety:

Wear gloves and lab coat. Avoid contact and inhalation of dyes and chemicals.

Bouin’s fluid contains formaldehyde, a known carcinogen; picric acid can become explosive when dry. It is toxic by skin absorption. Keep hot uncapped Bouin’s under the hood. Phosphotungstic acid, phosphomolybdic acid, and acetic acid solutions are skin and eye irritants and strong corrosives.

Procedure:

• Bring sections to water.

• Mordant in Bouin’s fluid, 60 °C for 1 h (or microwave 1 min at level 2), and allow to cool.

• Wash in running tap water to remove the picric acid for 5 min.

• Weigert’s working hematoxylin for 10 min.

• Blue in running tap water for 5 min; rinse in distilled water.

• Biebrich scarlet for 1 min.

• Rinse in distilled water.

• Phosphotungstic/phosphomolybdic acid for 10 min; discard the solution.

• Transfer directly into aniline blue for 1–3 min.

• Rinse in distilled water.

• 1% acetic acid for 1 min, discard solution, rinse in distilled water (to enhance the intensity and fix the trichrome stain).

• Dehydrate in ascending graded alcohol, clear in xylene, and mount in DPX.

End results:

• Nuclei: black

• Cytoplasm, erythrocytes: red

• Collagen, basement membrane: blue

Notes:

• Light green may be substituted for aniline blue.

• 5% phosphotungstic acid for 5 min must be used while using light green.

• Improperly prepared staining solutions will result in poor staining contrast.

• 5. Phosphotungstic Acid Hematoxylin (PTAH, Mallory’s) for Demonstration of Fibrin

Fixative:

Zenker’s or formalin

Reagents:

• Lugol’s iodine

• Sodium thiosulfate (5%): 5 g in 100 mL of distilled water

Caution:

Avoid contact and inhalation.

Potassium permanganate (1%):

• Potassium permanganate: 1 g.

• Distilled water 100 mL.

• Make fresh; discard after use.

Caution:

Strong oxidant

Oxalic acid solution (5%):

• Oxalic acid: 5 g

• Distilled water: 100 mL

• Mix well, label with initial and date. The solution is stable for 1 year.

Caution:

Avoid contact and inhalation

PTAH solution:

• Hematoxylin: 0.5 g

• Phosphotungstic acid: 10 g

• Distilled water: 500 mL

• Dissolve ingredients in separate portions of distilled water, hematoxylin with gentle heat.

• When cool, combine.

• Instant ripening of solution can be achieved by adding 0.1 g of potassium permanganate.

• Store at room temperature in an amber color bottle. The solution is stable for 6 months.

Procedure:

• Deparaffinize and hydrate to distilled water; wash with tap water.

• Lugol’s iodine for 5 min; wash in tap water.

• Sodium thiosulfate (5%) for 1–2 min

• Wash in tap water for 10 min.

• Oxidize in 1% potassium permanganate for 5 min: wash in tap water.

• Bleach in 5% oxalic acid until white.

• Wash in tap water; rinse in distilled water.

• PTAH stain overnight, room temperature.

• Dehydrate in ascending graded alcohol, clear in xylene, and mount in DPX.

End results:

• Muscle cross striations, fibrin, glial fibers: blue

• Nuclei: blue

• Collagen: reddish-brown

• Elastic fibers: purplish

Limitations:

• PTAH solution not working

• Over differentiation of stain in dehydrating grades of alcohols

Precautions:

• Avoid contact and inhalation.

• Zenker’s contains mercuric chloride, extremely toxic, severe skin and eye irritant, multiorgan systemic effects on ingestion.

• Potassium dichromate, iodine, sodium thiosulfate, potassium permanganate, and oxalic acid are all similarly toxic compounds, so accidental contact, inhalation, or ingestion should be avoided.

• 6. Mayer’s Mucicarmine for Mucin and Cryptococcus

As mentioned earlier, this is a very important stain for the GI Pathology laboratory.

Reagents:

Mucicarmine solution: Grind 1 g of carmine and 0.5 g of aluminum chloride in 2 mL of distilled water in a small beaker. Heat on an electric hot plate for 2 min. The solution becomes black and syrupy. Dilute with 100 mL of 50% alcohol and let it stand overnight. Filter. Store in the refrigerator. Stable for 4 months.

Working solution: Dilute 1 part of mucicarmine with four parts of tap water.

Caution:

Add aluminum chloride slowly and avoid accidental inhalation

• Harris hematoxylin

• Metanil yellow (0.25%): metanil yellow 0.25 g in 100-mL distilled water

• Glacial acetic acid 0.25 mL

Procedure:

• Bring section to water.

• Stain the nuclei with Harris hematoxylin: 10 min.

• Differentiate and blue in dilute lithium carbonate.

• Wash in running tap water.

• Mucicarmine stain for 30 min to an hour.

• Rinse briefly in distilled water.

• Counterstain in metanil yellow to give a light yellow background.

• Rinse quickly in distilled water.

• Dehydrate in ascending graded alcohol, clear in xylene, and mount in DPX.

End results:

• Mucin and Cryptococcus capsule: deep rose

• Nuclei: blue

• Background: light yellow

Caution:

Handle aluminum chloride and metanil yellow with care; potentially toxic.

• 7. Grocott methenamine silver stain

Reagents:

Chromic acid (5%):

• Chromium trioxide 25 g.

• Distilled water 500 mL.

• Mix; the solution is stable for 6 months.

Caution:

Corrosive, possible carcinogen, avoid contact and inhalation

Borax 5%:

• Sodium borate 5 g.

• Distilled water 100 mL.

• The solution is stable for 3 months.

Methenamine silver stock solution:

• Methenamine (hexamethylenetetramine) 3%: 100 mL.

• Silver nitrate (5%): 5 mL.

• Mix pour into an acid-cleaned brown bottle. Store in the refrigerator. The solution is stable for 3 months.

Caution:

Possible carcinogen

Working methenamine silver solution:

• Methenamine silver nitrate solution (stock) 25 mL.

• Distilled water: 25 mL.

• Borax 5% solution 2 mL.

• Prepare fresh before use.

Gold chloride (1%):

• Gold chloride 0.5 g.

• Distilled water 100 mL.

• Mix, store in acid-cleaned brown bottle, and refrigerate. The solution is stable for 1 year.

• Caution: avoid contact and inhalation.

Light green (1%):

• Light green SF yellow 1 g.

• Distilled water 100 mL.

• Glacial acetic acid 0.5 mL.

• Mix, stable for 6 months.

Precautions:

• Sodium metabisulfite, silver nitrate, and sodium thiosulfate are all toxic and irritant to skin, eyes, mucous membranes, which cause severe gastrointestinal discomfort.

Procedure:

• Deparaffinize and bring sections to water.

• Chromic acid 5% for 30 min at room temperature.

• Wash in tap water for 3 min, rinse in distilled water.

• Working methenamine silver solution, 60 °C water bath, for 1 h or until brown (alternatively microwave at level 3 for 3 min. Check for color development).

• Carefully agitate the slides in hot solution intermittently for even staining.

• Rinse in distilled water, four changes.

• Gold chloride (1%) for 15 s or until gray.

• Wash in distilled water.

• Hypo (5%) for 1 min.

• Wash in tap water for 1 min, rinse in distilled water.

• Light green for 2 min.

• Dehydrate in ascending graded alcohol, clear in xylene, and mount in DPX.

End results:

• Fungi: black

• Background: green

Notes:

• If the chromic acid turns brown, it needs to be changed.

• For the microwave technique, a lesser concentration of chromic acid (2%) is used.

• If fungi are not appearing black, check whether the reagents are fresh.

• If the working solution turns cloudy or shiny on the sides of the Coplin jar, prepare a fresh solution.

Giemsa Stain for H. Pylori

Reagents

0.25% Acetic acid:

• Acetic acid 1.0 ml

• Distilled water 400.0 ml

• Mix well, stable for 1 year

Caution: Mild corrosive acid

Giemsa stain working solution:

• 4 ml Giemsa stock solution

• 6 ml of distilled water

• Prepare fresh

Giemsa stock solution:

• Giemsa powder 4 g

• Glycerol 250 ml

• Methanol 250 ml

Procedure

Deparaffinize and hydrate to distilled water.

Place quickly in methanol for 2 minutes.

Preheat working Giemsa solution in microwave on medium to high power for 30 seconds. Filter the hot solution on the slides. Stain for 2 minutes (Coplin jar may be used).

Quickly rinse in distilled water, 2 dips, 1 second each.

Rinse in 0.25% acetic acid, 2 quick dips, 1 second each.

Quickly rinse in distilled water, 2 dips, 1 second each.

Rinse in 3 changes of methanol.

Clear in xylene and mount.

End Results

H. pylori, fungi, and other bacteria: Dark blue

Mast cells: Purple

Tissue elements: Lighter shades of blue

Note

The use of methanol to dehydrate stained sections prior to mounting seems to provide lighter staining of surrounding tissue, offering better contrast with stained organisms.

Warthin-Starry stain

For visualization of Spirochetes, Helicobacter pylori, Legionella pneumophila, and Cat scratch fever bacteria.

Reagents

Gelatin (4%), acidulated (with acetic acid)

Silver nitrate solution (0.5%), acidulated (with acetic acid)

Hydroquinone solution (0.1%), acidulated (with acetic acid)

Silver nitrate solution (2%), acidulated (with acetic acid)

Procedure (microwave)

Combine: 12.5 ml gelatin (4%), acidulated; 20–30 drops silver nitrate solution (2%), acidulated; and 7.5 ml hydroquinone solution (0.1%), acidulated to make the reducing solution.

Heat silver nitrate solution (0.5%), acidulated in microwave at full power for 30 seconds.

Place the slides in this heated silver nitrate solution (0.5%), acidulated for 5 min with repeated agitation.

Heat the previously prepared reducing solution in microwave at full power for 20 sec.

Place the slides in this heated reducing solution with repeated agitation, until tissue section is brown (approximately 2–5 min).

Rinse slide in hot tap water for 2 min.

Dehydrate through 3 changes of fresh absolute alcohol.

Clear in xylene and mount.

End Results

Helicobacter pylori: Black

Legionella pneumophila: Black

Spirochetes: Black

Cat scratch fever bacteria: Black

Klebsiella: Brown/black

Nuclei: Brown

Background: Yellow

Note

For Spirochete staining, a special sensitizing solution may be provided by the company in the kit, requiring one extra step at the beginning. The use of this step will not affect the staining quality for rest of the organisms.

1.2 Few Stains for Microbiology Laboratory

Modified Acid-Fast Staining

Various modifications have been tried, but the procedure and concentration which allows detection of broadest spectrum of organisms involve [50]:

• Kinyoun stain: no heating; contains high concentrations of phenol and basic fuchsin, allowing better penetration of the dye.

• Using 1% sulfuric acid without alcohol as decolorizer.

• Counterstaining by malachite green or methylene blue.

Another variation described [24, 51]:

• Addition of dimethyl sulfoxide (DMSO) to the phenol-basic fuchsin: DMSO was thought to allow better penetration of the fuchsin stain into the lipid layer.

• Acetic acid with malachite green as combined decolorizer-counterstain.

Fluorescent Acid-Fast Staining [51]:

• It is the fluorescent auramine-phenol stain.

• Detects mycobacteria and oocysts of Cryptosporidium.

• Auramine stains both lipids and DNA.

Its advantage being, due to fluorescence, large areas can be screened at lower magnification. However, staining artifacts may interfere with interpretation, and it has limited use for detection of other coccidian parasites.

Safranin Staining for Identifying Cryptosporidium [52]:

• Heat-fixed slides to be fixed again in an acid-alcohol solution (3% HCl in methanol).

• Slides were stained in 1% aqueous safranin with heating.

• Washed and counterstained with 1% methylene blue.

Oocysts of Cyclospora and Cystoisospora can also be stained by this method.

Calcofluor White Stain (Optical Brightener) for Cryptosporidium, Microsporidia [53]:

• Calcofluor white or Uvitex 2B solution (0.1%) in 10% NaOH, warmed to 60–65 °C.

• Evans blue as a nonfluorescent background stain.

• Slides treated with these solutions are examined in violet light using a 395- to 400-nm excitation filter and 460- to 520-nm barrier filters.

• Strong fluorescence associated with oocysts.

The correct combination of excitation and barrier filters in fluorescence microscopy helps to obtain fluorescence of optimum color and intensity. With the appropriate barrier filter, Microsporidia oocysts appear bluish white in ultraviolet light [50]. As with fluorescent stains, it allows quicker screening of large areas at lower magnifications.

Autofluorescence of Cyclospora/Cystoisospora/Cryptosporidium [24, 50, 54]:

• These organisms absorb light in the UV range and emit energy of longer wavelength.

• This property is possibly attributed to tyrosine and 3,4-dihydroxyphenylalanine (DOPA) cross-linking in oocyst cell walls, giving the appearance of fluorescent rings.

• Under 365 nm (UV light), Cryptosporidium and Cystoisospora appear violet, and Cyclospora appears blue; under 405 nm (violet light) or 436 nm (blue light), they all appear green.

Annexure IV

1.1 SOP for Immunohistochemistry

Reagents:

• Xylene, graded alcohol, Milli-Q water.

• Citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 9.0) for heat-induced antigen retrieval.

• 50 mM TBS (pH 7.6) wash buffer.

• Peroxidase block for endogenous blocking.

• Protein block or milk block for nonspecific binding sites.

• Primary antibody.

• Secondary antibody.

• 3,3′-Diaminobenzidine (DAB) as chromogen.

• Hematoxylin for counterstain.

• DPX.

1.2 Preparation of Buffers

Phosphate buffer saline 0.1 M (pH 7.2–7.4): 1000 mL

• Disodium hydrogen phosphate (Na₂HPO₄): 1.48 g

• Sodium dihydrogen phosphate (NaH₂PO₄): 0.43 g

• Sodium chloride (NaCl): 7.2 g

Preparation:

• Dissolve these reagents in 1000 mL of double-distilled water/Millipore water.

• Adjust pH with 1 N NaOH or 1 N HCl.

Tris buffer saline (pH 7.4–7.6) 1000 mL:

• Tris 6.05 g.

• Sodium chloride (NaCl) 8.7 g.

• 1 N HCl: 37 g.

• Dissolve Tris and NaCl in 800 mL of double-distilled/Millipore water.

• Add 37 mL of 1 N HCl and adjust pH to 7.4–7.6 with 1 N NaOH or 1 N HCl.

• Make the volume up to 1000 mL.

Antigen retrieval buffer: 0.1 M citrate buffer:

• Dissolve 2.11 g of citric acid in 1000 mL of double-distilled water/Millipore water.

• Adjust pH to 6.0 with 1 N NaOH.

Tris-EDTA buffer:

• 1.21 g TRIS.

• 0.4 g EDTA.

• Dissolve in 1000 mL of double-distilled Millipore water.

• Adjust pH to 9.0 with 1 N NaOH or 1 N HCl.

Procedure in brief:

• 5 μm paraffin sections collected in poly-L-lysine coated slides.

• Deparaffinize and bring sections to water.

• Antigen retrieval in microwave or pressure cooker using citrate (pH 6.1) or tris/EDTA buffer (pH 9).

• For pressure cooker, single whistle, and for microwave, high power for 10–20 min, depending upon the antibody concerned; usually, antibodies with nuclear expression take longer time.

• Peroxidase block for 10 min to block endoperoxides activity and protein block for 5 min to block nonspecific antibody uptake.

• Slides to be blot dried and covered with primary antibody (reconstituted, or recommended dilution, after standardization) and incubated overnight at 4 °C.

• Subsequent day washing in phosphate-buffered saline (PBS), pH 7.4.

• Biotinylated secondary antibody (some kits mention primary amplifiers) for 30 min.

• Tertiary antibody (usually streptavidin or histidine-rich protein-polymer) applied for 45 min.

• Freshly prepared DAB (3,3′-diaminobenzidine) solution used as chromogen for 10 min until the desired color is reached.

• The color reaction is stopped by dipping the slides in distilled water to avoid nonspecific staining.

• Washing thrice using PBS buffer after every step.

• Counterstaining by Harris hematoxylin and evaluation under a light microscope.

• Suitable positive and negative control slides applied with each batch of IHC.

1.3 Automated IHC

A generalized protocol for automated IHC has been given here, which should be modified as per the company’s recommendations and standardized in the department:

• Turn the system on.

• Slides are prepared for IHC and properly labeled with a barcode.

• Slides are loaded in the slide tray in the appropriate position as instructed.

• Prepare the reagents and reaction buffers as per the given instructions and load the required bottles and reagent trays.

• Remove caps from the tip of the dispenser and place them in the cap holder.

• Place uncapped dispensers on reagent trays, and load reagent trays in the instrument.

• Empty waste container if necessary.

• Start the Run after entering the number of slides and checking appropriate boxes.

• At the end of the Run, open the access door, take slides out of the instrument, and place them in the slide holder.

• Exit the program and continue with slide dehydration and mounting as usual.

Common antibodies:

Commonly used antibodies which should be made available (but not exclusively) in the GI Pathology laboratory have been mentioned in the following table, with suitable controls:

Commonly used antibodies in the GI Pathology laboratory with positive controls:

Antibody	Positive controls
Pan CK	Internal control in epithelial cells and glands
EMA	Internal control in epithelial cells and glands (membranous), the kidney
Vimentin	Kidney, prostate, skin
CK 5/6	Skin
CK7	Liver, kidney, pancreas
CK19	Esophagus, colon, skin
CK20	Colon
CDX2	Colon
MOC31	Colon and breast carcinoma
Calretinin	Testes, thymus
Desmin	Internal control; muscularis, prostate
SMA	Internal control; muscularis
S100	Nerve bundles, skin
Melanoma/PEComa panel HMB45, Melan-A, Mart-1	Skin, melanoma
MUC1	Pancreatico-biliary epithelium (bile duct, gall bladder)
MUC2	Colon
MUC5AC	Gastric foveolar (normal stomach)
MUC6	Gastric pyloric (normal stomach)
p53	Breast cancer, colon, skin
p63	Skin
p40	Skin
CD31	Blood vessel endothelium, kidney
CD34	Kidney
CD117	Skin, brain, lung, spleen, mast cells in the intestine
DOG1	Appendix (Cajal cells), GIST cases
Podoplanin (D2–40)	Tonsil (cytoplasmic), lymph node, lymphangioma
WT-1	Kidney (glomeruli), testis
Ki67	Breast carcinoma; tonsil
Synaptophysin	Neuroendocrine cells of gut (internal control), pancreas
Chromogranin	Neuroendocrine cells of gut (internal control), pancreas
Insulin	Pancreas
Glucagon	Pancreas
Gastrin	Stomach (pyloric mucosa)
CD56	Tonsil, neuroblastoma, pancreas
NSE	Pancreas, brain (cerebral cortex)
Lymphoma panela	Reactive lymph node, tonsils, spleen, thymus
IgG4	Spleen, tonsil
Microsatellite markers (MLH1, MSH2, MSH6, PMS2)	Reactive lamina propria lymphocytes, epithelial cell nuclei

1. ^aLymphoma panel includes the following antibodies: LCA, Tdt, CD3, CD20, CD5, Bcl2, Bcl6, CD10, cyclin D1, CD23, MUM1, CD38, CD138, IgG4, kappa, and lambda

Annexure V

Annexure VI

Annexure VII

1.1 SOP and Some Demo Specifications for Molecular Pathology Laboratory

• 1. Deep Freezer – 20 °C, 200 L:

  • Microprocessor-controlled temperature can be digitally controlled with audible and visual temperature alarms.

  • Ambient temperature display.

  • Suitable stabilizer.

  • Lockable insulated solid door.

  • Preferably stainless steel body and internal chamber.

  • A freezer must use a (1) ¾ hp compressor and R404a refrigerant, with at least a 2-year warranty.

  • Must have at least four baskets and two pull cover drawers.

  • A compressor on/off indicator on the control panel.

  • Low battery alarm.

  • Rapid freeze feature for fast cool down.

• 2. Deep Freezer – 80 °C:

  • Should record temperature excursions including actual temperature, warmest temperature, and coldest temperature. This indicator should be resettable by the user.

  • The freezer should notify the customer to perform preventative maintenance tasks including filter change and backup battery test.

  • A freezer should use only CFC-free, commercially available refrigerants are used: R404a in the first stage; R508b/R290 in the second stage.

  • The freezer must be constructed using 1″ thick vacuum panel insulation in conjunction with environmentally friendly water-blown foam.

• 3. Microcentrifuge (Refrigerated or Non-refrigerated):

  • Maximum g-force should not be less than 20,000 RCF.

  • Maximum speed should not be less than 14,500 rpm.

  • Rotor number and tube volume should be specified.

  • Large LED display for time, speed, and temperature.

  • Max noise levels: 50 dBs.

  • Acceleration/deceleration time 15–20 s.

  • Should be fitted with timer; time set range 1–99 min, 1 min increments.

  • Buzzer system when run is over.

  • User-friendly touch keypad for control of speed and programming.

  • Motorized lid latch.

  • 230 V, 50/60 Hz.

  • Preferably 5-year warranty and 5-year AMC.

• 4. Laboratory Centrifuge:

  • Soft-touch keypads/knob for fast and accurate setting of run parameters.

  • Overheating safety cutoff circuit to be provided for motor protection.

  • Rotor head for 16× 5- to 10-mL tubes.

  • Safety lid interlock so that the rotor does not run with the cover open, and the cover cannot be opened unless the rotor comes to a complete halt.

  • Speed range 0–10,000 rpm.

  • User-friendly microprocessor control with interactive LCD.

  • Choice of high brake, low brake, and coasting.

  • Speed holding accuracy 100 rpm.

  • Minimum 1-year warranty

• 5. Ductless Fume Hood

  Chemical fume hoods are recommended when there is a risk of exposure to hazardous fumes or splashes, while chemical solutions are being prepared or dispensed. Airflow is generally controlled by a movable sash and should be in the range of 80–120 ft/min.

  Staining setup (trichrome and/or iron hematoxylin) and preparation of formalin solutions should be carried out in a fume hood.

  • Should have external dimensions of approximately 52 × 29″ × 57″ (W × D × H).

  • Internal dimensions 50″ × 23″ × 30″ WDH approximately.

  • Should be able to eliminate toxic gases, fumes, odors, and powders.

  • Should efficiently recirculate air within the room and release highly purified air back into the room free of any toxic vapors or odors.

  • The main body should be made up of galvanized steel.

  • Sidewalls should be made up of safety glass.

  • Horizontal laminar airflow should be auto bypass type.

  • The cabinet should have activated carbon filter with prefilter.

  • Corrosion-resistant epoxy powder-coated electrogalvanized steel airfoil which should safely ventilate fumes generated toward the front of the hood for superior operator protection.

  • Slanted sash constructed of tempered safety glass enables optimal visibility and ergonomic workspace.

  • Hood should have an air velocity of >75 ft/min.

  • The exhaust volume should be >200 cfm.

  • Should have fitted fluorescent lights with vapor-proof fittings with illumination greater than 1000 lux.

  • Worktop should be of 16-gauge stainless steel and have an oval-shaped sink with a suitable drainage system.

  • There should be one water connection and two electric sockets, prefitted.

  • Low sound levels less than 60 dBA.

  • 220 V, 50 Hz.

  • Germicidal UV lamp to be provided.

  • Base cabinet for storage with wheels and height-adjustable lab chair preferably should be provided.

  • Minimum 3-year warranty

• 6. Biological Safety Cabinet (BSC)

  BSCs operate on the principle of negative air pressure, where air passes through a HEPA filter. Two variants are being used: class I (open-face) and class II (laminar flow). It is usually recommended for laboratories performing cultures for parasite isolation (Microbiology Review).

  However, BSCs should not be used as fume hoods, as toxic, radioactive, or flammable vapors are not removed by HEPA filters.

  • (i) Automated nucleic acid extractor

    The nucleic acid extraction kits manufactured by different companies presently provide reliable quality nuclear material. Fully automated extractors use the principle of magnetic separation. Disposable reagent cartridges should be used in automated extraction. However, automated nucleic acid extraction is a costly affair and may not suit our moderate resource centers. Manual extraction may be managed in centers receiving 50–100 samples per day. Switching to automated nucleic acid extraction would be justified in centers performing 200 or more samples a day; however, this is entirely based on the personal choice and affordability of any institution.

  • (ii) PCR/thermocycler:

    • Block format: two to three blocks, 0.2-mL well capacity, independent control, Peltier-based heating, and cooling system.

    • VeriFlex/gradient blocks.

    • The block’s ramp rate of 6 °C/s or better ramping rate.

    • Should have a temperature range of 0–100 °C, accuracy and uniformity of at least ±0.25 °C across the block.

    • Should have auto restart after power failure.

    • Intuitive graphical screen/LCD and touch screen.

    • At least 500 programs memory through USB and on board.

    • The system should have stimulation mode.

  • (iii) Real-time PCR:

    • The system should be an automated and integrated system for both real-time PCR and post-PCR (endpoint) analysis.

Hardware:

• The system should be a 96-well silver block system with Peltier-based heating and cooling, along with an optional 384-well block, which can increase the throughput.

• The system should have at least six Peltier elements for uniform heating and cooling.

• The system should be an open platform in terms of reagents as well as consumables, i.e., support microwell plates, individual tubes, and eight-tube strips.

• The instrument should have a 96-well format that is capable to do the multiplexing for at least six colors, able to run fast, and standard runs on the same block.

• The excitation source should be white LED/multiple LEDs.

• The detection system should be simultaneous and scan-free for all wells. Detection should be photodiode/PMT.

• Should support the temperature range from 25 °C temperature to 100 °C.

• The maximum ramp of the system rate should be more than 4 °C/s so that it should be able to finish 40 cycles in 40 min at a fast mode.

• The system should support a reaction volume of 10–30 μL.

• The system should have a robust touch screen LCD feature.

• Fast-PCR (40 cycles in less than 40 min) should be an integral feature.

Software:

• Should be pre-calibrated for commonly used dyes like FAM™/SYBR^® Green, VIC^®/JOE™, NED/TAMRA, and ROX™.

• Can analyze multiple perspectives in the multiple plot view, including the amplification plots, standard curve, multicomponent data plots, and raw data.

• Support applications including absolute quantitation, relative quantitation, multiplex-PCR, allelic discrimination (SNP), melt curve analysis, as well as pathogen detection, an assay using internal positive control and high-resolution melting analysis for gene and mutation screening.

• The desktop should have preloaded windows of the latest version with Microsoft office and robust antivirus software, RAM 4 GB, hard disc 1 TB

Supporting Chemistries and Applications:

• Support all real-time PCR chemistry like TaqMan, SYBR Green, simple and hydrolysis probes, molecular beacons, etc.

• (iv) Electrophoresis unit with universal power supply:

  • Should be capable of running two mini gels side by side simultaneously.

  • Buffer chamber requirement for each gel running should be around 300–400 mL.

  • Cell size should be around 11 × 12 × 16 cm (height with lid on), 26- and 51-well combs, gel caster.

  • Bromophenol blue migration: ~4.5 cm/h (at 75 V).

  • At the recommended conditions and constant voltage, proteins should be transferred to nitrocellulose or PVDF membranes typically in 30–60 min.

  • Fully adjustable timer control.

  • Pause/resume function.

  • Automatic recovery after power failure.

  • Power input – 220 V AC, 50/60 Hz.

  • Display – LCD graphic type

Universal power supply:

• 100–120/220–240 V, power supply for a broad range of apparatus, including thermocyclers

• Suitable UPS for 30 min power backup

• (v) Western blotting assembly (semidry transfer apparatus):

  • Should be able to blot a maximum gel size (W × L) 24 × 16 cm.

  • Should have a gel capacity of four mini hand-cast gels.

  • Should have a buffer requirement of no more than 200 mL.

  • Should have platinum-coated titanium anode plate electrode and stainless steel cathode plate electrode.

  • A programmable semidry protein transfer system with preprogrammed methods and the facility to create save and edit customized methods.

  • The system should be compatible with both precast and lab-made polyacrylamide gels. The entire system should be made of inert, chemically resistant material. The system should support the use of traditional transfer buffer as well as proprietary transfer buffers.

  • The system should be capable of transferring proteins ranging from 10 to 300 kDa from polyacrylamide gels to nitrocellulose or PVDF membranes in a short time (<60 min). The system should be capable of transferring four mini gels or two midi gels simultaneously.

  • The system should have an LCD/LED display and indicate warnings in case of lack of passage of current or power failure.

With accessories:

Nylon membrane – Pkg of 1 roll, 30 cm × 3.3 m, high-strength nylon membrane positively charged with quaternary amine groups.

• In addition to western blotting, the Trans-Blot semidry cell should also transfer DNA and RNA using the agarose gel semidry blotting support frame.

• (vi) Qubit specification or nano-spectrophotometer:

  • An analytical instrument for DNA, RNA, RNA IQ, and protein quantification with a small benchtop footprint and 5.7″ touchscreen intuitive user interface.

  • Monochromator-based xenon flash lamp as the light source for better performance.

  • The instrument should use as little as 0.5–1 μL of sample.

  • The instrument should have a sensitivity range for dsDNA of 0.5 ng/mL (10% CV).

  • Surface retention technology for microvolume measurement.

  • Measurement time less than 5 s.

• (vii) Specifications of chemi documentation system:

  • Systems should image and analyze chemiluminescent western blots, visible and near-infrared fluorescence-based western blots and gels across five separate channels (RGB and near-infrared (NIR)), and stained protein (Coomassie, silver, Sypro, etc.), DNA (EtBr, Sybr, etc.) gels.

  • Image resolution >4 MP.

• (viii) Fully automated elisa system:

  • Fully automated walk away Elisa Processor (with capability for upgradation for fluorescence and luminescence) with following features:

  • No of plates: six or more with the possibility of having additional plates for future upgrading.

  • Facility for both disposable (optional) and Teflon tips (Standard) with two arms for dispensing reagents and samples and one arm for moving plates from one station to another.

  • Modular system all parts, i.e., robotic sampler, reader, washer, and incubator, should be usable together and individually.

  • Up to 256 samples should be assayed and more than one Elisa type at one time.

• (ix) Cooling plate:

  • Temperature can be digitally controlled with 1 °C increments.

  • Must have easy cleaning Teflon-coated surface which allows a minimum ice formation.

  • The cooling surface should have the capacity to cool minimum of 70 cassettes at a time.

  • 220 V, 50 Hz.

  • Two-year warranty.

• (x) Diatome diamond knife for ultrathin sectioning:

  • 3/3.5-mm 45° cutting angle.

• (xi) pH meter with detachable electrode:

  • pH range 1.00–14.00

  • Resolution 0.01

  • Automatic temperature compensation preferred

  • Minimum 1-year warranty

• (xii) Digital balance (wide weight range denominations):

  1 mg–200 g; 1 μg–5 g; 0.1 μg–2 g

  • Electronic weighing with LCD and complete with metal casing and transparent wind shielded cover.

  • Reproducibility

    • For 1 mg–200 g balance: 0.1 mg

    • For 1 μg–5 g balance: 1 μg

    • For 0.1 μg–2 g balance: 0.4 μg

  • Pan size – minimum 80 mm

  • Electrical adapter for mains 230–240 V/50–60 HZ.

  • Minimum 1-year warranty

• (xiii) Mini shaker:

  • Tube size 1.5/2 mL, block for 0.5 mL to be provided

  • Microprocessor-controlled temperature 15 °C below ambient temp to 100 °C

  • Temperature uniformity: ±0.5 °C

  • Digital LCD-display

  • Security switch, which stops the shaking process when opening the lid

  • Speed: 250–1250 rpm, amplitude: 3 mm

  • Heating power: 200 Watt

  • Minimum 1-year warranty

• (xiv) Vortex:

  • Type of movement: orbital

  • Voltage 240 V, 50 Hz

  • Speed 100–3500 rpm

  • Orbital diameter 4–5 mm

  • Supplied with cup attachment and microtube insert

• (xv) Ice maker:

  • 120 kg max. production/24 h

  • 32 kg max. storage

  • Space-saving “Up & Over” bin door

• (xvi) Cooling water bath (4 °C):

  • Double-walled, puff insulated.

  • The working temperature should be −20 to +120 °C

  • Fitted with Kirloskar cooling compressor.

  • Copper cooling coils are welded.

  • Caster wheels for easy movability.

  • Digital temp controller.

  • Provided with circulating pump, stirrer.

  • Single-speed pressure pump with an external circulation capacity.

  • Capacity to blow liquid for homogeneous heating or cooling throughout.

  • Front-mounted drain.

  • Electrical: 230 V/50 Hz.

• (xvii) Magnetic stirrer without hot plate:

  • Should be microprocessor-controlled

  • Digital speed indicator for displaying string speed

  • Speed range 100–650 rpm

  • Stirring capacity: 1000 mL

  • Should have direct-drive motor and magnet system to permit quiet stirring

  • Should be provided with three magnets

  • Electrical: 230 V/50 Hz

• (xviii) Fully autoclavable variable volume micropipettes and multichannel pipettes and tips:

  • Lightweight and ergonomic design

  • Super blow out for microvolume pipetting

  • Should have separate soft-touch tip ejector

  • 3-year warranty

  • Size specifications:

    • 0.2–2.0 μL

    • 0.5–10 μL

    • 2.0–20 μL

    • 5–50 μL

    • 10–100 μL

    • 20–200 μL

    • 15–300 μL

    • 1–5 mL

• (xix) Vertical autoclave:

  • Should have a triple-walled construction.

  • The working chamber, steam jacket, outer chamber, and lid should be made of stainless steel.

  • Should have water inlet and outlet valves.

  • Should have a water level gauge and gauges for measuring inner and outer steam pressure.

  • Should have an inner temperature indicator.

  • Should have automatic pressure control switch, safety valve, and eject valve.

  • Should have a jointless silicone gasket.

  • Should have 121 °C working temperature at 15–20 psi pressure.

  • Should be operated in mains 220–240 V AC 50 Hz input power supply.

• (xx) Vortex mixer for molecular biology:

  • Should be ideal for continuous operation and touch on modes for gentle to vigorous mixing and vortexing with a variable speed controller.

  • Should have durable plastic housing to resists acids and alkalis.

  • Should have heavy-duty cast metal base with rubber feet assures stability and eliminates creep during use.

  • Anti-spill technology for controlled mixing and no cross-contamination (2D mix control).

  • A variety of interchangeable attachments should be provided to accommodate almost any sample vessel size or shape.

  • Should be supplied to vortex for single tube, four tubes, 3-inch platform suitable for beakers, flasks, and multiple tubes. Additional accessories should be supplied to accommodate a range of vessels including microtubes and microplates.

  • Speed range 100–3000 rpm.

  • Timer to be provided 15 s–99 h.

  • Electrical: 230 V/50 Hz.

• (xxi) Laboratory Thermometers to Measure Room Temperature:

  • Vertical with stand

  • Should be fixable to wall, suitable fixtures to be provided.

  • Temperature range 0–100 °C

• (xxii) Laboratory thermometers to measure ultralow temperatures:

  • Vertical with stand

  • Temperature range −90 to 40 °C

Annexure VIII

1.1 Tentative List of Consumables for GI Pathology Laboratory

Some of the regularly used consumables will be highlighted here; the rest should be decided by the user laboratory as per the tests and stains being run over time. The initial pack size and the amount specified are intended for initial setup only. This may be modified subsequently when the exact need can be accurately estimated by the individual laboratories as tests are standardized and regularly implemented in due course.

• Formalin jars, xylene, grossing instruments, biopsy cassettes, reagents for H&E, and special stains.

• Glass wares (graduated cylinders, flasks, bottles, funnels, centrifuge tubes, slides and coverslips, and pipettes).

• Depending on the relevant procedures, sterile glassware may be required.

• Gauze, culture tube racks, applicator sticks, storage boxes, filter paper, lens paper, forceps/scissors.

• Appropriate biohazard containers.

• A list of some consumables with initial requirements for the surgical pathology and cytology of the GI tract has been provided in the following table.

Name of items	Initial requirement
Eosin	100 g
Hematoxylin crystalline	25 g
Light green	10 g
Sliver nitrate (molecular grade)	25 g
Pararosaniline hydrochloride	25 g
Basic fuchsin	25 g
Periodic acid	25 g
Giemsa stain	25 g
Toluidine blue	25 g
Azure B	10 g
Giemsa stain	25 g
Slide holder for 25 slides stainless steel	10
Staining trough horizontal with lid glass (14 × 10 × 8 cm)	20
Cyto-filter card for cytospin; single/double holes	5
Cyto-slides for cytospin	Number of boxes as required
Cyto-funnels for cytospin	5
Cyto-clips for cytospin	10
Cryo-embedding medium for cryostat (polyvinyl alcohol, polyethylene glycol)	2 bottles
High-profile blades for microtomy	10
Low-profile blade for microtomy	10
Disposable scalpel blades for grossing	20
Scalpel and knife handle compatible with used microtomy blades for grossinga	2
Tissue marking dyes – blue/green/red	1 bottle each
Dissecting grossing board – white cutting board of approximately 30 × 20 cm: Noncorrosive, unbreakable, nonabsorbent, scratch-resistant Easy to fix (prevent sliding) and clean Preferably with XY scales in centimeters for sizing of specimens	1
Electronic digital weighing scale for weighing of specimens	1
High-resolution camera with a zoom lens and LED lighting, mounted overhead for grossing station	1

1.2 Tentative List of Molecular Biology Consumables

Name of items with specification	Usual pack size
Tissue DNA extraction kit	50 prep
DNA extraction kit (whole blood)	50 prep
cDNA synthesis kit	50 prep
Total RNA extraction kit	50 prep
Agarose-molecular biology grade	500 g
SYBR Green master mix for QPCR	200 × 25 μL
DNA methylation kit	50 prep
PCR master mix (2×)	200 × 50 μL rxns
DreamTaq PCR master mix (2×)	250 prep
Ethidium bromide	10 g
Bromophenol blue	25 g
EDTA molecular biology grade	500 g
Restriction enzymes	As necessary
Ethanol AR	500 mL
6× loading dye for DNA agarose electrophoresis	Per 6 × 1 mL
DNA bisulfite modification kit for methylation-specific PCR stations)	48 Prep
Micro RNA cDNA synthesis kit	50 rxns
Serum microRNA isolation kit	50 Prep
Guanidinium thiocyanate	100 g
Random hexamer for RTPCR	120 μL
Oligo (dT) 18 primer for RTPCR	Per base
M-MuLV Reverse Transcriptase for RT-PCR	5000 units
Taq DNA polymerase 3 U/L with 10× buffer and separate vial of 25-mM MgCl2	1000 U
Amplitaq Gold DNA Polymerase	100 U
Proteinase K	Pack of 1 mL
50-bp DNA ladder	50 μg
100-bp DNA ladder	50 μg
Micro RNA ladder	5 × 20 μL
dNTP mix 10 mM adjusted to neutral pH	1000 μL
TRIS base AR	1 kg
Sodium dodecyl sulfate AR	500 g
Acrylamide AR	500 g
N,N′-Methylenebisacrylamide AR	100 g
TEMED	100 mL
Diaminobenzidine (DAB)	10 × 6 mg
Hot Start Taq DNA polymerase with 10× buffer and	250 U
Hot Start PCR master mix	100 × 50 μL
RNAse inhibitor human placental or RiboLock TM or RNAsin	2500 units
Genomic DNA purification kit for fresh and frozen blood/tissues	50 prep
Protease-, Dnase-, and Rnase-free water for molecular Biology Provision for Milli-Q water	As required
Protein molecular weight marker Low range Medium range	50 μg
Dithiothreitol	5 g
Bradford reagent	950 mL
DMSO for molecular biology	500 mL
Glycerol for molecular biology	1 L
Sodium acetate for molecular biology	500 g
Sodium hydroxide pellets extra pure for mol. biology	500 g
10×TBE buffer	1 L
Hydrochloric acid	500 mL
Boric acid for mol. biology	1 kg
Acetic acid glacial extra pure for molecular biology	500 mL
Tris chloride for mol. biology	500 g
ECL prime blocking reagent	1 L
Full-range rainbow molecular weight marker	2 × 250 μL
ECL dual Vue western blotting markers	200 mL/kit
ECL prime western blotting detection reagent	500 mL/kit
Triton X-100	1 L
Tween 20	1 L
PCR tubes with ultrathin walls
Cryobox for storage of cryogenic vial (1–2 mL size)	1–2
Microcentrifuge tube (1.5- to 2-mL size) freezer storage boxes with 80 wells	1
Microtip box 96 positions 0.2–10 μL	3–5
Microtip box 96 positions 20–200 μL	3–5
Microtip box 96 positions 200–1000 μL	3–5
Nitrocellulose membrane for western blotting 0.45-μm pore size	20 × 20 cm

Annexure IX

1.1 E-Tender for Equipment

Schedule of Tender (Proforma)

Disclaimer: Bidders are requested to attend the pre-bid meeting. If any query or clarification is required on the terms & conditions of tender or on the specification of item(s), it may be raised in the meeting. Queries raised after the pre-bid meeting shall not be entertained.

Validity of tender:

Validity of rate contract:

Total (EMD)/bank guarantee submitted:

Annexure X

1.1 Pre-qualification Bid (Checklist)

Name of the bidder _____________________________

	Details of documents
1	Bid security (also known as earnest money deposit) detailed at above Name of the bank/date and its validity Amount of bid security
2	Income tax-related verification: Submit a self-attested copy of filing income tax return for the last 3 years, along with self-attested copies of identity proofs issued by the income tax department
3	Satisfactory performance certificate of the firm: The firm is required to submit original or self-attested photocopies, issued by an authorized person, from any two reputed hospital/institution/laboratory (of more than 200 bedded/samples) to whom similar supplies of equipment/machinery have been made during the last 3 years, certifying that the performance of the firm was found to be satisfactory.
4	Undertaking: only on a nonjudicial stamp paper to abide by the terms and conditions of tender, duly signed and stamped by the bidder
5	The bidder must furnish the name of his authorized distributor, if any, along with address, mobile/telephone number, fax number, or email to supply, install, and submit the bills to this institute and take responsibility for AMC/CMC of this item
6	Any other relevant document(s) which the firm wishes to enclose

Annexure XI

(Technical Bid)

Technical Bid Proforma

Note:

• The quantity of items mentioned is tentative and may increase or decrease depending upon the requirement of the user department.

• The hospital does not make any commitment to the procurement of the quantity mentioned against each item.

• The list of equipment and detailed specifications are given below. All the bidders are requested to carefully read the technical specifications and tender terms and conditions before submitting their e-bids. For any clarification, the prospective bidders are advised to attend the pre-bid meeting.

Annexure XII

1.1 Financial Bid Proforma

Name of the bidder ___________________________

Note:

• 1. The Financial Bid Proforma should be filled up for only that equipment offered in the Technical Bid Evaluation. All the columns are mandatory.

NAME OF BIDDER
	SIGNATURE and STAMP of the BIDDER

Chapter Summary

• A good pathology laboratory should have well-developed basic facilities including histopathology, cytopathology, histochemistry, and immunohistochemistry.

• In surgical pathology, grossing is the most important step which forms the basis of any histopathological diagnosis. It should be taken care of by trained personnel and newer residents, or staff should be given enough training before giving them independent responsibility of grossing.

• Wherever affordable and possible, a molecular laboratory comprising largely Real-time PCR and sequencing facilities should be added.

• Emphasis should be put on sectioning and staining quality, particularly about histochemical stains.

• Automation should be practiced, especially in immunohistochemistry depending on caseload as it has the additional advantage of providing better-quality staining.

• The hospital information system (HIS) has become an essential component of a hospital providing patient care, teaching, and research. It increases efficiency, decreases errors, and improves the service quality of an institution.

• Meticulous planning, assessing the requirement, and having knowledge of updated technology information should be gathered before implementing HIS in a hospital.

• Repeated discussions and running trials of different software should be sorted before completely switching on to HIS.

• Before making the module windows mandatory in HIS, the staff including doctors, residents, nursing, and clerical personnel should work on their specific modules to become aware of the problems faced and ask for customization of the modules according to the requirements based on their nature of work.

MCQs and Fill in the Blanks

• 1. Among the mismatch repair proteins, which is true?

  • (a) MSH2 heterodimerizes with MSH6

  • (b) MLH1 heterodimerizes with PMS2

  • (c) MSH3 heterodimerizes with PMS2

  • (d) MLH1 heterodimerizes with MSH6

• 2. Rapid processing of endoscopic biopsy requires a minimum time of ___________, while microwave processing requires a time of ___________.

• 3. In the molecular pathology laboratory, areas designated for sample preparation and PCR amplification should have:

  • (a) Positive pressure with outward airflow

  • (b) Negative pressure with inward airflow

  • (c) Positive pressure with space for reagent preparation

  • (d) Negative pressure with space for reagent preparation

• 4. The implementation of the hospital information system includes all except

  • (a) Software requirement

  • (b) Knowledge about specifications of hardware and software required

  • (c) Information on manpower strength working in the institution

  • (d) Awareness of functional design and needs of different divisions of the institution

• 5. The following is true about APLIS and CPLIS in a laboratory information system (LIS).

  • (a) Distinct from each other in software and hardware requirements.

  • (b) Distinction between the two is not possible due to overlapping of investigations between the two components.

  • (c) Contain a separate set of investigations to be followed in each type of LIS.

  • (d) Functional design of both is different.

• 6. Integration of hospital information system includes

  • (a) Data and functional integration

  • (b) Workflow, data, and function integration

  • (c) Functional and workflow integration

  • (d) Data integration only

Answers and Explanations

Answer 1

(a) and (b)

During DNA mismatch repair, MLH1 works in conjunction with PMS2 to recognize the replication errors, and a heterodimer composed of MSH2 and MSH6 directs the repair of those errors. In addition, MLH1 promoter methylation has also been described in sporadic tumors, leading to transcriptional silencing and absent expression of both MLH1 and PMS2 [1]. Absence of nuclear expression of these proteins in the tumor, with retained nuclear expression in adjacent nonneoplastic glands and lymphocytes is considered as microsatellite instability due to deficient mismatch repair.

In addition, demonstration of BRAF V600E mutation through DNA-based PCR or more recently by IHC favors sporadic MSI-high colorectal carcinoma due to MLH1 promoter methylation [17].

Answer 2

4–5 h; 15–60 min

Rapid processing provides the opportunity of a shortened duration of 4–5 h processing schedule for small biopsies, like endoscopic biopsies, by omitting various alcohol baths and using vacuum infiltration for the paraffin-permeation stages. Microwave method of tissue processing can reduce the processing time even further to 15–60 min, using alcohol, isopropanol, and formalin, at temperature ranging from 67 to 75 °C [12].

Answer 3

(b)

Separate areas should be dedicated for different activities like reagent preparation, sample preparation, PCR amplification, and post-PCR analysis of amplified product. Unidirectional workflow should be maintained through these work areas to avoid contamination. Positive pressure with outward airflow is recommended in the reagent preparation compartment, whereas negative pressure with inward airflow in sample preparation, PCR amplification, and post-amplification (detection and analysis of amplified product) compartments.

Answer 4

(c)

The HIS functions with the coordination of different modules which are different components of a centralized information system. The components of module consist of software requirement, specification, and functional design documents for each division of the hospital. Each module is designed to serve the need of different divisions of a hospital such as the registration of patients, outpatient department, ward, operation theatres, procedure rooms, radiology, laboratories, billing, and drug distribution.

Answer 5

(b)

The LIS for pathology is broadly categorized into anatomic pathology (APLIS) and clinical pathology (CPLIS). The distinction between APLIS and CPLIS is not very useful as many tests are included in both APLIS and CPLIS such as reporting of bone marrow aspiration and biopsies in hematopathology.

Answer 6

(b)

Integration of data, function, and workflow aspects are required in hospitals for HIS.

For clinical events, integrated data is stored in the actual data center in the format of 6W1H (who, when, where, to whom, what, why, how).

To manage operations of all divisions of the hospital, function integration of web services needs to be implemented.

To monitor and manage processes and flow of work between an individual and department, the workflow is also integrated into the client-server model.