CellBlockistry: Chemistry and art of cell-block making – A detailed review of various historical options with recent advances

Lysis of blood interference

If the specimen has a significant proportion of blood contamination as compared to the diagnostic cell-tissue component, then treat the blood contaminated concentrated specimen with lysing reagent such as proprietary lysing reagent BloodLyz™ (ammonium chloride based lysing reagent similar to that used for flow cytometry, so that immunohistochemistry results are not affected).[58] Acetic acid-based and alcohol-based lysing reagents may compromise results of ancillary tests such as immunohistochemistry and should be avoided.[959]

Mix the working lysing reagent with blood contaminated concentrated specimen and let the lysis be completed by keeping at room temperature for up to 10 minutes. Then, centrifuge the mixture with lysing reagent for 3 minutes at 2500 rpm to sediment the cell-tissue components in the concentrated sediment suspension. Discard the relatively clear (transparent) pink to red supernatant with lysed red blood cells and use the whitish-buff-colored sediment with concentrated nucleated diagnostic cells to make the cell-block by adding to the Nano unit [Figure 10]. The portion of this sediment with nucleated cell rich diagnostic cells may also be used for making cytology preparations.

Simple sedimentation method

1. Spin the concentrated specimen for 3 minutes at 2500 rpm

2. Decant the supernatant, and add about 1 ml of 10% tinted formalin and spin again for 3 minutes at 2500 rpm

3. Decant the supernatant formalin and discard it appropriately. Transfer the conglomerated partially fixed sediment button on a 2 inch × 2 inch lens paper piece inside the labeled tissue cassette

4. Wrap the pellet gently in lens paper, prestain the button (with eosin or hematoxylin as per institutional preference), and close the tissue cassette by snapping the top cover

5. Transport the cassette in 10% formalin to be processed after fixing in 10% formalin for at least 2 hours with protocol for paraffin embedding.

The diagnostic cells are present as randomly and indiscriminately distributed cells and may be admixed with blood and proteinaceous material.[60]

Preparation of Celloidin bags (under a vented fume hood)

Material required

1. 15 ml Pyrex conical glass tubes (Corning 8060, #05-505, Fisher Scientific, Pittsburgh, PA)

   or 15 ml polypropylene conical plastic centrifuge tubes

2. Celloidin Flexible USP (Mallinckrodt, #4580-500-NY, Baxter Scientific, McGraw Park, Ill)

   or prepare Celloidin solution (by soaking Celloidin flakes in absolute alcohol and then dissolving the soaked flakes in ether to get 10% solution in absolute alcohol-ether (1:1) (store in a well-stoppered bottle at room temperature), and

3. Vertical fume hood.

Method of preparation of Celloidin bags (needs experience and skill and has some risk related to the handling of inflammable, volatile material)

1. Place the 15 ml conical centrifuge tubes in the rack and top with Celloidin solution under a vented fume hood

2. Wait for 10 minutes (Celloidin bags would be thicker if this time is longer)

3. The top surface of Celloidin would harden with the evaporation of the alcohol-ether solvent. Puncture and remove the hardened top surface with wooden applicator stick

4. Pour out the Celloidin solution (back into the bottle for reusing later [Celloidin solution may be reused multiple times until the dry bags are very thick and difficult to fold]) and drain all Celloidin solution by inverting the conical tubes until the tubes dry under a vented fume hood. Initially, the tubes may appear cloudy, but final dried tubes will be clear

5. The celloidin tubes with thin film (about 20–50 μ thick) on the inner surface of the tubes can be stored upside down in a dry, cool condition up to 6–8 weeks until used.

Other alternative, although less practical, is to store these coated tubes (with about 20–50 μ thick layer of celloidin) partly filled with chloroform (which hardens the celloidin and prevents excessive drying of the film) and stoppered. The chloroform is discarded just before use.

Preparation of Cell-block with Celloidin bags

1. Add concentrated specimen to the celloidin tubes and fill the tube nearly to the top with the saline

2. Centrifuge the tube at 1500 rpm for 8 minutes

3. Discard the supernatant (with a Pasteur pipette without disturbing the cell button or by inverting the tube if the pellet is not loose)

4. Remove the softened Celloidin sac gently with pointed tip forceps by separating out the bag-like cast from the tube wall by peeling it away gradually and gently without significant jerking to avoid disturbing the cell button (this step needs skill and practice)

5. Fold the bag-like an egg role as close to the cell button as possible and trim the excess bag with scissors without disturbing the cell button

6. The folded bag is wrapped in lens paper and placed in B-5 fixative for 2 minutes

7. The wrapped Celloidin bag with concentrated cytology specimen is transferred from B-5 fixative (without exposure to any metal structure) to the labeled plastic tissue cassette

8. The cassette is placed in 70% ethanol for 2 minutes to rinse off excess B-5 fixative before transferring to 10% formalin for final tissue processing and paraffin embedding.

The diagnostic cells are present mostly along the wall of Celloidin bag and may be admixed with blood and proteinaceous material.[60]

Hot methods

These are gels such as Agar, gelatin, and HistoGel™ have to be molten with heat, and the molten gel is mixed with sediment and then allowed to solidify leading to a button which can be handled for processing. Heating of gel with melting at optimum temperature may be clumsy in addition to the problems related to controlling solidification stage with proper alignment of sediments along the cutting surface of final FFPE of cell-block before solidification.

Routine indiscriminate procedures using molten gels [Figure 16][121319]

1. Specimens with relatively abundant sediments may be processed by using gels which are liquid when hot and solidify after cooling

2. Melt small amount of gel in a tube until it melts completely

3. Cool the gel to the temperature which will not be damaging to the specimen but will still keep the gel molten (e.g., −60°C for Agar and HistoGel™)

4. A few drops (about 1–2 drops) of molten gel are added to the sediment. The button should not have extra water (excess water may dilute the molten gel and prevent or delay solidification)

5. Mix by vertexing or with wooden applicator stick

6. Place the tube with its pellet mixed with gel into a refrigerator for 5–10 minutes or until solid

7. With small spatula, carefully transfer the pellet embedded in the gel from the tube onto a lens paper inside the tissue cassette

8. Fold the lens paper gently over the pellet. With pellet wrapped in paper, snap the top cover of tissue cassette to close it

9. Drop the cassette into 10% formalin and process for paraffin embedding.

Shidham's method

This method concentrates the diagnostic cells along the cutting surface with dark-colored AV marker which aligns during the procedure at the level of concentrated diagnostic cells. This addition of discriminatory component to the entire procedure results in quantitative improvement. It also introduces precision to the routine random technique of cell-block making and grants ultimate control over paraffin block sectioning by histotechnologist.[20]

Shidham's method for cell-block preparation using HG is reproduced from the original open access publication in entirety after minor modifications below.[20] This video publication in ‘Open Access’ also includes a step-by-step video demonstrating the procedure. As compared to other random approaches, the following are the two critical features of this protocol for preparing cell-blocks from relatively hypocellular specimens with singly scattered loose cells.

1. This protocol involves steps to concentrate the diagnostic cells along the plane parallel to the cutting surface of the cell-block

2. It also includes a beacon-like dark inclusion of AV marker, which serves two of the following purposes:

   • To visualize the level at which the cells are concentrated. The area of the cell-block with the cells of interest now could be visualized by the histotechnologist when the dark-colored beacon is exposed during cutting. This ability to monitor would prevent one from cutting through the level with most of the cells, or not cutting too superficial into the level with the highest concentration of sample cells

   • To provide a locator reference point in serial cell-block section on different slides. This reference point acts as a beacon to help locate particular cells or groups of cells for evaluation of a coordinate immunoreactivity pattern with the SCIP approach.

Preparation of the sample

1. Transfer the concentrated specimen to a flat bottom glass tube (15 mm diameter × 45 mm). Place the glass tube into a larger plastic carrier tube (28 mm × 85 mm) and centrifuge. Remove the glass bottom tube from the carrier tube and pour off the supernatant

2. The glass tube is then capped (to prevent spillage of heating water in the next step) and placed back inside a larger flat bottom carrier plastic tube

3. The carrier plastic tube containing the glass tube is then capped, placed in a centrifuge (with swiveling cups and not fixed angle cups so that the cells fall perpendicularly to the flat bottom of the glass tube), and spun at 1805 G (3000 rpms, rotor radius-17 cm) for 5 minutes

4. The tubes are then removed vertically from the centrifuge, and the smaller glass tube is removed with forceps from the larger carrier plastic tube without disturbing the sedimented pellet with cells

5. The glass tube with the specimen is uncapped, and the supernatant is poured off taking care not to disturb the flat layer of sediment cells at the bottom.

Inclusion of the reference coordinate AV marker and addition of gel

1. A dark beacon AV marker (about 2 mm × 2 mm in size, flat surfaced, fragment of dark-colored, sectionable material) is added as a signpost to the glass tube

2. Melt an aliquot of HG by melting it in the microwave for 10 s at medium power

3. Add 0.5 ml of molten HG to the tube, mix with the sediment quickly, and recap it (Proceed to the next step quickly without allowing the HG to begin solidifying)

4. Add about 2.5 ml of warm (45°C) water to the carrier plastic tube

5. The smaller capped glass tube is placed inside the plastic tube with warm water. (This step is necessary to keep the HG from solidifying during the next steps)

6. The carrier plastic tube is placed in the centrifuge (with swiveling cups and not fixed angle cups so that the cells fall perpendicularly to the flat bottom of the glass tube), and spun at 1805 G (3000 rpms, rotor radius-17 cm) for 5 minutes. The purpose of this centrifugation step is to push the AV marker and to concentrate the cells into a layer closer to the cutting surface of the final paraffin embedded cell-block

7. The tubes are then removed gently and vertically from the centrifuge taking care not to disturb the sedimented thin layer with sample cells at the bottom

8. The larger plastic tube is uncapped, and the smaller glass tube is removed vertically by a forceps without disturbing the sediment layer of specimen cells

9. The small glass tube is refrigerated in vertical position for 15 minutes to cool and solidify the HG.

Removal of the cell-block as a button of gel with the specimen for final processing

1. The solidified HG disk, with the layer of concentrated/sediment specimen at the bottom, is dislodged from the flat bottom glass tube by squirting 10% formalin through a 23 G needle with the syringe

2. The needle is inserted along the side of the tube at the periphery of solidified HG disc with the specimen

3. The needle is rotated along the side of the tube while formalin is slowly pushed through the syringe. This results in the separation of the HG button along with trapped dark-colored beacon AV marker and the concentrated specimen in it from the flat bottom of the glass tube

4. The cell-block (gel button with specimen cells) is then placed in a labeled cassette and submitted for tissue processing to prepare paraffin embedded cell-blocks.

Embedding and cutting of the specimen

1. The disk is embedded in paraffin with the dark beacon marker side down as cutting surface

2. The block is sectioned until the dark-colored AV marker as a beacon is exposed and clearly visible

3. 3–4 μ sections are cut from this level which should contain most of the singly scattered cells from the specimen

4. The sections are collected on the glass slide for further staining, immunohistochemical staining, or other tests as indicated. The protocols for these tests including the type of slides to use for mounting the sections may vary. In general for immunostaining, coated slides are used to prevent floating and loss of sections from the slides during the immunostaining steps.

The diagnostic cells are present as randomly and indiscriminately distributed cells and may be admixed with blood and proteinaceous material with supporting gel.[20]

Cold methods

Plasma/fibrinogen-thrombin method: Plasma (pooled plasma from a blood bank may be used) and thrombin (1000 NIH U/ml) are used to prepare the cell-block. The stability of the reagents should be checked periodically by adding two drops of thrombin solution to two drops of plasma, which should clot in about 30 seconds.

Procedure for plasma/fibrinogen-thrombin method [Figure 15][2829]

1. The sediment is mixed with 2–3 drops of plasma/fibrinogen solution

   (If the specimen is not in isotonic medium but in fixative such as formalin, the sediment with fixative will interfere with the enzymatic activity of thrombin and may not achieve proper coagulation of plasma/fibrinogen. Such sediments should be washed in saline by centrifuging and discarding the supernatant twice. The final saline washed suspension of fixed sediments can be used similar to the sediments of the unfixed specimen)

2. Put 3–4 drops of thrombin solution (5000 units in 10 ml distilled water)

3. Mix by tapping. The mixture usually clots in a few seconds

4. Transfer the clot from the tube on to a 2 inch × 2 inch lens paper piece inside the labeled tissue cassette

5. Wrap the pellet gently in lens paper, prestain the button (with eosin or hematoxylin as per institutional preference), and close the tissue cassette by snapping the top cover of

6. Transport the cassette in 10% formalin to process with the protocol for paraffin embedding.

Microscopic evaluation of the HE-stained sections shows randomly and indiscriminately distributed diagnostic cells which may be admixed with blood and proteinaceous material in the background.

Procedure for cell-block preparation with proprietary instruments/methods

• Procedure for cell-block preparation with Shandon Cytoblock™ (using proprietary reagent #1 and reagent #2. This method require Cytospin instrument with proprietary kit and reagents).[26]

  1. Add 10% Formal saline to 0.5 ml sediment suspension, re-suspend the sediments, and allow to be fixed for at least 30 minutes at room temperature

  2. Centrifuge formalized cell suspensions at 800 rpm for 5 minutes. Discard the supernatant

  3. Add sufficient reagent #2 to the above sediment to give a concentration of approximately 5 × 10⁷ cells per 100 μl, and mix

  4. Each 100 μl cell mixture will produce one Cytoblock. Prepare the required number of Cytoblock cassettes as follows

  5. Apply three drops reagent #1 to the well in the Cytoblock board

  6. Assemble Cytoblock cassettes and cytofunnels in the cytospin clips, and load into the cytospin head

  7. Add 100 μl of the cell mixture from step #e to the cytofunnels and spin for 5 minutes at 1500 rpm on “Lo” acceleration

  8. Open the clips, and remove the Cytoblock carefully, checking that the cell button is retained in the well. Add one drop of reagent #1 to the center of the well and close cassette

  9. Immerse the cytospin clips and funnels in disinfectant and leave overnight, then rinse and allow to air dry to be reused

  10. Process the cassettes for tissue processing

  11. Embed the cell buttons in paraffin wax.

• Cellient[25]

  Cellient™ is proprietary automated cell-block system which achieves concentration, processing, and embedding of sediments in cytology specimen for making a paraffin block. A special cassette, used by the instrument, concentrates sediments in cytology specimen by suction. This sediment is processed by treating with alcohol followed by xylene and then infiltrated with paraffin in the same cassette.[25] Please follow the instructions from the manufacturer.

  The resultant cell-block sections show good morphology, both as HE-stained sections and as immunostained sections. This approach, however, has most important limitation related to exposure and fixation with alcohol leading to potential interference with immunoprofiles affecting the final interpretation and even the results of prognostic/therapeutic tests with liability issues unless specifically validated for each test [Table 2].[913] Recently, a protocol for formalin fixation is available as option to overcome issue related to immunohistochemistry. However, this step cannot be matched with requirement of fixing in 10% formalin for specified duration such as 6 to 72 hours for some immunomarkers such as Her2 and some tests such as FISH per ASCO/CAP guidelines.[25a] In addition, to initial capital investment and recurring kit cost, the instrument allows processing of only one block at a time, limiting the throughput, unless laboratories with high turnover invest in multiple instruments. Similar to other random methods, the cell-blocks made with this method do not allow monitoring of the depth of section cutting by histotechnologists with potential for nonreproducible final cell-block sections due to either undercutting with lack of diagnostic material on slides or overcutting leading to loss of diagnostic material from the cell-block without diagnostic material on the slides.

  The diagnostic cells are present as randomly distributed diagnostic cells admixed with blood and proteinaceous material.[60]

• Methods using preformed supporting media such as premade gel and sponge discs with wells for the accumulation of sediments (proprietary Nano and Micro NextGen CelBloking™ kits (kits based on Shidham's method with built-in precisely set dark AV marker) [Figures 13,14,19–26]:[35]

  Nano version (with premade gel discs with wells) is for specimen of any cellularity.

  Micro version (with premade sponge discs with wells) is for specimens with >1 ml concentrated sediment suspension with >50% Cytocrit/Tissuecrit. The results with Micro version on low cellularity specimens may not be optimum.

  1. Prepare concentrated sediment button by spinning enough quantity of specimen by centrifuging for 3 minutes at 2500 rpm

  2. Pool all the sediments as final concentrated sediment with maximum Cytocrit/Tissuecrit and re-suspend the sediment.

  3. At this stage, if the final concentrated specimen is >1 ml and has >50% Cytocrit/Tissuecrit then Micro version of the NextGen CelBloking™ kits may be used. However, Nano kits[36] could be used for specimens generating final concentrated specimens with any cellularity. In case of doubt, it is recommended to choose Nano units for optimum results.

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Figure 19

Cell-block prepared with Nano NextGen CelBloking™ unit. (a) Gel disc with preformed wells loaded with diagnostic material transferred to the tissue cassette for tissue processing. (b) Embedding of tissue processed sponge disc of Nano NextGen CelBloking™ unit with diagnostic material. The tissue paper cover is opposite the cutting surface, and the bottoms of the wells are the cutting surface. (c) The cutting surface of the final cell block with gel disc of Nano NextGen CelBloking™ unit after rough cut remove the bottom layer of the disc exposing the precisely set dark-colored AV marker which corresponds with the bottoms of the wells with concentrated diagnostic material[67]

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Figure 20

Cutting of paraffin block prepared with Nano NextGen CelBloking™ unit[67]

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Figure 21

(a) Final paraffin block; (b) Scanning power view of HE-stained section of cell-block prepared with Nano NextGen CelBloking™ kit. The preformed Nano gel disc is made of the proprietary medium which allows the processing reagents to be exchanged freely, but the diagnostic cells are retained and concentrated in the wells. The gel medium has clean transparent property as a clean background. (pleural fluid)

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Figure 22

Comparison of the morphological details and quantitative enhancement by Nano NextGen CelBloking™ kit (Metastatic adenocarcinoma, pleural fluid). (a and b) Cell-block section with very scant cellularity (conventional random, indiscriminatory, plasma-thrombin method); (c and d) very cellular cell-block section with many diagnostic cells in the wells (cell-block prepared with enhancement method-Nano NextGen CelBloking™ kit (AV BioInnovation, based on Shidham method http://www.jove.com/index/Details.stp?ID = 1316)

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Figure 23

Cell-block prepared with Micro NextGen CelBloking™ unit. (a) Sponge disc with preformed wells loaded with diagnostic material transferred to the tissue cassette for tissue processing. (b) Embedding of tissue processed sponge disc of Micro NextGen CelBloking™ unit with diagnostic material. The tissue paper cover is opposite the cutting surface, and the bottoms of the wells are the cutting surface. (c) The cutting surface of the final cell block with sponge disc of Micro NextGen CelBloking™ unit after rough cut removing the bottom layer of the disc exposing the precisely set dark colored AV marker which correspond with the bottoms of the wells with concentrated diagnostic material[67]

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Figure 24

Cutting of paraffin block prepared with Micro NextGen CelBloking™ unit[67]

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Figure 25

(a) Final paraffin block; (b) Scanning power view of HE-stained section of cell-block prepared with Micro NextGen CelBloking™ kit. The preformed Micro sponge disc is made of the proprietary porous medium which concentrates the diagnostic cells predominantly in the wells, but the small groups of cells and singly scattered cells wandered around during concentration process may also be seen in the sponge spaces*. The sponge disc medium stains faintly. (Pleural fluid)

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Figure 26

Comparison of the morphological details and quantitative enhancement by Micro NextGen CelBloking™ kit (Metastatic adenocarcinoma, pleural fluid). (a and b) Cell-block section with very scant cellularity (conventional random, indiscriminatory, plasma-thrombin method); (c and d) Relatively cellular cell-block section with many diagnostic cells in the wells and in small spaces in the sponge disc (cell-block prepared with enhancement method-Micro NextGen CelBloking™ kit

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After this, the processing steps for both Nano and Micro kits are as mentioned below:

1. Cover the mouth of the wells with sediments in the gel or sponge disc with tissue paper cover provided with the kit.[66] This step minimizes the potential for cross contamination

2. Then lay over this tissue paper cover, the second sheet of tissue sponge (moistened with 10% formalin) and close the labeled tissue cassette

3. Transport the cassette horizontally with the bottom down in container with 10% formalin to process with the protocol for paraffin embedding after fixing in 10% formalin for at least 2 h (or for more duration as required by individual laboratory/institution protocol)

4. The processed gel/sponge disc (with sediments in the wells) is embedded along with lens paper in such a way that the bottom of the discs corresponding with the bottoms of the wells will be the cutting surface in the paraffin block (and top surface with mouths of the wells covered with lens paper is deep in the paraffin block) [Figure 19 and 23][67]

5. Rough cut the paraffin blocks until the dark-colored dot of AV marker is seen on the paraffin section (this is the level at which the constituent cells in the specimen have sedimented and aligned)[67]

6. Then, cut the block as usual (preferably only one level at which the AV marker is initially visible). Additional levels may be cut later as indicated for elective studies after studying the HE-stained initial level with reference to clinical details and findings in cytology preparations.

The diagnostic cells are present as concentrated, focal accumulation in the wells of the supporting disc medium [Figures 22 and 26]. In the cell block sections prepared using Micro version of NextGen CelBloking™ kit, the cells are also present in the spaces in the sponge disc [Figures 25 and 26]. The gel disc medium in the cell block sections prepared using Nano version of NextGen CelBloking™ kits is clear and do not show cells in the areas other than in the wells. The gel disc medium in the cell block sections prepared using Micro version of NextGen CelBloking™ kits is seen as gray-bluish sponge material with intervening spaces which may have diagnostic cells in addition to those concentrated in the wells [Figures 25 and 26].

The precisely set, built-in AV marker, in addition to objectively guiding to select the first level with diagnostic cells, also allows orientation of individual sections on the glass slides in identical fashion in serial order for proper application of SCIP approach for the evaluation of coordinate immunoreactivity of various diagnostic components in the cell-block sections. This approach is especially critical for evaluation of immunostains on hypocellular cell-blocks.

These methods allow precise selection of wells with maximum cellularity for diagnostic material to be cored out with device similar to skin punch biopsy (trephine-like) device. Such cores could be submitted as FFPE material for ancillary tests such as molecular testing. Most of the cell-blocks are rich in diagnostic cells such as tumor cells without significant stroma as contaminant. This provides proportionately more diagnostic component without significant stromal contamination for various molecular pathology material in contrast to core biopsies, which usually have proportionately more stromal component.