Chapter 22

Making Formalin-Fixed, Paraffin Embedded Blocks

Alireza Sadeghipour and Pegah Babaheidarian

Abstract
Paraffin embedding is a standard technique used in clinical and research laboratories to create a formalin-
fixed, paraffin-embedded (FFPE) block of tissue. Formalin-fixed tissue undergoes tissue processing and
then is embedded in paraffin (wax) to create a FFPE block or paraffin block. The paraffin block can be cut
using a microtome to generate thin sections of tissue contained in paraffin to be stained or paraffin tissue
ribbons suitable for nucleic acid extraction. In addition, the FFPE blocks can be stored at room temperature
for years. Herein, we provide a basic knowledge, and introduce common methods of the paraffin embed-
ding process.

Key words FFPE, Formalin, Paraffin, Embedding, Paraffin blocks, Tissue orientation

1 Introduction

With the advent of microscopy and the groundbreaking work of

Morgagni, Bichat, and Virchow, the specialty of pathology entered
a new era in the second half of the nineteenth century. The micro-
scope revolutionized concepts of disease, moving the focus from
whole organs to cells. It enabled the practice of histopathology and
spawned numerous advances in techniques necessary for modern
medical and scientific practice. In the beginning, slices of fresh
tissue were cut by hand and examined unstained. Gradually, in the
last decades of the twentieth century, this crude approach evolved
to employ fixed tissue, embedding techniques, microtomy, a pleth-
ora of biological stains, and greatly improved microscopes. The
man who introduced paraffin embedding in 1869 was Edwin
Klebs (1834–1913) [1]. Embedding is the process in which cor-
rectly oriented tissues are completely enclosed in a firm, supporting
medium such as paraffin to facilitate cutting thin sections. Some
samples such as tumor tissue may not need to be oriented but
tissues with layered components such as skin may require orienta-
tion to visualize the desired layers. The supporting medium also is
called an embedding medium. This medium should provide

William H. Yong (ed.), Biobanking: Methods and Protocols, Methods in Molecular Biology, vol. 1897,
https://doi.org/10.1007/978-1-4939-8935-5_22, © Springer Science+Business Media, LLC, part of Springer Nature 2019

253
254 Alireza Sadeghipour and Pegah Babaheidarian

elasticity, resisting section distortion while facilitating sectioning

[2]. Nowadays, pathology laboratories have a choice of techniques
for paraffin embedding. In this chapter, we illustrate preparing
paraffin block preparations using either a dedicated embedding
center or a combination of simple equipment that are described in
Subheading 4.

1.1 Paraffin Although various substances such as celloidin, ester wax, synthetic
Embedding Media resins, and gelatin have been introduced, paraffin or wax continues
to be the most popular infiltration and embedding medium in
histopathology laboratories. It is compatible with most routine
and special stains as well as immunohistochemistry protocols.
Solid paraffin wax is a mixture of long chained hydrocarbons
derived from petroleum. It is white or colorless, usually odorless,
and commercially available with varying melting points, ranging
from 40  C to 70  C [3–5]. Based on the type of tissue to be cut,
you can use soft or hard paraffin. For example, for working with a
soft tissue you can choose a paraffin with melting point of 45  C or
lower and, for hard tissues such as uterine cervix or bone, the
optimal melting point for paraffin is 60  C or more. In routine
pathology laboratories, it is impractical to separate the specimens
for embedding in paraffin of varying melting points, therefore, to
be able to produce ribbons of sections consistently, it is necessary to
select a wax of suitable hardness at room temperature; so, a paraffin
with a melting point of about 56 to 57  C is recommended for
general purposes [3–6] (see Note 1). The paraffin wax should be
free from dust, grit or other foreign material. Using ordinary filter
paper, the wax has to be filtered before use (see Note 2). Liquid
paraffin wax permeates the tissue and solidifies rapidly when cooled.
For a successful impregnation the wax oven has to be kept at high
temperature (see Note 3).

1.2 Paraffin Wax Several different substances can be added to paraffin wax in order to
Additives change its plasticity, consistency and melting point to improve its
effectiveness during microtomy. These additives can vary the paraf-
fin wax hardness so that it is compatible with the tissue to be
embedded. These substances can increase stickiness of the medium
so that a better ribbon can be obtained. In comparison with paraf-
fin, these substances typically have a higher melting point and
consequently make the tissue more brittle [2, 3] (see Note 4).
Commonly used additives are described below:
1. Ceresin is a wax purified from ozocerite. Its melting point is
between 61  C and 70  C. Addition of 0.3–0.5% ceresin is
usually sufficient to reduce the crystalline structure of paraffin
wax and to increase its firmness.
2. Plastic Polymers are added to paraffin in order to increase its
elasticity and consistency. Plastic polymer reduces compression
of sections to obtain better ribbons.
 Paraffin Blocks 255

3. Rubber increases the stickiness and elasticity of wax, making it

easier to obtain continuous ribbons. This additive is particularly
suitable for circular specimens as it allows the original shape of
the sample to be regained after sectioning [4, 7].
4. Dimethyl sulfoxide is a colorless hygroscopic liquid which pro-
motes rapid paraffin infiltration.
5. Beeswax is a yellow substance with a melting point of 64  C.
This additive is usually mixed with paraffin in a 10–20% propor-
tion of beeswax so as to reduce the crystalline structure of the
paraffin wax promoting a uniform cutting consistency and facil-
itating continuous wrinkle-free ribbons [2, 4].
6. Bayberry wax is a vegetable wax additive extracted from the peal
of bayberries. Its melting point is 45  C. Bayberry wax not only
prevents the crystallization of paraffin but also lowers its melting
point [7].
Depending on the tissue and the need, various mixtures of
paraffin wax and its additives can be created (see Notes 4 and 5).

1.3 Embedding There are several devices, specialized materials and equipment that
Equipment facilitate paraffin embedding.

1.3.1 Embedding Molds Embedding molds are used in order to shape or cast liquid paraffin
into blocks. Many types of molds are used for embedding.
1. Stainless steel molds are probably the most widely used molds.
These are suitable for most embedding purposes and are manu-
factured in different sizes to accommodate various sizes of tissue
specimens (10  10  5 mm; 15  15  5 mm;
24  24  5 mm; 30  24  5 mm, etc.) (Fig. 1).
2. Plastic molds are relatively inexpensive, convenient and user
friendly. These are disposable molds and, therefore, the need
for cleaning after use is eliminated. These types of molds are very
shallow and must be used in conjunction with plastic embedding
rings or cassettes to give the block support during sectioning (see
Note 6) (Fig. 2).
3. L molds (Leuckhard mold) is an adjustable mold made up of
metal and consists of two L-shaped pieces resting on a flat
metal plate (Fig. 3). The two L pieces are jointed to form sides
of a rectangular box and can be moved to adjust the size of the
mold depending on the size of the tissue. These types of molds
are easy to procure and are reusable.
4. Pop-out embedding molds are made up of two hinged together
aluminum alloy pieces. In a closed position, they hold paraffin
allowing formation of a paraffin block. For removal of the block,
the mold is swung open [4] (Fig. 4a–c).
256 Alireza Sadeghipour and Pegah Babaheidarian

Fig. 1 Stainless steel embedding molds

Fig. 2 Disposable plastic cassettes

Fig. 3 Leuckhard mold, consists of two L-shape metal pieces and a flat metal plate
 Paraffin Blocks 257

Fig. 4 Pop-out embedding (a) mold, (b) with its open, and (c) closed positions

5. Multiblock embedding units: A compound embedding unit com-

poses of a square shaped brass or metal plate with a series of
interlocking plates. These interlocking plates can be used to
make several compartments—each of which may be used as
a mold.

1.3.2 Embedding Center An embedding center consists of several units that are involved in
paraffin block preparation steps. The integrated form of these units
is available and can be purchased as one streamlined, modular unit
(Fig. 5). An embedding machine contains the following parts:
1. Paraffin reservoir and dispenser with adjustable temperature of
45–75  C (see Note 7).
2. Warm plate for orienting the specimen in melted paraffin, mold
warmer and cassette bath (see Note 8).
3. Cold plate which is a high efficiency refrigeration system with a
temperature of 4 or 5  C and a capacity of about 50–60
blocks (see Note 9).
4. Forceps warmer or an alcohol lamp.
258 Alireza Sadeghipour and Pegah Babaheidarian

Fig. 5 Tissue embedding center contains paraffin reservoir and dispenser, warm and cold plates

2 Materials

1. Paraffin wax.
2. Embedding center.
OR
1. Paraffin wax.
2. Paraffin additives.
3. Paraffin dispenser.
4. Cold plate.
5. Warm plate.
6. Molds.
7. Forceps.
8. Forceps warmer.

3 Methods

The quality and suitability of a tissue section depends on every step

in processing, and each step’s quality depends on the preceding
step. Once tissue samples are infiltrated by paraffin, they are
removed from the cassettes, carefully examined, and based on
their structure, positioned in the paraffin blocks. This step is critical
as incorrect orientation of tissues may result in diagnostically
important tissue elements being missed or damaged during
microtomy.
 Paraffin Blocks 259

3.1 Specimen 1. Tissue sections are embedded flat against the mold surface with
Orientation enough pressure in order to obtain a complete section (see
Note 10).
2. All sides of the tissue must be surrounded by at least 2 mm of
paraffin wax for maximum cutting support (see Note 11).
3. Orientation should be such that the resistance the tissue puts
forward on the knife gradually increases. For example, a trian-
gular piece of tissue should be oriented so that the knife
touches the apex of the triangle first.
4. Large rectangular, dense or hard specimens such as uterus and
bone should be embedded at a slight angle to the knife edge. In
this situation, the knife starts the cutting with less resistance
[4].
5. Elongated tissues are placed diagonally across the block.
6. Multiple soft tissue fragments such as Tru-Cut biopsies of
breast lesions and core needle biopsies of lung or lymph
nodes should be placed side by side with a space in between
(see Note 12).
7. Tubular structures such as ureter, vas deferens, vessels, fallopian
tube, small cysts, and appendix are embedded so that the knife
cut across the lumen and provide a transverse section showing
all tissue layers.
8. The orientation of the tissues with an epithelial surface such as
skin, bronchus, and urinary bladder is such that the plane of
section crosses all tissue layers. The epithelial surface should be
at the top of the block so that it will be cut at the end [4] (see
Note 13).
9. Multiple pieces of a tissue are oriented side by side with the
epithelial surface facing in the same direction [3].
10. Cystic structures should be embedded with the cut surfaces
down so that the knife goes through all layers of the cyst wall.
For small bisected cysts, try to cut the upper most part of the
dome in order to make a hole on it. This technique prevents air
bubbles trapping in the dome of the cysts during paraffin
embedding.
11. For muscle biopsy both transverse and longitudinal planes are
included in sections.
12. Orientation of pure tumor tissue is generally not critical.
13. Several commercial products are available for ensured proper
orientation: marking systems, tattoo dyes, biopsy bag, sponges,
papers, sectionable cassettes, sectionable filters, silicon pads,
etc. The sectionable cassettes are made of unstainable material
with sectioning characteristics similar to paraffin. Tissue
260 Alireza Sadeghipour and Pegah Babaheidarian

Fig. 6 Different types of (a) sectionable cassettes, (b) Sectionable cassettes with outer plastic frames and
corresponding stainless-steel base molds for paraffin embedding

orientation is well preserved inside the sectionable cassettes.

Each cassette is composed of two parts: (1) Interior part with
an attached lid, (2) Outer frame. Both must be placed in a
stainless-steel base mold for paraffin embedding (Fig. 6a, b).
The advantage of this system is quick embedding with preser-
vation of the specimens’ orientation (i.e., no mixed-up sam-
ples), but its disadvantage is higher expense and a more
frequent need to change disposable blades during microtomy.

3.2 Embedding All steps of tissue embedding are carried out at room temperature.
Technique
1. Check that the different parts of embedding station or embed-
ding center are at the appropriate temperature. Paraffin should
be in liquid form in the paraffin reservoir. Warm plate or work
surface should be warm, and the cold plate should be cold. If
you use the metallic type of molds such as stainless-steel molds,
they should be kept warm.
2. Use forceps that is warmed with some commercial forceps
warmer, a Bunsen burner or an alcohol lamp to prevent paraffin
collection at the tip.
3. Remove the cassettes from the last tissue processor melted
paraffin bath and transfer to the cassette bath or warm com-
partment of the embedding center.
4. Transfer one cassette onto the warm plate (see Note 14).
5. Snap off the cassette lid in order to view the tissue sample.
6. Check gross information entry to ensure the correct number of
tissue pieces is present.
7. Select a mold that best corresponds to the size of the tissue.
The specimen should not be in contiguity with the edge of the
 Paraffin Blocks 261

Fig. 7 Pour melted paraffin until the mold is partially filled

Fig. 8 Transfer the paraffin infiltrated tissue into the bottom of the mold after transferring the mold onto the
warm plate

mold. A minimum 2 mm surrounding wax margin is acceptable

(see Note 15).
8. Transfer the mold onto the warm plate or its equivalent.
9. Pour melted paraffin from the paraffin dispenser or its con-
tainer until the mold is partially filled (Fig. 7).
10. Rewarm the forceps, remove the tissue from the cassette and
place it at the bottom of the mold (Fig. 8) (see Note 16).
262 Alireza Sadeghipour and Pegah Babaheidarian

Fig. 9 Orient the tissue carefully

Fig. 10 Press the tissue mildly, using forceps or histopress in order to hold on to complete section

11. Orient the tissue inside the mold to obtain the best position. A
small amount of pressure may be used in order to hold on to
complete section (Fig. 9) (see Note 17).
12. Carefully transfer the mold onto the cold plate. Allow a few
seconds for the paraffin to solidify (it turns white in color),
meanwhile orient the tissue and gently press the surface of
 Paraffin Blocks 263

Fig. 11 Place the labeled plastic cassette on the top of mold

Fig. 12 Cool the blocks on the cold plate

tissue against the solid plate surface. If multiple pieces of tissue

should be embedded, this process must be carried out rapidly
[4] (Fig. 10) (see Note 18).
13. Immediately place the original labeled embedding ring or cas-
sette on top of the embedding mold. Using the ring or original
embedding cassette which is typically labeled with the speci-
men identification number helps you to trace the specimen
identification being as a holder during sectioning (Fig. 11).
264 Alireza Sadeghipour and Pegah Babaheidarian

Fig. 13 Snap off the molds

14. Carefully fill the combined mold and embedding cassette or

ring with paraffin to the level of the upper edge of the cassette
or ring (see Note 19).
15. Cool immediately by transferring the mold and cassette onto
the cold plate. Wait approximately 15 min until the paraffin has
hardened (Fig. 12) (see Note 20).
16. Separate the mold from the embedding cassette (Fig. 13).
17. The tissue and solidified wax remain attached to the embed-
ding cassette or ring.
18. Paraffin block is ready for sectioning.
19. Store all blocks at room temperature.
20. If you use other types of molds you must go through the same
steps (see Note 21).

4 Notes

1. Climate should be considered for choosing your paraffin. Soft

paraffin is not suitable for use in hot climates because of their
low melting point. Low melting point paraffin may not be firm
enough to support the tissue during cutting. In addition, when
the room temperature exceeds 40  C such blocks may melt.
Paraffin blocks made from soft paraffin should be stored in a
cool, dry place. Paraffin waxes with high melting points (60  C
or more) can be difficult to be ribbon [3, 5].
2. Presence of water causes paraffin wax to crystallize and turn
white.
 Paraffin Blocks 265

3. Heating the paraffin wax to a high temperature alters the

property of the wax. Overheating paraffin to 10–20  C above
the melting point could evaporate some fractions of the mix-
ture with lower melting points. Also, further oxidation of wax
turns it yellow with a soap-like consistency. If the wax is over-
heated for the long period of time, it will lose its original
crystalline structure [3, 5].
4. Mixtures of highly purified paraffin wax and various types of
additives are commercially available, providing a selection that
is available for most laboratories (e.g., Paraplast and Bioloid
tissue embedding medias). If you do not have any access to
commercially available paraffin waxes (e.g., Paraplast and etc.)
you can prepare your own mixture. Three common and practi-
cal mixtures are Hance’s rubber paraffin, Maxwell embedding
wax, and Gray’s formula [5, 7]. Cut 20 g of crude rubber into
small pieces and dissolve, with constant stirring, in 100 g of
paraffin heated to steaming to make a stock rubber solution.
Hance’s rubber paraffin can be made with 100 g paraffin, 4 g
stock rubber solution, and 1 g beeswax. Maxwell embedding
wax consists of 100 g paraffin, 4 g stock rubber solution, 7 g
Bayberry wax, and 1 g beeswax. Gray’s formula uses 70 g
paraffin wax (melting temperature 58  C), 5 g rubber, 5 g
beeswax, 5 g spermaceti, and 15 g Nevillite “5” (Clarite).
5. Studies have shown that molecular, proteomic, and morpho-
logical characteristics can be altered in formalin-fixed paraffin
embedded specimens by suboptimal processing conditions.
There has been concern that high temperatures in molten
paraffin may contribute to degrading tissue components. In
general, lower melting-temperature paraffin where practicable
is recommended for the impregnation of tissue especially if
molecular analysis is desired. Use of additives such as beeswax
must be avoided. These additives contain pollen and other
contaminants which interfere with the recovery of biomole-
cules [8, 9].
6. Two commercially available systems of embedding molds are
Tissue-Tek system 1(Mark 1 system) and Tissue-Tek system
2 (Mark 2 system). In the former, rapid embedding and cutting
of tissues are enabled using plastic embedding rings and
stainless-steel molds. The advantage of this method is that
blocks are stored with the plastic rings and, therefore, the
angle does not change for further sections. For small tissue
samples where multiple recuts are needed such an approach can
mitigate tissue loss or waste. In the Mark 2 system tissue is
processed in plastic embedding cassettes with a stainless-steel
lid. The cassette has a rough slope for writing the accession
number [2].
266 Alireza Sadeghipour and Pegah Babaheidarian

7. In order to use liquid wax for embedding, you have to maintain

the temperature of the wax at just above its melting point. Two
degrees above the melting point typically suffices. For this
purpose, you can use a simple thermostatically controlled
oven or even make a simple practical cheap device [7]. Care
must be taken to prevent excessive heat or boiling of the wax.
8. If you don’t have a warmer, you can gently heat a stainless-steel
mold in the flame of an alcohol lamp to a temperature above
the melting point of wax and then use forceps with a warm tip
for transferring the tissue to the mold.
9. You can use a block of melting ice instead of the cold plate for
this purpose.
10. Take care not to make artifact by rough handling and excessive
pressure.
11. Molds of different sizes are available for a variety of specimens.
A suitably sized mold is always chosen for each specimen. If the
mold is small, in comparison to size of the specimen, then the
specimen is in contiguity with the edge of the block and
therefore sectioning may become difficult [10].
12. If tissues are placed one above the other, there is the chance
that the lower tissue may distort the upper one [4].
13. Cutting the hard keratin layer of skin last minimizes compres-
sion and scratches in the subcutaneous structures.
14. The temperature of the warm plate should be regularly checked
otherwise excessive heat can damage the specimen [10].
15. If you use the traditional L-molds or multiblock embedding
units you can adjust size of molds according to the shape and
size of the tissue.
16. Forceps must be heated to the point where the wax just melts.
Using excessive heat can cause local heat damage to the tissue
and a change in its morphology in the vicinity of the forceps
contact point.
17. Tiny friable pieces of tissue must go through double embed-
ding process in which fragments of tissue are embedded in
melted agar after formalin fixation, allowed to solidify and
trimmed for routine processing, then agar pellet is embedded
in paraffin wax.
18. If the specimen moves during this step and loses its orientation,
put the mold back onto the warm plate, wait until the whole
paraffin liquefies and after that start again.
19. Overfilling of the mold requires scraping of the back and edges
of the cassette prior to microtomy. Overfilled blocks may lie
unevenly in the microtome chuck causing instability that may
 Paraffin Blocks 267

Fig. 14 Using L molds for embedding: (a) Adjust L mold according to the shape and size of the tissue and
prepare melted paraffin in a metal or glass cup, (b) Place the tissue in the mold and fill it with paraffin, (c)
Reorient the tissue and make small amount of pressure with a previously warmed forceps, (d) After cooling the
block on the cold plate or a piece of melting ice remove it from the mold

lead to the tissue becoming damaged or lost during microtomy

[10].
20. The quick cooling of the wax ensures a small crystalline struc-
ture, producing fewer artifacts when sectioning the tissue [3].
21. If L-molds or similar types of molds are used, embedding
cassette or rings are not required. After orientation of the
specimen the mold should be filled with paraffin. After the
insertion of the identifying label place it onto the cold plate
or a block of melting ice until the paraffin has solidified. After
approximately 30 min, the mold should be separated from the
paraffin block that is now ready for sectioning (Fig. 14a–d).
268 Alireza Sadeghipour and Pegah Babaheidarian
Acknowledgments
We are grateful to Nima Sotoudeh, a graphic designer and student
of animation at the University of Technology Sydney (UTS),
Australia, for his artful illustrations used in this chapter.
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