Feature Article
What is the Best Procedure to Remove
Formalin Pigment from Formaldehyde-Acetic
Acid-Alcohol Fixed Tissues?
Anthony Henwood
Histopathology Department, The Children’s Hospital at Westmead, Westmead, Australia
Abstract
There has been some disagreement as to whether formalin
pigment is bleached by potassium permanganate/oxalic
acid (PPO). This study analyzed the bleaching characteristics of PPO on tissues fixed in formaldehyde-acetic acidalcohol and compared PPO with other bleaching agents and
conventional formalin pigment removal methods. It was
found that PPO removed formalin pigment. Other bleaching
methods (hydrogen peroxide, periodic and chromic acids)
also removed the pigment to some extent. Garvey’s pigment
removal procedure was more effective than alcoholic picric
acid for the routine removal of formaldehyde-acetic acidalcohol associated formalin pigment. (The J Histotechnol
33(3):109–111, 2010)
Submitted April 26, 2010; accepted July 20, 2010.
Key words: FAA fixation, formalin pigment, removal
Introduction
Several authors have reported that bleaching with potassium permanganate/oxalic acid (PPO) removes formalin
pigment (1,2). Culling (3), however, has recorded that formalin pigment (acid formaldehyde hematin) is not bleached.
Bleaching is commonly used to remove melanin from heavily melanocytic lesions (4). The aim of this study was to
ascertain whether PPO removes the pigment present in formaldehyde-acetic acid-alcohol (FAA)-fixed tissues.
Methods
Four-micrometer paraffin sections from placental specimens fixed in FAA were used. FAA fixative was prepared as
follows:
absolute ethanol, 1680 mL;
glacial acetic acid, 128 mL;
Address reprint requests to Anthony Henwood, Histopathology Department, The Children’s Hospital at Westmead, Locked Bag 4001, Westmead,
2145, Australia. E-mail: anthonyh@chw.edu.au
One continuing education contact hour can be earned by reading this
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The Journal of Histotechnology / Vol. 33, No. 3 / September 2010
concentrated formalin (37%), 200 mL; and
distilled water, 2000 mL.
Four-micrometer paraffin sections from tissues containing
a range of pigments fixed in neutral-buffered formalin also
were examined. These included a lymph node fixed in B5
(mercury pigment), liver with bile pigment, liver with hemochromatosis (iron), lymph node containing a metastatic pigmented melanoma (melanin), substantia nigra (neuromelanin),
and lateral geniculate body (lipofuscin) (5).
Techniques for formalin pigment removal included:
• 1.8% picric acid in absolute alcohol (15 min), followed
by a 15-min wash in water.
• Garvey’s formalin pigment removal technique (6).
Sections were placed in alkylphenol ethoxylate (APE)
(phenol 5 mL, HCl 5 mL, absolute ethanol 90 mL) for
5 min followed by washing in water for 5 min.
• PPO: sections treated with 5% potassium permanganate
for 2 min, washed in water 2 min, and decolorized in
5% oxalic acid, 2 min (1,2).
For heavily pigmented cases, treatment times were
extended as follows: picric acid 1 hr, APE 30 min, and PPO
10 min in KMnO4. A few cases also were treated with concentrated (30%) hydrogen peroxide for 10 min, 1% periodic
acid (15 min), or 5% chromic acid (10 min), followed by
2% sodium bisulfite.
All treated slides, including nontreated (controls), were
stained with hematoxylin and eosin (H&E). Duplicate treated
sections of bile-containing liver was stained with Fouchet’s
stain for bile, liver with hemochromatosis was stained with
Perl’s stain (7), and lateral genticulate body was stained with
periodic acid-Schiff (after diastase) for lipofuscin (8).
Results
Results are summarized in Tables 1 and 2. PPO was effective in removing the formalin pigment present in FAA-fixed
tissues (Figures 1 and 2). When used for the recommended
times, PPO performed better than alcoholic picric acid,
although it was not as efficient as Garvey’s APE (Figure 3).
109
Table 1. FAA fixed tissues (5+ = heavily pigmented)
Case
Untreated
3829
5529
5411
5536
3502
3502 E
1+
3+
5+
5+
5+
Alcoholic
picric acid
Garvey’s
APE
PPO
0
2+
4+
4+
5+
5+ (1 hr)
0
0
1+
1+
3+
1+ (30 min)
0
0
2+
2+
4+
0 (10 min)
E, extended times.
Table 2. Nonformalin pigments
Pigment
Mercury
Bile
Iron
Dermal melanin
Neuromelanin
Lipofuscin
Alcoholic
picric acid
Garvey’s APE
PPO
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
0
1+
0
0
1+
Figure 2. Placenta fixed in FAA, stained with H&E after bleaching with
PPO. The pigment has been removed, but the resultant basophilia is not
acceptable.
2+, pigment unaffected; 1+, pigment partially removed; 0, pigment
removed.
Figure 3. Placenta fixed in FAA, stained with H&E after treatment with
Garvey’s APE. The pigment present in Figure 1 has been removed.
Figure 1. Placenta fixed in FAA, stained with hematoxylin and eosin
(H&E). Dark brown extracellular pigment granules are present.
For heavily pigmented sections, PPO performed better than
Garvey’s APE and alcoholic picric acid when treatment
times were extended.
PPO removed formalin pigment, bile, dermal, and neuromelanin. Iron, demonstrated with the Perl’s stain, was
partially removed. Lipofuscin, as demonstrated by periodic
acid-Schiff reaction (after diastase), was partially removed.
PPO caused an increased basophilia of the tissues and diminished affinity for the eosin counterstain (Figure 2).
Garvey’s APE removal technique was by far the most efficient method for the removal of formalin pigment. Alcoholic
picric acid was not as efficient and sections often required
prolonged washing to remove the yellow coloration. Both
Garvey’s APE and alcoholic picric acid removed formalin
pigment, whereas mercury, bile, iron, lipofuscin, dermal,
and neuromelanin were unaffected.
110
Thirty percent hydrogen peroxide treatment for 10 min
removed most of the formalin pigment in most cases but the
procedure often needed to be extended to 30 min to be as effective as Garvey’s or PPO treatment. Much pigment was also
removed by periodic acid and chromic acid, but the efficiency
of removal was not as good as Garvey’s or PPO treatment.
Discussion
Formalin pigment, also known as acid hematin pigment,
is commonly associated with the use of nonbuffered or acidic
formalin fixatives (9,10). The extracellular pigment is often
seen associated with congested tissues and especially close
to blood vessels (10). The pigment appears as microcrystalline dark brown birefringent needles or rhomboids (1,11).
A similar pigment occurs in congested blood vessels and
interstitial hemorrhages in Carnoy-fixed tissues, which have
not been exposed to formaldehyde (10).
Several authors have reported that bleaching with potassium permanganate/oxalic acid removes formalin pigment
Removal of Formalin Pigment from Formaldehyde-Acetic Acid-Alcohol Fixed Tissues / Henwood
(1,2). Culling (3), however, has recorded that formalin pigment is not bleached. Our results indicate that the formalin
pigment present in FAA-fixed tissues is bleached by PPO
and prolonged hydrogen peroxide bleaching. Periodic acid
and chromic acid will also remove significant amounts of
formalin pigment. PPO treatment will adversely affect
H&E staining, causing increased basophilia and decreased
eosinophilia.
Of the techniques available for formalin pigment removal,
Garvey’s APE was by far the most efficient. Alcoholic picric
acid was disappointing. This finding is at odds with other
investigators recommending alcoholic picric acid as the
removal method of choice (1,3,12). Waldrop and coworkers
(10) found that the critical alcohol concentration of the saturated alcoholic picric acid solution for complete removal of
formalin pigment lies between 90% and 95%. It is feasible
that picric acid treatment would be improved if the recommendations of Waldrop and coworkers (10) are followed.
Ammoniacal ethanol solutions have also been successfully used to remove formalin pigment (2,11), and Tseng
(13) recommended 30 min of treatment in a solution of 30
mL of acetone and 9 mL of 28% ammonium hydroxide.
A matter of concern is the number of studies that use PPO
as a definitive procedure for the identification of melanin
(14). The current study shows that, apart from formalin pigment, bile, neural, and dermal melanins are also removed.
PPO will also remove, to some degree, lipofuscin and iron.
It is important to include Garvey’s APE or alcoholic picric
acid treatment to exclude formalin (or acid hematin) pigment when considering the nature of any pigment since,
apart from malarial pigment, no other known pigments are
removed.
Conclusions
The formalin pigment resulting from FAA fixation can be
bleached with PPO. Garvey’s APE is recommended for the
routine removal of this pigment.
The Journal of Histotechnology / Vol. 33, No. 3 / September 2010
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