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What is the Best Procedure to Remove Formalin Pigment from Foraldehyde-Acetic Acid-Alcohol Fixed Tissues?

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Tony Henwood at Children's Hospital at Westmead
Tony Henwood
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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.
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The Journal of Histotechnology / Vol. 33, No. 3 / September 2010 109
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Abstract
There has been some disagreement as to whether formalin
pigment is bleached by potassium permanganate/oxalic
acid (PPO). This study analyzed the bleaching characteris-
tics of PPO on tissues fixed in formaldehyde-acetic acid-
alcohol 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 acid-
alcohol 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 potas-
sium permanganate/oxalic acid (PPO) removes formalin
pigment (1,2 ). Culling (3), however, has recorded that for-
malin pigment (acid formaldehyde hematin) is not bleached.
Bleaching is commonly used to remove melanin from heav-
ily melanocytic lesions (4). The aim of this study was to
ascertain whether PPO removes the pigment present in form-
aldehyde-acetic acid-alcohol (FAA)-fixed tissues.
Methods
Four-micrometer paraffin sections from placental speci-
mens fixed in FAA were used. FAA fixative was prepared as
follows:
absolute ethanol, 1680 mL;
glacial acetic acid, 128 mL;
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 hemo-
chromatosis (iron), lymph node containing a metastatic pig-
mented 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 KMnO
4 . A few cases also were treated with con-
centrated (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 effec-
tive 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 ).
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
Removal of Formalin Pigment from Formaldehyde-Acetic Acid-Alcohol Fixed Tissues / Henwood
110
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 neu-
romelanin. 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 dimin-
ished affinity for the eosin counterstain ( Figure 2 ).
Garvey’s APE removal technique was by far the most effi-
cient 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.
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 effec-
tive 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 microcrystal-
line 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 potas-
sium permanganate/oxalic acid removes formalin pigment
Table 2. Nonformalin pigments
Pigment
Alcoholic
picric acid Garvey’s APE PPO
Mercury 2+ 2+ 2+
Bile 2+ 2+ 0
Iron 2+ 2+ 1+
Dermal melanin 2+ 2+ 0
Neuromelanin 2+ 2+ 0
Lipofuscin 2+ 2+ 1+
2+, pigment unaffected; 1+, pigment partially removed; 0, pigment
removed.
Figure 1. Placenta fixed in FAA, stained with hematoxylin and eosin
(H&E). Dark brown extracellular pigment granules are present.
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.
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.
Table 1. FAA fixed tissues (5+ = heavily pigmented)
Case Untreated
Alcoholic
picric acid
Garvey’s
APE PPO
3829 1+ 0 0 0
5529 3+ 2+ 0 0
5411 5+ 4+ 1+ 2+
5536 5+ 4+ 1+ 2+
3502 5+ 5+ 3+ 4+
3502 E 5+ (1 hr) 1+ (30 min) 0 (10 min)
E, extended times.
The Journal of Histotechnology / Vol. 33, No. 3 / September 2010 111
(1,2). Culling (3), however, has recorded that formalin pig-
ment 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 satu-
rated 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 recom-
mendations of Waldrop and coworkers (10) are followed.
Ammoniacal ethanol solutions have also been success-
fully 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 pig-
ment, 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) pig-
ment 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.
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Article
  • Sep 1991
A modified Mayer hematoxylin stain that can be combined with either phloxinelsaffron or eosin for general staining 01 as a counterstain is presented. This method uses 10% less alum, and chloral hydrate is replaced by absolute ethanol, a more effective penetrator and bacteriostat. Phenol is recommended for formalin pigment removal in formaldehyde fixed tissues to effect a more esthetically pleasing section with most techniques. Saffron bought through the health food stores is 8 times less expensive than that bought commercially. (The J Histotechnol 14:163, 1991)
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An Amylase Reagent with a Long Shelf Life for the Removal of Glycogen from Tissue Sections
Article
  • Sep 2002
An amylase reagent is described for the removal of glycogen from tissue sections. The reagent has a long shelf life and uses an incubation time of 10 min at room temperature. It is suitable for formalin and Brazil's fixed paraffin sections as well as air-dried and ethanol fixed frozen sections. (The J Histotechnol 25:153, 2002)Submitted: May 9, 2002; Accepted with revisions July 23, 2002
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Intracellular Crystalline Material in Visceral Adipose Tissue: “Rediscovery” of a Common Artifact
Article
  • Mar 1993
Intracellular crystalline deposits were observed in visceral adipose tissue in nearly one-third of stillborn autopsies reviewed. These spicular brown crystals are a frequent finding in stillbirth autopsies. The artifact, sometimes prominent and extensive, should be recognized as formalin pigment and without pathologic consequence. (The J Histotechnol 16:69,1993)
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Formalin deposition as artifact in biopsies from patients affected by a new variant of endemic pemphigus foliaceus in El Bagre, Colombia, South America
Article
  • Dec 2009
  • J CUTAN PATHOL
Most autoimmune diseases occur sporadically; however, endemic pemphigus foliaceus (EPF) is present in specific locales restricted to some geographic rural regions mostly in South America, Central America and in Tunisia (Africa). Its geographic restriction makes it an invaluable natural model for studying how the environment, genetic background and host response contribute to the development of autoimmunity. We described a new variant of EPF in El Bagre, Colombia, (El Bagre-EPF). When we examined the skin biopsies from 10 patients and controls from the endemic area, we detected in a systematic manner several types of pigmentation, sometimes intracellular, and sometimes in the extracellular matrix in most biopsies. Aim: We aim to determine the nature of this pigment in these skin biopsies. We studied 10 patients and 10 controls matched by sex, age and work activity living in the endemic area by routine hematoxylin and eosin (H&E). We were unable to find any bacteriological or parasitic organism. Specifically, we searched for several tropical disease agents as possible causative agents of this pigment. Iron stains and melanin pigment bleaching techniques failed to determine the etiology of this pigment. We then tried the removal of formalin pigment using picric acid. The pigment was removed after very strong treatment with different acids including picric acid. Formalin pigment shares many properties with hemozoin. In this case, the authors recommend the use of neutral buffered formalin to prevent the formation of formalin pigment especially after long periods of fixation when taking biopsies under extreme temperature and environmental humidity.
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Acid Hematin Pigmentation of the Cornea in Stillborn Fetuses
Article
  • Feb 1990
  • Pediatr Pathol
During the postmortem histopathologic evaluation of eyes from stillborn fetuses we noted the presence of a prominent undescribed corneal pigment in 18 of 55 stillborn fetuses. The corneal pigment was frequently associated with documented meconium-stained amniotic fluid, and in no instance was a stained cornea coupled with recorded clear amniotic fluid. Pigmented corneas came from stillborn fetuses with a longer duration of intrauterine death than nonstained corneas. The pigment stained black with the Fontana-Masson stain, was birefringent, and treatment of tissue sections with 5% potassium permanganate and 5% oxalic acid as well as with saturated alcoholic picric acid solution removed the pigment indicating that it is acid hematin. The most likely cause of the acid hematin-stained corneas was tissue acidity created in utero with prolonged intrauterine death.
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