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Microscopic Quality Control of Haematoxylin
and Eosin – Know your Histology
Anthony Henwood, MSc, CT(ASC)
Senior Scientist and Laboratory Manager
The Children’s Hospital at Westmead
Locked Bag 4001, Westmead, 2145, Australia
Producing quality microscopic sections goes a long way to
achieving a confident, accurate diagnosis. Unfortunately tissues
can be distorted post-operatively, through fixation, during processing,
microtomy and staining. A competent histological scientist needs to be
able to minimise the distortion and correct it when the quality process
goes out of control. Quality staining is highly dependent on fixation
and processing and it is difficult to appreciate histochemical quality
control (QC) in isolation (1). Recognising microscopic quality is directly
connected with knowledge of basic histology and recognition of the
common microscopic disease entities. This review analyzes the quality
requirements for Hematoxylin and Eosin (H&E) staining.
Hematoxylin and eosin (H&E) is the “bread and butter” stain in most
histopathology laboratories. It is appreciated that the H&E stain varies
from laboratory to laboratory and that personal preference plays a large
part in the final results. Some general guidelines in assessing a good
1. Nuclear detail – how effective is the stain in demonstrating nuclear
membranes, nucleoli, chromatin and nuclei of both vesicular (open)
and hyperchromatic (small, dense) nuclei?
2. Cytoplasm & background substance – can cell cytoplasm, collagen,
muscle, red blood cells and mucin be easily discerned?
3. In sections of appendix, lung or gut is the mucin of epithelial cells
blue or clear? The mucin staining is a function of the Hematoxylin
pH. Lowering the pH (usually by adding acetic acid) can significantly
reduce mucin staining.
4. Do the haematoxylin-stained elements of the section appear brown
(indicating an over-oxidised solution)?
Luna (1991) (2) believes that an H&E-stained slide should contain the
following tinctorial qualities:
1. The nuclear chromatin should stain blue to bluish purple and be
2. Nucleoli should appear reddish purple if the hematoxylin is well
decolourised. If too much hematoxylin remains in the nucleoli, there
is no doubt, that there is excess hematoxylin in the section. Excess
eosin will produce a purple monochromatic nuclear chromatin and
3. Collagen and muscle should stain well but in a delicate fashion. One
should be able to see some form of fibrillar pattern by moving the
microscopic focal plain up and down. The same pattern is observed
by reviewing muscle except that muscle should have a slightly
redder colour than collagen.
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Figure 1. High power H&E showing plasma
cells with characteristic cart-wheel nuclei.
Figure 2. Colon section stained with H&E
showing blue mucin staining. Decreasing the
hematoxylin pH should remove this staining.
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4. Eosinophilic granules should be well-defined and appear orange-
red. Lack of well-defined eosinophilic granules suggests overstaining
by either hematoxylin or eosin, or both.
These are general guidelines but are there guidelines for individual
When assessing H&E-stained sections from differing organs, features
that are usually present are arteries. One should be able to
differentiate muscle and collagen with muscle appearing as dark
red. Red blood cells should be bright red. Assessment of nuclear
staining will depend on the cell-types in the tissue being stained and
the following is an attempt to set some standards according to the
tissue being assessed.
The Nissl substance of neurones and Purkinje cells should be well
demarcated and stain dark blue. The large nuclei should be open with a
visible nucleolus. The fibres should be delicately eosinophilic which will
allow easier demonstration of senile plaques if present. The pale pink
staining of neurones will also allow easier detection of “red” neurones,
an important indicator of antemortem injury (3).
The pituitary contains three principle cell types that should be apparent
with a good H&E: acidophils (40%) with pink cytoplasm, basophils
(10%) with a pale blue cytoplasm and chromophobes (50%) with clear
cytoplasm. The nuclei of acidophils should have relatively prominent
Figure 3. Skin section stained H&E. A (Left) pH of eosin too high, B (Right) acidic eosin showing differentiation of collagen and neural tissue.
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Colloid should be pale pink. The cytoplasm of follicular cells should
be a slightly darker pink. The nuclei should be delicately stained with
discernible small darker chromatin. The basement membrane binding
the follicular cells should be visible and appear as deep red. In well
fixed and processed tissue, Clear cells may be seen admixed with the
follicular cells (4).
The two cell types should be recognisable. Chief cells should have clear
to faint pink cytoplasm with even blue chromatin, and rare small nucleoli.
The cell membrane should be poorly defined. Oxyphil cells should show
an eosinophilic granular cytoplasm, pyknotic nuclei and should have a
prominent cell membrane (3).
The bronchioles contain smooth muscle and collagen in their walls. The
lung parenchyma is rich in small blood vessels and thus eosin balance
between muscle and collagen can be assessed. In the alveoli, type
II pneumocytes should have pale nuclei with prominent nucleoli. The
nuclei of type I pneumocytes are small and dense (4).
The heart is rich in blood vessels as well as cardiac muscle and collagen
enabling good eosin differentiation. Purkinje fibres, if present, should
have pale cytoplasm with open vesicular nuclei. Cardiac nuclei should
have evenly distributed dust-like chromatin with a clear (non-staining)
perinuclear sarcoplasm. The eosin should demonstrate the cross-
striations adequately (5).
In the germinal centres, centroblasts have large, round, vesicular
nuclei with 1-3 small but conspicuous nucleoli. Small lymphocytes have
dark staining, irregular nuclei. Plasma cells with their “spoke-wheel”
chromatin pattern caused by small clumps of chromatin on the nuclear
membrane in a clear nucleus may be present (Fig 1). A clear “hof”
should be seen (3).
The gastrointestinal (GI) tract contains epithelial, connective, neural and
lymphoid tissues that need to be easily recognisable using a routine
H&E. Lymphoid nuclei, especially those of plasma cells, should be
assessed. The usual colour differentiation should exist between muscle
and collagen. Ganglion cells should be apparent in between the muscle
layers. The nuclei should contain a single, prominent, eosinophilic
nucleolus and the cytoplasm should be pink (a lighter hue than that of
the surrounding muscle). It is expected that the epithelial mucin should
be clear. Blue staining of the mucin usually indicates that the pH of the
Hematoxylin is too high (Fig. 2). It has often been found that in these
cases, the hematoxylin often appears over-stained resulting in difficulty
in assessing the nuclear component. Throughout the GI tract there are
unique cells that exist and it important that they be recognised in a
In the stomach, the parietal cell cytoplasm should be light pink, where
as that of the chief cells should appear purplish. Small nucleoli should
also be apparent in the chief cells (3).
Paneth cells, showing intensely eosinophilic cytoplasmic granules,
should be present in the small intestine.
In the large bowel, the nuclei of the goblet cells should appear darker
than those of the absorptive cells. Endocrine and Paneth cells should
be discernible by the eosinophilic granules present in the cytoplasm.
The predominant cell is the hepatocyte. The nuclei should have small
nucleoli visible. The pale red-pink cytoplasm of the hepatocytes should
contrast well with the pink of collagen around blood vessels. There
should also be small blue granules (RER) in the cytoplasm. Cuboidal bile
duct cells have open nuclei and dark pink cytoplasm (3).
H&E stain varies from laboratory to laboratory
and that personal preference plays a large part
in the final results.
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The cell population in the spleen is similar to the lymph node so you can
recognise similar microscopic quality control features. Both medium
sized and small lymphocytes are present. Plasma cells are often found
near the arteries. The endothelial cells lining the sinus should have bean-
shaped nuclei containing a longitudinal cleft (3).
The pancreas has a wide variety of cells to meet the exocrine and
endocrine functions of the organ:
Acinar cells have dark nuclei with a few tiny clumps and strands of
chromatin. Nucleoli should be evident. The cytoplasm should have
intense eosinophilia at the apical with strong hematoxylinophilic
staining at the basal end.
Duct cells have clear to pale eosinophilic cytoplasm and lightly
stained nuclei (less dense than those of acinar cells).
Islet cell nuclei should have evenly stained nuclei and cytoplasm
a paler pink than acinar cells. Within the islet, differing cells should
be discerned with darker cytoplasm cells probably being glucagon
containing cells (3).
The kidney contains a diverse population of cells from those exhibiting
dense chromatin (in the glomerular tufts) to those exhibiting a dusting
of chromatin material (cuboidal cells of the collecting tubules) (2).
The cytoplasm of the cells of the Proximal Convoluted Tubules have
eosinophilic cytoplasm where as those of the Distal Convoluted Tubules
have paler cytoplasm (fewer granules). The cells of the Proximal
Convoluted Tubules should have a discernible brush border. The
basement membranes of both proximal and efferent tubules should
be visible (5).
Under low magnification, the cortex and medulla of the adrenal gland
should be apparent with acidophilic (pink) cortex and basophilic
In the medulla, the chromaffin cells should have an overall blue
cytoplasm. The nuclei should have coarsely clumped chromatin with
visible clear areas. The nuclear material of the ganglion cells should be
speckled and have prominent nucleoli.
Three zones should be visible in the cortex. The zona glomerulosa
(closest to the capsule), the cells should have clear cytoplasm
containing a scattering of pink granules. The nuclei should have a
longitudinal groove present in many of the cells. In the middle zone
(zona fasciculata) the nuclei should be more vesicular and less
chromatic than in the previous zone. A single small nucleolus should be
apparent and the cytoplasm should be clear. In the zona reticularis, the
cells have cytoplasm that is solid, granular and eosinophilic. Lipofuscin
should be discernible especially in cells adjacent to the medulla (3).
The stroma of the prostate contains both collagen and smooth muscle
and these should be easily differentiated on an H&E. The secretary
epithelial cells of the central zone of the prostate should have
darker cytoplasm and slightly darker nuclei compared to those in the
peripheral zone (3).
The main features of the testis that need to be recognised include (3):
Sertoli cells – nuclei should have a slightly wrinkled nuclear
membrane and have prominent nucleoli.
Germ cells – You should be able to easily distinguish each of the
differentiating elements based on the presence or absence of
nucleoli and chromatin pattern. A good H&E is imperative.
Leydig cells – have prominent nucleoli, the cytoplasm is intensely
eosinophilic and the eosinophilic crystalloid of Renke should
The endometrium contains epithelial and mesenchymal elements
and these need to be differentiated with a good H&E. Prominent
nucleoli should be apparent in the proliferating glandular epithelium.
The smooth muscle of the myometrium should contrast well with the
supporting collagen (4).
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Bone, apart from the rigors of fixation and processing, is also subjected
to acidic decalcifying solutions. This will adversely affect H&E quality. As
a guide, osteoblasts have a clear to blue cytoplasm often containing a
perinuclear halo. The nuclei often have a prominent nucleolus (3).
Skin, apart from a squamous cell covering, contains hair, sebaceous
and sweat glands, melanocytes, nerves, collagen and other cells that
should be seen in a good H&E (Fig. 3a and 3b) (3):
Keratohyalin granules in the cytoplasm of keratinocytes in the
Stratum Granulosum should appear densely basophilic.
Basal cells should show a coarse chromatin.
Collagen, nerves and muscle should show differing shades of
The basement membrane should be seen.
Within the Eccrine glands, three cell types should be present:
Clear cells, Dark cells bordering the lumen of the glands and
Breast tissue contains blood vessels, collagenous stroma, myoepithelial
cells and acinar epithelial cells. The latter cells are affected by the
menstrual cycle, for example, myoepithelial cells have clear cytoplasm
and small dense nuclei during the follicular phase and such changes
need to be considered when assessing H&E quality control (3).
H&E Quality Control Material
For the daily QC of the H&E stain I would suggest the following:
Colon – differentiation of muscle and collagen, inappropriate
staining of mucin, clear staining of epithelial vesicular nuclei.
Assessing plasma cell staining is quite helpful in that a clear clock
face arrangement of nuclear chromatin will indicate an adequate
differentiation of the haematoxylin.
Skin – demonstration of blue keratohyaline granules, differentiation of
keratin from collagen and nerves, definition of the reticular/papillary
border of the dermis.
Kidney – identification of basement membranes and both proximal
and efferent tubules. The kidney contains a wide diversity of cells
from those exhibiting dense chromatin (glomerular tufts) to those
having a dusting of chromatin material (cuboidal cells of collecting
tubules) (Luna 1988).
It is apparent that quality H&E sections are important in obtaining a
confident, accurate diagnosis. The above review is an attempt to list
those microscopic features that histotechnologists should assess in
order to obtain a quality stain. Unfortunately, fixation and processing
play a huge role in section quality and it is difficult to assess staining
quality in isolation.
Many of the above recommendations are based on personal experience
and as such should be open for debate. Critical discussion is
appreciated and encouraged. It is also apparent that knowledge of
routine histology and reference to classic histology texts (such as those
listed in the References) is important.
1. Llewellyn BD (2009) “Nuclear staining with alum hematoxylin” Biotechnic and Histochemistry
2. Luna LG (1991) “The quality control dilemma in Histotechnology: A possible answer”
3. Sternberg SS (1996) “Histology for Pathologists” Lippincott-Raven, Philadelphia- U.S.A,
p68, 153, 238, 254-257, 289, 316, 332-333, 382-391, 538, 627-629, 658-661, 732-736,
4. Young B, Heath JW (2002) “Wheater’s Functional Histology” 4th ed Churchill Livingstone,
Edinburgh-UK, p231, 316, 353-357.
5. Eroschenko VP (2005) “diFiore’s Atlas of Histology” 10th Edition, Lippincott Williams &
Wilkins, Philadelphia-U.S.A, p162-4, 314, 322.