ArticlePDF Available

Connection 2010 | 115 Microscopic Quality Control of Haematoxylin and Eosin – Know your Histology

Authors:

Abstract and Figures

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 H&E are: 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 very distinct. 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 ill-defined nucleoli. 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. H&E 
Content may be subject to copyright.
Connection 2010 |115
Microscopic Quality Control of Haematoxylin
and Eosin – Know your Histology
Anthony Henwood, MSc, CT(ASC)
Senior Scientist and Laboratory Manager
Histopathology Department
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
H&E are:
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
very distinct.
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
ill-defined nucleoli.
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.
H&E
| Connection 2010
116
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.
Connection 2010 |117
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
tissues?
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.
Nervous Tissue
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).
Pituitary
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
nucleoli (3).
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.
| Connection 2010
118
Thyroid
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).
Parathyroid
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).
Respiratory
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).
Heart
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).
Lymph Node
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).
Gastrointestinal Tract
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
good H&E:
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.
Liver
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.
Connection 2010 |119
Spleen
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).
Pancreas
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).
Kidney
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).
Adrenal Gland
Under low magnification, the cortex and medulla of the adrenal gland
should be apparent with acidophilic (pink) cortex and basophilic
(blue) medulla.
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).
Prostate
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).
Testis
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
be visible.
Endometrium
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).
| Connection 2010
120
Bone
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
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
red-pink.
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
Myoepithelial cells.
Breast
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).
Conclusion
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.
References
1. Llewellyn BD (2009) “Nuclear staining with alum hematoxylinBiotechnic and Histochemistry
84(4):159-177.
2. Luna LG (1991) “The quality control dilemma in Histotechnology: A possible answer”
Histologic 21(3):245-250.
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,
756-757, 898.
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.
... The hematoxylin and eosin (H&E) procedure is the 'bread and butter' stain in histopathology [1]. The diagnosis as well as other histochemical tests all follow on from a good H&E [2]. ...
... Both cytoplasmic counter stains stained equally well, allowing tinctorial separation of muscle, collagen, and red blood cells. Paneth cell and eosinophil granules were clearly stained [1]. ...
Article
An infrequent observation of assessing hematoxylin and eosin sections is the blue staining of mucins (for example those in goblet cells). This is believed to be due to a low concentration of alum and high pH of the hematoxylin staining solution. This study examines the incidence of blue mucin in various sites of the gastrointestinal tract using a low alum, high pH hematoxylin solution. The results are compared with a conventional hematoxylin solution, iron alum celestine blue method and an alcian blue (pH 2.5)-periodic acid-Schiff (AB-PAS) stain to characterize the type of mucin demonstrated. This study is the first to offer evidence that blue-stained mucin with low alum, high pH hematoxylin corresponds with carboxylated mucins as shown by the AB-PAS stain in the gastrointestinal tract. Iron alum celestine blue was also found to stain the mucin of a proportion of rectal biopsies and appendix as well as the carboxylated mucin of one duodenal biopsy.
... When preparing a digital slide, a variety of errors can occur during staining 4 (folded tissue due to bad mounting, partial wax removal, elevated eosin pH, hematoxylin break down, insufficient dehydration, etc.) and/or scanning 5 (e.g., autofocus failures causing regional blur, or stitching error causing a visible border between scanned tiles in high-power views). There are also many permissible variations in the staining procedures from lab to lab 6 or in behaviors of scanners from different vendors (e.g., illumination, resolution, and image composition algorithm). ...
Conference Paper
Slide quality is an important factor in pathology workflow and diagnosis. We examine the extent of quality variations in digitized hematoxylin-eosin (H&E) slides due to variations and errors in staining and/or scanning (e.g., out-of-focus blur & stitching). We propose two automatic quality estimators by adapting image quality assessment (IQA) methods that are originally developed for natural images. For the first estimator, we assume a gold-standard reference digital pathology slide is available. Quality of a given slide is estimated by comparing the slide to such a reference using a full-reference perceptual IQA method such as VIF (visual information fidelity) or SSIM (structural similarity metric). Our second estimator is based on IL-NIQE (integrated local natural image quality evaluator), a no-reference IQA, which we train using a set of artifact-free H&E high-power images (20× or 40×) from breast tissue. The first estimator (referenced) predicts marked quality reduction of images with simulated blurring as compared to the artifact-free originals used as references. The histograms of scores by the second estimator (no-reference) for images with artifact (blur, stitching, folded tissue, or air bubble artifacts) and for artifact-free images are highly separable. Moreover, the scores by the second estimator are correlated with the ratings given by a pathologist. We conclude that our approach is promising and further research is outlined for developing robust automatic quality estimators.
Article
The hematoxylin and eosin stain is the most common method used in anatomic pathology, yet it is a method about which technologists ask numerous questions. Hematoxylin is a natural dye obtained from a tree originally found in Central America, and is easily converted into the dye hematein. This dye forms coordination compounds with mordant metals, such as aluminum, and the resulting lake attaches to cell nuclei. Regressive formulations contain a higher concentration of dye than progressive formulations and may also contain a lower concentration of mordant. The presence of an acid increases the life of the solution and in progressive solutions may also affect selectivity of staining. An appendix lists more than 60 hemalum formulations and the ratio of dye to mordant for each.
The quality control dilemma in Histotechnology: A possible answer
  • L G Luna
Luna LG (1991) "The quality control dilemma in Histotechnology: A possible answer" Histologic 21(3):245-250.
Histology for Pathologists" Lippincott-Raven, Philadelphia-U.S.A, p68
  • S S Sternberg
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, 756-757, 898.
diFiore's Atlas of Histology
  • V P Eroschenko
Eroschenko VP (2005) "diFiore's Atlas of Histology" 10th Edition, Lippincott Williams & Wilkins, Philadelphia-U.S.A, p162-4, 314, 322.
Wheater's Functional Histology
  • B Young
  • J W Heath
Young B, Heath JW (2002) "Wheater's Functional Histology" 4th ed Churchill Livingstone, Edinburgh-UK, p231, 316, 353-357.