Microscopic Quality Control of Haematoxylin and Eosin ... - Dako

H&EMicroscopic Quality Control of Haematoxylinand Eosin – Know your HistologyAnthony Henwood, MSc, CT(ASC)Senior Scientist and Laboratory ManagerHistopathology DepartmentThe Children’s Hospital at WestmeadLocked Bag 4001, Westmead, 2145, AustraliaProducing quality microscopic sections goes a long way toachieving a confident, accurate diagnosis. Unfortunately tissuescan be distorted post-operatively, through fixation, during processing,microtomy and staining. A competent histological scientist needs to beable to minimise the distortion and correct it when the quality processgoes out of control. Quality staining is highly dependent on fixationand processing and it is difficult to appreciate histochemical qualitycontrol (QC) in isolation (1). Recognising microscopic quality is directlyconnected with knowledge of basic histology and recognition of thecommon microscopic disease entities. This review analyzes the qualityrequirements for Hematoxylin and Eosin (H&E) staining.Hematoxylin and eosin (H&E) is the “bread and butter” stain in mosthistopathology laboratories. It is appreciated that the H&E stain variesfrom laboratory to laboratory and that personal preference plays a largepart in the final results. Some general guidelines in assessing a goodH&E are:1. Nuclear detail – how effective is the stain in demonstrating nuclearmembranes, 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 cellsblue or clear? The mucin staining is a function of the HematoxylinpH. Lowering the pH (usually by adding acetic acid) can significantlyreduce 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 thefollowing tinctorial qualities:1. The nuclear chromatin should stain blue to bluish purple and bevery distinct.2. Nucleoli should appear reddish purple if the hematoxylin is welldecolourised. If too much hematoxylin remains in the nucleoli, thereis no doubt, that there is excess hematoxylin in the section. Excesseosin will produce a purple monochromatic nuclear chromatin andill-defined nucleoli.3. Collagen and muscle should stain well but in a delicate fashion. Oneshould be able to see some form of fibrillar pattern by moving themicroscopic focal plain up and down. The same pattern is observedby reviewing muscle except that muscle should have a slightlyredder colour than collagen.Connection 2010 | 115

Figure 1. High power H&E showing plasmacells with characteristic cart-wheel nuclei.Figure 2. Colon section stained with H&Eshowing blue mucin staining. Decreasing thehematoxylin pH should remove this staining.116 | Connection 2010

4. Eosinophilic granules should be well-defined and appear orangered.Lack of well-defined eosinophilic granules suggests overstainingby either hematoxylin or eosin, or both.These are general guidelines but are there guidelines for individualtissues?When assessing H&E-stained sections from differing organs, featuresthat are usually present are arteries. One should be able todifferentiate muscle and collagen with muscle appearing as darkred. Red blood cells should be bright red. Assessment of nuclearstaining will depend on the cell-types in the tissue being stained andthe following is an attempt to set some standards according to thetissue being assessed.Nervous TissueThe Nissl substance of neurones and Purkinje cells should be welldemarcated and stain dark blue. The large nuclei should be open with avisible nucleolus. The fibres should be delicately eosinophilic which willallow easier demonstration of senile plaques if present. The pale pinkstaining of neurones will also allow easier detection of “red” neurones,an important indicator of antemortem injury (3).PituitaryThe pituitary contains three principle cell types that should be apparentwith a good H&E: acidophils (40%) with pink cytoplasm, basophils(10%) with a pale blue cytoplasm and chromophobes (50%) with clearcytoplasm. The nuclei of acidophils should have relatively prominentnucleoli (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 | 117

ThyroidColloid should be pale pink. The cytoplasm of follicular cells shouldbe a slightly darker pink. The nuclei should be delicately stained withdiscernible small darker chromatin. The basement membrane bindingthe follicular cells should be visible and appear as deep red. In wellfixed and processed tissue, Clear cells may be seen admixed with thefollicular cells (4).“H&E stain varies from laboratory to laboratoryand that personal preference plays a large partin the final results. ”ParathyroidThe two cell types should be recognisable. Chief cells should have clearto faint pink cytoplasm with even blue chromatin, and rare small nucleoli.The cell membrane should be poorly defined. Oxyphil cells should showan eosinophilic granular cytoplasm, pyknotic nuclei and should have aprominent cell membrane (3).RespiratoryThe bronchioles contain smooth muscle and collagen in their walls. Thelung parenchyma is rich in small blood vessels and thus eosin balancebetween muscle and collagen can be assessed. In the alveoli, typeII pneumocytes should have pale nuclei with prominent nucleoli. Thenuclei of type I pneumocytes are small and dense (4).Gastrointestinal TractThe gastrointestinal (GI) tract contains epithelial, connective, neural andlymphoid tissues that need to be easily recognisable using a routineH&E. Lymphoid nuclei, especially those of plasma cells, should beassessed. The usual colour differentiation should exist between muscleand collagen. Ganglion cells should be apparent in between the musclelayers. The nuclei should contain a single, prominent, eosinophilicnucleolus and the cytoplasm should be pink (a lighter hue than that ofthe surrounding muscle). It is expected that the epithelial mucin shouldbe clear. Blue staining of the mucin usually indicates that the pH of theHematoxylin is too high (Fig. 2). It has often been found that in thesecases, the hematoxylin often appears over-stained resulting in difficultyin assessing the nuclear component. Throughout the GI tract there areunique cells that exist and it important that they be recognised in agood H&E:HeartThe heart is rich in blood vessels as well as cardiac muscle and collagenenabling good eosin differentiation. Purkinje fibres, if present, shouldhave pale cytoplasm with open vesicular nuclei. Cardiac nuclei shouldhave evenly distributed dust-like chromatin with a clear (non-staining)perinuclear sarcoplasm. The eosin should demonstrate the crossstriationsadequately (5).Lymph NodeIn the germinal centres, centroblasts have large, round, vesicularnuclei with 1-3 small but conspicuous nucleoli. Small lymphocytes havedark staining, irregular nuclei. Plasma cells with their “spoke-wheel”chromatin pattern caused by small clumps of chromatin on the nuclearmembrane in a clear nucleus may be present (Fig 1). A clear “hof”should be seen (3).In the stomach, the parietal cell cytoplasm should be light pink, whereas that of the chief cells should appear purplish. Small nucleoli shouldalso 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 darkerthan those of the absorptive cells. Endocrine and Paneth cells shouldbe discernible by the eosinophilic granules present in the cytoplasm.LiverThe predominant cell is the hepatocyte. The nuclei should have smallnucleoli visible. The pale red-pink cytoplasm of the hepatocytes shouldcontrast well with the pink of collagen around blood vessels. Thereshould also be small blue granules (RER) in the cytoplasm. Cuboidal bileduct cells have open nuclei and dark pink cytoplasm (3).118 | Connection 2010

SpleenThe cell population in the spleen is similar to the lymph node so you canrecognise similar microscopic quality control features. Both mediumsized and small lymphocytes are present. Plasma cells are often foundnear the arteries. The endothelial cells lining the sinus should have beanshapednuclei containing a longitudinal cleft (3).PancreasThe pancreas has a wide variety of cells to meet the exocrine andendocrine functions of the organ:Acinar cells have dark nuclei with a few tiny clumps and strands ofchromatin. Nucleoli should be evident. The cytoplasm should haveintense eosinophilia at the apical with strong hematoxylinophilicstaining at the basal end.Duct cells have clear to pale eosinophilic cytoplasm and lightlystained nuclei (less dense than those of acinar cells).Islet cell nuclei should have evenly stained nuclei and cytoplasma paler pink than acinar cells. Within the islet, differing cells shouldbe discerned with darker cytoplasm cells probably being glucagoncontaining cells (3).KidneyThe kidney contains a diverse population of cells from those exhibitingdense chromatin (in the glomerular tufts) to those exhibiting a dustingof chromatin material (cuboidal cells of the collecting tubules) (2).The cytoplasm of the cells of the Proximal Convoluted Tubules haveeosinophilic cytoplasm where as those of the Distal Convoluted Tubuleshave paler cytoplasm (fewer granules). The cells of the ProximalConvoluted Tubules should have a discernible brush border. Thebasement membranes of both proximal and efferent tubules shouldbe visible (5).In the medulla, the chromaffin cells should have an overall bluecytoplasm. The nuclei should have coarsely clumped chromatin withvisible clear areas. The nuclear material of the ganglion cells should bespeckled and have prominent nucleoli.Three zones should be visible in the cortex. The zona glomerulosa(closest to the capsule), the cells should have clear cytoplasmcontaining a scattering of pink granules. The nuclei should have alongitudinal groove present in many of the cells. In the middle zone(zona fasciculata) the nuclei should be more vesicular and lesschromatic than in the previous zone. A single small nucleolus should beapparent and the cytoplasm should be clear. In the zona reticularis, thecells have cytoplasm that is solid, granular and eosinophilic. Lipofuscinshould be discernible especially in cells adjacent to the medulla (3).ProstateThe stroma of the prostate contains both collagen and smooth muscleand these should be easily differentiated on an H&E. The secretaryepithelial cells of the central zone of the prostate should havedarker cytoplasm and slightly darker nuclei compared to those in theperipheral zone (3).TestisThe main features of the testis that need to be recognised include (3):Sertoli cells – nuclei should have a slightly wrinkled nuclearmembrane and have prominent nucleoli.Germ cells – You should be able to easily distinguish each of thedifferentiating elements based on the presence or absence ofnucleoli and chromatin pattern. A good H&E is imperative.Leydig cells – have prominent nucleoli, the cytoplasm is intenselyeosinophilic and the eosinophilic crystalloid of Renke shouldbe visible.Adrenal GlandUnder low magnification, the cortex and medulla of the adrenal glandshould be apparent with acidophilic (pink) cortex and basophilic(blue) medulla.EndometriumThe endometrium contains epithelial and mesenchymal elementsand these need to be differentiated with a good H&E. Prominentnucleoli should be apparent in the proliferating glandular epithelium.The smooth muscle of the myometrium should contrast well with thesupporting collagen (4).Connection 2010 | 119