Atlas of Clinical Hematology || Light Microscopic Procedures
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Light Microscopic Procedures
1 Staining Methods for the Morphologic and Cytochemical Differentiation of Cells 8
1.1 Pappenheims Stain (Panoptic Stain) 8
1.2 Undritz Toluidine Blue Stain for Basophils 8
1.3 Mayers Acid Hemalum Nuclear Stain 8
1.4 Heilmeyers Reticulocyte Stain 8
1.5 Heinz Body Test of Beutler 8
1.6 Nile Blue Sulfate Stain 9
1.7 Kleihauer-Betke Stain for Demonstrating Fetal Hemoglobin in Red Blood Cells 9
1.8 Kleihauer-Betke Stain for Demonstrating Methemoglobin-Containing Cellsin Blood Smears 10
1.9 Berlin Blue Iron Stain 10
1.10 Cytochemical Determination of Glycogen in Blood Cells by the Periodic Acid SchiffReaction and Diastase Test (PAS Reaction) 11
1.11 Sudan Black B Stain 13
1.12 Cytochemical Determination of Peroxidase 13
1.13 Hydrolases 13
1.13.1 Cytochemical Determination of Leukocyte Alkaline Phosphatase (LAP)in Blood Smears 13
1.13.2 Cytochemical Determination of Acid Phosphatase 141.13.3 Detection of Esterases with Naphthyl Acetate or Naphthyl Butyrate
(Neutral Esterases) 14, Acid Esterase (ANAE) 151.13.4 Naphthol AS-D Chloroacetate Esterase (CE) 15
1.14 Appendix 16
2 Immunocytochemical Detection of Cell-Surface and Intracellular Antigens 18
3 Staining Methods for the Detection of Blood Parasites 19
3.1 Thick Smear Method 19
3.2 Bartonellosis 19
3.3 Detection of Blood Parasites in Bone Marrow Smears 19
3.4 Toxoplasmosis 19
3.5 Microfiliariasis 19
3.6 Mycobacterium Species (M. tuberculosis, M. leprae) 19
1. Staining Methods for theMorphologic and CytochemicalDifferentiation of Cells
1.1 Pappenheims Stain (Panoptic Stain)
The hematologic stain that we use most fre-quently, and which was used in most of the platespictured in this atlas, is Pappenheims panopticstain. It is based on a combination of the Jen-ner-May-Grunwald stain and Giemsa stain.
Method. Place the air-dried slide with the film sideup in prepared May-Grunwald eosin-methyleneblue solution for 3 min. Dilute with water or buf-fer solution (phosphate buffer pH 7.3, see below)for an additional 3 min. Pour off this solution andapply Giemsa stain immediately, without inter-mediate rinsing. The stock Giemsa stain is dilutedwith neutral distilled water by adding 10 mLwaterper 10 drops of Giemsa solution. Stain the speci-men for 15 to 20 min. The dilution ratio andGiemsa staining time should be individually ad-justed to allow for inevitable variations in thecomposition of the solution. After Giemsa stain-ing, wash the slide with neutral water and tilt toair-dry. Fixation is effected by the methyl alcoholalready contained in the May-Grunwald solution.The quality of the stain depends greatly on the pHof the water that is used. The smear will be too redif the water is too acidic and too blue if the wateris too alkaline. Standard pH strips can be used totest the water for proper acidity. Water left stand-ing in the laboratory can easily become too acidicthrough exposure to acid fumes, especially fromcarbon dioxide. The latter problem is solved bypreboiling. A more accurate way to ensure correctacidity for staining is to use a pH 7.3 buffer solu-tion (22.3 mL of 1/15 mol/L KH2PO4 + 77.7 mL of 1/15 mol/L Na2HPO4) instead of water.
1.2 Undritz Toluidine Blue Stain for Basophils
Reagent. Saturated toluidine blue-methanol: dis-solve 1 g toluidine blue in 100 mL methanol. Thesolution will keep indefinitely.
Method. Fix and stain the air-dried smears on thestaining rack by covering with the toluidine blue-methanol for 5 min. Wash in tap water, air dry.
Interpretation. The granulations in basophils andmast cells stain a red-violet color owing to thestrong metachromatic effect of the sulfate presentin the heparin. As a result, these cells are easilyidentified even at moderate magnification. Bycontrast, azurophilic granules (even in severe
toxic granulation) and the coarse granules inleukocytes affected by Adler anomaly showvery little violet transformation of their blue col-or.
1.3 Mayers Acid Hemalum Nuclear Stain
This is used for the blue contrast staining of nu-clei in assays of cytoplasmic cell constituents (gly-cogen, enzymes; pp. 9 ff.) and in immunocyto-chemistry.
Reagents. Dissolve 1 g hematoxylin (Merck) in 1 Ldistilled water and add 0.2 g sodium iodate(NaIO3) and 50 g aluminum potassium sulfate(KAl(SO4)2 12H2O). After these salts are dis-solved, add 50 g chloral hydrate and 1 g crystal-lized citric acid. The hemalum will keep for atleast 6 months at 20 8C with no change in stainingproperties. The solution can also be purchased inready-to-use form.
Method. The necessary staining time in the hema-lum bath varies with the method of specimen pre-paration and must be determined by progressivestaining. After staining, wash the slide for at least15 min in several changes of tap water (acid resi-dues may reduce the intensity of the stain).
1.4 Heilmeyers Reticulocyte Stain
Draw a 1 % brilliant cresyl blue solution in phy-siologic saline to the 0.5 mark of a white cellcounting pipet, and draw up the blood to the1.0 mark. Expel the mixture carefully, withoutforming air bubbles, into a paraffinated watch-glass dish, mix carefully with a paraffinated glassrod, and place in a moist chamber for 1520 min.Then remix carefully with a paraffinated glassrod. With the rod, transfer 1 or 2 drops of the mix-ture to a microscope slide and smear in standardfashion using a ground coverslip. Examine theair-dried slides under oil-immersion magnifica-tion, and count the number of reticulocytes per1000 red cells at multiple sites in the smear.Very high-quality films can be obtained by Giem-sa counterstaining.
1.5 Heinz Body Test of Beutler1
This test is used to detect defects of red cell me-tabolism that do not allow glutathione to be
1 After Huber H, Loffler H, Faber V (1994) Methoden der diag-nostischen Hamatologie. Springer, Berlin Heidelberg NewYork Tokyo.
8 Chapter II Light Microscopic Procedures
maintained in a reduced state. The defect may re-sult from a glucose-6-phosphate dehydrogenasedeficiency, a glutathione reductase deficiency,diseases with unstable hemoglobin, or an idio-pathic Heinz body anemia. The test involves theoxidative denaturation of hemoglobin to intra-erythrocytic Heinz bodies following incubationof the red cells with acetylphenylhydrazine.
1. Sorensen phosphate buffer, pH 7.6, 0.67 M:1/15 M KH2PO4 13 parts.1/15 M Na2HPO4 87 parts.
2. Glucose phosphate buffer: dissolve 0.2 g glu-cose in 100 mL phosphate buffer. The solutionmay be stored frozen or at 4 8C (watch forclouding!).
3. Acetylphenylhydrazine solution: dissolve20 mg acetylphenylhydrazine in 20 mL glucosephosphate buffer at room temperature. Thissolution is prepared fresh and should beused within 1 h.
4. (a) Dissolve saturated alcohol solution of bril-liant cresyl blue; or (b) 0.5 g methyl violet in100 mL of 0.9 % NaCI, and filter; blood: hepar-inized, defibrinated, or treated with EDTA.
Method. Centrifuge the blood lightly for 5 min. Pi-pet 0.05 mL of test erythrocytes into 2 mL of theacetylphenylhydrazine solution. Suspend normalerythrocytes in an identical solution to serve as acontrol. Aerate the suspensions by drawing themup into the pipet and carefully blowing them outwith a small quantity of air; repeat several times.Incubate for 2 h at 37 8C, aerate again, and incu-bate 2 h more.
To stain with brilliant cresyl blue: spread asmall drop of stain solution 4(a) onto a clean, de-greased slide and dry the thin stain film rapidly inair. Place a small drop of the incubated erythro-cyte suspension on a coverglass and invert theglass onto the stain; examine with the micro-scope.
To stain with methyl violet: mix a small drop ofthe erythrocyte suspension with 2 or 3 drops ofstain solution 4b on the slide and cover with acoverslip. Let the mixture stand for 5 10 minand examine with the microscope.
Interpretation. The percentage of erythrocytesthat contain more than four Heinz bodies is de-termined. Normal values range from 0 % to 30 %.The number of Heinz bodies is elevated in the dis-eases listed above.
1.6 Nile Blue Sulfate Stain
This stain is used for the visualization of Heinzinclusion bodies. A 0.5 % Nile blue sulfate solu-tion in absolute alcohol is transferred to theend of a slide with a glass rod until about 1/3of the slide is covered. The slide is dried by blow-ing on it, and the stain film is spread out evenlywith a cotton swab. Slides prepared in this way areplaced face-to-face and wrapped in paper for sto-rage. Staining is performed by dropping 2 or 3large drops of blood onto the prepared part ofthe slide and covering with the prepared partof the second slide. The slides, held by their un-stained outer ends, are separated and placed backtogether several times to thoroughly mix theblood with the stain. Finally the slides are left to-gether for 3 to 5 min, separated, and a groundcoverslip is used to collect the blood from eachslide and smear it onto another slide, which is al-lowed to dry. The Heinz bodies appear as small,dark blue bodies situated at the margin of the yel-low to bluish erythrocytes.
1.7 Kleihauer-Betke Stainfor Demonstrating Fetal Hemoglobinin Red Blood Cells
Principle.Normal adult hemoglobin (HbA) is dis-solved out of the red cells by incubating air-driedand fixed blood smears in citric acid phosphatebuffer (of McIlvain), pH 3.3, at 37 8C. Fetal hemo-globin (HbF) is left undissolved in the red cellsand can be made visible by staining.
Ethyl alcohol, 80 % McIlvaine citric acid-phosphate buffer, pH 3.3 Stock solution A:
Sorensen citric acid, 21.008 g in 1 L water 0.1 M
Stock solution B:Disodium hydrogen phosphate Na2HPO42H2O, 27.602 g in 1 L water 0.2 M
For pH 3.3:266 mL of solution B + 734 mL of solution A,Ehrlich hematoxylin, 0.1 % erythrosin solution
Method. Prepare thin blood smears, air dry, andfix in 80 % ethyl alcohol for 5 min. Wash in waterand dry. If further processing is delayed, the slidesmay be stored in a refrigerator for 45 days. Forelution, place the slides upright in a beaker con-taining the buffer in a 37 8C water bath for 3 min,moving the slides up and down after 1 and 2 minto keep the buffer mixed. Then wash in runningwater.
1 Staining Methods for the Morphologic and Cytochemical Differentiation of Cells
Staining. Stain in Ehrlich hematoxylin for 3 min,then poststain in 0.1 % aqueous erythrosin solu-tion for 3 min. Examine at 40 using dry or oil-immersion magnification.
Interpretation. Erythrocytes that contain HbA ap-pear as unstained shadows, while cells that con-tain HbF will stain a bright red color.
The method can be used for the diagnosis ofthalassemia major and for the detection of fetalerythrocytes that have entered the maternal circu-lation.
1.8 Kleihauer-Betke Stainfor Demonstrating Methemoglobin-Containing Cells in Blood Smears
Principle.Methemoglobin combines with KCN toform cyanmethemoglobin, while oxyhemoglobindoes not react with cyanides. Oxyhemoglobinfunctions as a peroxidase, whereas cyanmethe-moglobin has very low perixodase activity.
Method. Add 1/50 vol of a 0.4 M KCN solution toblood anticoagulated with heparin or sodium ci-trate. Prepare thin smears from this mixture, dry,and immerse in the following mixture at roomtemperature: 80 mL of 96 % ethyl alcohol + 16mL of 0.2 M citric acid + 5 mL of 30 % H2O2.Move the smears rapidly in the solution for about1 min, then leave them in the solution for 2 min.Wash the smears first in methyl alcohol, then indistilled water, and stain with hematoxylin anderythrosin (see stain for HbF). Examine at 40using dry or oil-immersion magnification.
Interpretation. Oxy-Hb-containing cells stain abright red. Cells that contain met-Hb (convertedto cyanmet-Hb) are eluted and appear as sha-dows.
The same staining procedure can be used todifferentiate erythrocytes with a glucose-6-phos-phate dehydrogenase (G-6-PDH) deficiency bycombining it with the Brewer test (method ofBetke, Kleihauer and Knotek). This test is basedon the principle that hemoglobin converted tomet-Hb by the addition of nitrite reduces tooxy-Hb in the presence of methylene blue andglucose. Red blood cells with a G-6-PDH deficitcannot undergo this reduction. Even after severalhours, when all the methemoglobin in normalerythrocytes has converted back to oxy-Hb, cellswith a G-6-PDH deficiency retain all or most oftheir met-Hb. This causes the deficient cells to ap-pear blank with appropriate staining (see top ofthis section).
1.9 Berlin Blue Iron Stain
The Berlin blue reaction is used for the histo-chemical demonstration of trivalent iron. Ironin protein compounds can also be demonstratedby the addition of dilute hydrochloric acid. Ironin hemoglobin is not detected.
Methanol Potassium ferrocyanide (potassium hexacya-
noferrate), 2 % HCl, 37 % Pararosaniline solution inmethanol, 1 % (alter-
native: nuclear red stain)
Fix the air-dried smears in formalin vapor for30 min (alternative: fix in methanol for 1015min).
Wash in distilled water for 2min and air dry. Place the specimens in a cuvet that contains
equal parts of a 2 % solution of potassium fer-rocyanide and a dilute HCl solution (1 part37 % HCl mixed with 50 parts distilled water)for 1 h.
Wash in distilled water. Nuclear stain in pararosaniline solution: 300
lL of 1 % pararosaniline solution in methanoldiluted with 50 mL distilled water.Alternative. Stain nuclei with nuclear true redsolution (which yields a fainter nuclear stain).
All materials should be iron-free, and metal for-ceps should not be introduced into the solution.Pappenheim- or Giemsa-stained smears can sub-sequently be used for iron staining. They are firstprepared by destaining them for 1224 h in puremethanol. These smears do not need to be fixedprior to staining.
Iron is stained blue, appearing either as diffuselyscattered granules or as clumps in the cytoplasm.There are two applications for iron staining in he-matology:(a) demonstrating sideroblasts and siderocytes,
and(b) demonstrating iron stored in macrophages
and endothelial cells.
Regarding (a): sideroblasts and siderocytes are,respectively, erythroblasts and erythrocytes thatcontain cytochemically detectable iron. Thisiron can be demonstrated in the form of smallgranules that may be irregularly scattered
10 Chapter II Light Microscopic Procedures
throughout the cytoplasm or may encircle the nu-cleus of erythroblasts like a ring. Normally thegranules are very fine and can be identified in ery-throblasts only by closely examining the smearswith oil-immersion microscopy in a darkenedroom. Generally 1 to 4 fine granules will beseen, rarely more. When iron deficiency is pre-sent, the percentage of sideroblasts is reducedto less than 15 %. Sideroblasts containing coarseiron granules that form a partial or complete ringaround the nucleus (ringed sideroblasts) are de-finitely abnormal. The detection of siderocyteshas little practical relevance: they are increasedin the same diseases as sideroblasts, and theyare elevated in the peripheral blood followingsplenectomy, as the spleen...