Detection of Esterases with Naphthyl Acetate or Naphthyl Butyrate Neutral Esterases

Solution a mix 1 drop (0.05 mL) sodium nitrite solution (4 ) + 1 drop (0.05 mL) pararosaniline solution (4 in 2 mol L HCl) for about 1 min (yields a pale yellow solution), then dissolve in 5 mL of 0.2 mol L phosphate buffer, pH 7.0-7.1 (250 mL Na2HPO4+130 mL NaH2 PO4). Solution b dissolve 10 mg a-naphthyl acetate in 0.2-0.3 mL chemically pure acetone add 20 mL of 0.2 mol L phosphate buffer pH 7.0 - 7.1 while stirring vigorously. Mix solutions a and b and filter into small cu-vets. Fix the...

Nile Blue Sulfate Stain

This stain is used for the visualization of Heinz inclusion bodies. A 0.5 Nile blue sulfate solution in absolute alcohol is transferred to the end of a slide with a glass rod until about 1 3 of the slide is covered. The slide is dried by blowing on it, and the stain film is spread out evenly with a cotton swab. Slides prepared in this way are placed face-to-face and wrapped in paper for storage. Staining is performed by dropping 2 or 3 large drops of blood onto the prepared part of the slide...

Cells of the Monocyte Macrophage System Fig 13 ah

Typical monocytes with pleomorphic nucleus, pale blue cytoplasm, and fine, barely perceptible azurophilic granules (Fig. 13 a, b). Monocyte, with a metamyelocyte at upper left (Fig. 13 c). Monocyte with a small nucleus and, below it, a segmented neutrophil (Fig. 13 d). Two promonocytes in a bone marrow smear (Fig. 13 e). Two promonocytes with nucleoli, and a mono-cyte at upper left (Fig. 13 f). Monocyte with phagocytized nuclear residue Monocyte with phagocytized material in the cytoplasm...

Microfiliariasis

Wet preparation (thick smear method) Examine a drop of fresh (anticoagulated) blood under a coverslip on a microscope slide (bearing in mind the periodicity in microfilarial activity, see p. 403). The highly motile organisms are clearly visible even at low magnification (250 x). 2. Concentrating the sample To 3 - 5 mL of drawn venous blood, add 10 -15 mL of a mixture of 95 mL formalin (5 ), 5 mL acetic acid, and 2 mL of an alcoholic gentian violet solution (4 g per 100 mL 96 alcohol)....

Berlin Blue Iron Stain

The Berlin blue reaction is used for the histo-chemical demonstration of trivalent iron. Iron in protein compounds can also be demonstrated by the addition of dilute hydrochloric acid. Iron in hemoglobin is not detected. Potassium ferrocyanide potassium hexacya-noferrate , 2 Pararosaniline solution in methanol, 1 alternative nuclear red stain Fix the air-dried smears in formalin vapor for 30 min alternative fix in methanol for 10 -15 min . Wash in distilled water for 2 min and air dry. Place...

Illustrations

Mycobacteria Bone Marrow

III Overview of Cells in the Blood, Bone Marrow, and Lymph Nodes 23 VI Tumor Aspirates from Bone Marrow Involved by Metastatic Disease 385 VII Blood Parasites and Other Principal Causative Organisms of Tropical Disease 399 Overview of Cells in the Blood, Bone Marrow, and Lymph Nodes Figure 1 presents an overview of the various cells of hematopoiesis. The figure does not attempt to answer unresolved questions of cell origins and is intended only as an introductory scheme to help the beginner...

Light Microscopic Procedures

1 Staining Methods for the Morphologic and Cytochemical Differentiation of Cells 8 1.1 Pappenheim's Stain Panoptic Stain 8 1.2 Undritz Toluidine Blue Stain for Basophils 8 1.3 Mayer's Acid Hemalum Nuclear Stain 8 1.4 Heilmeyer's Reticulocyte Stain 8 1.5 Heinz Body Test of Beutler 8 1.7 Kleihauer-Betke Stain for Demonstrating Fetal Hemoglobin in Red Blood Cells 9 1.8 Kleihauer-Betke Stain for Demonstrating Methemoglobin-Containing Cells in Blood Smears 10 1.10 Cytochemical Determination of...

Cytochemical Determination of Peroxidase

Benzidine or diaminobenzidine more often used is converted, in the presence of peroxide, from the leuko form into a high-polymer form that is detectable by cytochemical staining. Fixative methanol 37 formalin 10 1 . DAB solution 5 mg diaminobenzidine tetrahy-drochloride in 20 mL of 0.05mol L tris-HCl buffer pH 7.6 with 50 iL of 1 H2O2 added Tris-HCl 50 mL of solution A 121.14 g trishy-droxymethylaminomethane dissolved in 1 L distilled water 40 mL of solution B 1 mol L HCl 960 mL distilled water...

Naphthol ASD Chloroacetate Esterase CE

Methanol-formalin solution, 9 1 v v . 0.1 mmol L Michaelis buffer, pH 7.0. Naphthol AS-D chloroacetate. Sodium nitrite solution, 4 . Pararosaniline solution, 4 , in 2 mol L HCl. Staining solution A mix 0.1 mL sodium nitrite solution and 0.1 mL pararosaniline solution with 30 mL Michaelis buffer. Staining solution B dissolve 10 mg naphthol AS-D chloroacetate in 1 mL dimethylforma-mide. Staining solution C mix solutions A and B , adjust to pH 6.3 with 2 mol L HCl, and filter into a cuvet. Use...

Staining Methods for the Morphologic and Cytochemical Differentiation of Cells

1.1 Pappenheim's Stain Panoptic Stain 8 1.2 Undritz Toluidine Blue Stain for Basophils 8 1.3 Mayer's Acid Hemalum Nuclear Stain 8 1.4 Heilmeyer's Reticulocyte Stain 8 1.5 Heinz Body Test of Beutler 8 1.7 Kleihauer-Betke Stain for Demonstrating Fetal Hemoglobin in Red Blood Cells 9 1.8 Kleihauer-Betke Stain for Demonstrating Methemoglobin-Containing Cells in Blood Smears 10 1.10 Cytochemical Determination of Glycogen in Blood Cells by the Periodic Acid Schiff Reaction and Diastase Test PAS...

Containing Cells in Blood Smears

Methemoglobin combines with KCN to form cyanmethemoglobin, while oxyhemoglobin does not react with cyanides. Oxyhemoglobin functions as a peroxidase, whereas cyanmethemoglobin has very low perixodase activity. Method. Add 1 50 vol of a 0.4 M KCN solution to blood anticoagulated with heparin or sodium citrate. Prepare thin smears from this mixture, dry, and immerse in the following mixture at room temperature 80 mL of 96 ethyl alcohol 16 mL of 0.2 M citric acid 5 mL of 30 H2O2. Move...

Blood and Bone Marrow

4.1 Light Microscopic Morphology and Cytochemistry 28 4.1.1 Cells of Erythropoiesis Fig. 4a-f 28 4.1.2 Granulocytopoiesis and Mast Cells Tissue Basophils 38 4.1.3 Degenerate Forms, Toxic Changes and Artifacts Fig. 9 a-d 44 4.1.4 Congenital Anomalies of Granulocytopoiesis Fig. 10a-f 46 4.1.5 Cells of the Monocyte-Macrophage System Fig. 13 a-h 54 4.1.6 Megakaryocytes Fig. 15 a-e 60 4.1.7 Osteoblasts and Osteoclasts Fig. 16 a-f 63 4.1.8 Lymphocytes and Plasma Cells Fig. 17a-g 66 5.1 Composition of...

Bone Marrow

Percutaneous aspiration of the posterior iliac spine is the current method of choice for obtaining a bone marrow sample. It is a relatively safe procedure, and with some practice it can be done more easily and with less pain than sternal aspiration. Marrow aspirate and a core sample can be obtained in one sitting with a single biopsy needle e.g., a Yamshidi needle . When proper technique is used, the procedure is not contraindicated by weakened host defenses or thrombocytopenia. However, there...

Degenerate Forms Toxic Changes and Artifacts Fig 9 ad

Bone Marrow Aspiration Site Infant

Rarely, degenerate leukocyte forms Fig. 9 a may be found in the peripheral blood of patients exposed to certain irritants. They are more commonly seen in smears prepared from long-stored blood previously treated with EDTA or citrate solution. Most of these cells have the same diameter as segmented forms, but many are considerably smaller 4-8 im . Usually their cytoplasm is slightly more basophilic than in segmented forms, and their granules are coarser and often smudged. Pronounced nuclear...

Fixation Suitable for Esterase Acid Phosphatase DAP IV

The fixative solution is composed of 30 mL buffer solution 20 mg disodium hydrogen phosphate 12H20 and 100 mg potassium dihydrogen phosphate dissolved in 30 mL distilled water pH should be 6.6 45 mL analytical grade acetone Fix air-dried smears in this solution for 30 s at 4 -10 0C, wash in three changes of distilled water, and dry at room temperature for 10 - 30 min. Schaefer universal fixative. Mix 0.5 mL of 25 glutardialdehyde solution and 60 mL analytical grade acetone in distilled water to...

Undritz Toluidine Blue Stain for Basophils

Saturated toluidine blue-methanol dissolve 1 g toluidine blue in 100 mL methanol. The solution will keep indefinitely. Method. Fix and stain the air-dried smears on the staining rack by covering with the toluidine blue-methanol for 5 min. Wash in tap water, air dry. Interpretation. The granulations in basophils and mast cells stain a red-violet color owing to the strong metachromatic effect of the sulfate present in the heparin. As a result, these cells are easily identified even at...

Cytochemical Determination of Acid Phosphatase

Staining solution mix together 0.8 mL hexazo-tized pararosaniline mix equal parts 4 sodium nitrite and 4 pararosaniline in HCl, see Appendix 30 mL Michaelis buffer pH 7.4 58 mL of 0.imol L sodium barbital 41.9 mL of 0.imol L HCl 10 mg naphthol-AS-BI phosphate, dissolved in 1 mL dimethyl-formamide. Adjust the solution to pH 4.9-5.1 and filter before use. Fix the air-dried smears at 4 0C for 30 s. Incubate in stain solution for 3 h at room temperature. Place in Mayer's hemalum for 3 min. Blue in...

Cells of Erythropoiesis Fig 4af

Mast Cells Bone Marrow

The proerythroblasts, called also pronormoblasts or rubriblasts, are the earliest precursors of erythropoiesis. They range from 15 to 22 im in size and do not yet contain hemoglobin. They typically have a darkly basophilic, often shadowy cytoplasm that sometimes shows pseu-dopodia. The nucleus has a dense, finely honeycombed chromatin structure Fig. 4 a - c . Most proerythroblasts have several at most five indistinct pale blue nucleoli, which disappear as the cell matures. Like all...

G

Grained Bone Marrow Megakaryocytes

Macrophages in the Bone Marrow Fig. 14 a-h Macrophage with nuclei, erythrocytes and platelets in the cytoplasm nucleus at lower right Fig. 14 a . Two macrophages with cellular residues Fig. 14 b . Macrophage showing high acid phosphatase activity red and nuclear residues bluish gray Fig. 14 c . Binucleated macrophage with predominantly fine-grained hemosiderin in the cytoplasm yellow-gold Fig. 14 d . Abundant hemosiderin, some coarsely granular, in the cytoplasm Fig. 14 e . Clumped and...

Granulocytopoiesis and Mast Cells Tissue Basophils

Hymenal Tissue

Myeloblasts and Promyelocytes Fig. 7 a-h Myeloblasts are the earliest precursors of granulocytopoiesis that can be identified by light microscopy. Today it is believed that they also function as precursors of monocytes, i.e., as myelomo-noblasts. They range from 12 to 20 im in diameter Fig. 7a-c . The cytoplasmic rim is basophilic but may show a range of hues from soft pale blue to dark blue. The cytoplasm is agranular on ordinary panoptic staining, although older cells frequently show...

Pappenheims Stain Panoptic Stain

The hematologic stain that we use most frequently, and which was used in most of the plates pictured in this atlas, is Pappenheim's panoptic stain. 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 side up in prepared May-Grunwald eosin-methylene blue solution for 3 min. Dilute with water or buffer solution phosphate buffer pH 7.3, see below for an additional 3 min. Pour off this solution and apply Giemsa stain...

Congenital Anomalies of Granulocytopoiesis Fig 10 af

Detect Eosinophils Tissue

This is an inherited anomaly involving the nuclei of granulocytes. The heterozygous form predominates in man while the homozygous form, characterized by small round or oval nuclei Fig. 10 c , is extremely rare. The nucleus of neutrophils is indented and resembles the band form, giving rise to a pseudoregenerative white blood picture. When nuclear segmentation lobulation occurs, the neutrophils acquire two nuclear lobes and rarely three. These lobes are exceptionally short, thick, and...

Mayers Acid Hemalum Nuclear Stain

This is used for the blue contrast staining of nuclei in assays of cytoplasmic cell constituents glycogen, enzymes pp. 9 ff. and in immunocyto-chemistry. Reagents. Dissolve 1 g hematoxylin Merck in 1 L distilled water and add 0.2 g sodium iodate NaIO3 and 50 g aluminum potassium sulfate KAl SO4 2 12H2O . After these salts are dissolved, add 50 g chloral hydrate and 1 g crystallized citric acid. The hemalum will keep for at least 6 months at 20 0C with no change in staining properties. The...

Splenic Aspiration

Splenic aspiration is rarely practiced nowadays and is always performed under some form of radiologic guidance. Today it is indicated only in certain forms of hypersplenism or unexplained splenic enlargement. We consider the Moeschlin technique to be the safest. Splenic aspiration is contraindicated in patients with hemorrhagic diathesis, septic splenomegaly, splenic cysts, or painful splenomegaly due to excessive capsular tension or infarction. The procedure should be used with caution in...

Pararosaniline Solution

Dissolve 2 g Graumann pararosaniline Merck in 50 mL of 2 mol L HCl by gentle heating. Cool and filter the solution. The sodium nitrite and pararosaniline solutions will keep for several months when stored in a dark bottle under refrigeration. Most of the reagents and even commercial staining kits can be ordered from pharmaceutical houses Merck, Serva, Sigma, etc. . Before kits are used for routine tests, they should be compared against solutions prepared by the methods indicated. The...

Hematology

Begemann With 199 Figures, in 1056 separate Illustrations, Mostly in Color, and 17 Tables Ehem. Direktor der II. Medizinischen Klinik und Poliklinik der Universit t Kiel im St dtischen Krankenhaus Seelgutweg 7, 79271 St. Peter, Germany Ehem. Leiter der Abteilung fur Hamatologie und Onkologie 1. Medizinische Klinik und Poliklinik Klinikum rechts der Isar der Technischen Universit t M nchen Westpreu enstra e 71, 81927 Munchen, Germany Professor Dr. med. Dr. phil....