haematology DDKItalia Stains for blood and bone marrow classic staining methods, fast stain and foil staining haematology

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1 DDKItalia Stains for blood and bone marrow classic staining methods, fast stain and foil staining haematology

2 Haematological staining methods All reagents from one source Fast and safe processes Easy to use Quick and sure results in less than 30 minutes All stainings at room temperature First-class and reproducible staining pictures The same careful fixation for all stainings According to the standard literature

3 Hematological staining methods Hematological staining Staining of blood and bone marrow smears are made whit a mixtures of acid (eosines) and basic (methylene blue, azure) dyes especially suitable. With the basic dyes the acid parts of cells such as nucleic acid are stained. The acid dyes stains above all basic proteins, such as lysosomal enzymes in the granula. The staining picture with the staining intensity and color is determined from the dye solution, the quality of the staining is determined by the chosen staining method as well as their performance. Beside that the ph of the used water and the chosen staining times play an important role. From the great number of the hematological staining methods some have due to their excellent results become widespread. There are the classic staining methods according to Giemsa and May-Grünwald as well as the combination of both (Pappenheim), the staining according to Wright, to Fast Color fast stain. The water quality at the blood smears staining The quality of the staining pictures becomes determined in high grade through the quality of the used water. If the water reacts acid, the eosines-effect of the dye solution becomes raised.

4 reddish shades dominates. If the water is alkaline, it comes to one reinforcement of the azure-metachrome and simultaneously to an extenuation of the eosin-effect so that a bluish-grey shade prevails. Determining is the ph, which should be as neutral as possible, i.e. the ph of the water Should be in the range of 6.8 to 7.2. Optimal reproducible staining pictures are accomplished by the application of buffer solutions according to Weise. According to desired staining results the ph of the used buffer solution is to choose. Storage and documentation of preparations For the documentation of the preparations DdMount low viscosity is highly recommended. As soon as the slide has dried, few drops of DdMount are brought up on the preparation. With a coverslip glass under avoidance of air bubbles the slide is covered. After a drying time of about minutes the smear can examined under the microscope and stored in an archive. The so preserved preparations remain over 10 years colorstable.

5 Pappenheim May-Grünwald Staining rack May-Grünwald solution 3 minutes Buffer solution (1ml) add, mix, stain 3 5 minutes Giemsa solution minutes Rinse with buffer solution Coplin jar Blood smears Air dried May-Grünwald's solution Giemsa solution Wash in buffer solution 3 5 minutes minutes 2 x 1 minutes Staining rack May-Grünwald solution 3 minutes Buffer solution (1ml)add, mix, stain 6 minutes Rinse with buffer solution Coplin jar Blood smears air dried May-Grünwald solution 3 minutes Dilute May-Grünwald's solution 6 minutes Wash in buffer solution 2 x 1 minutes Staining automat May-Grünwald's solution Giemsa solution Wash in buffer solution Tap water 4 minutes 20 minutes 1 minutes 2 minutes 3 minutes Staining automat May-Grünwald's solution Dilute May-Grünwald solution Wash in buffer solution Tap water 3 minutes 6 minutes 1 minutes 2 minutes 3 minutes Nuclei Lymphocytes plasma Monocytes plasma Granulocytes neutrophilic eosinophilic basophilic Thrombocytes Erythrocytes purple to violet light blue dove-violet granules light violet granules brick-red to red-brown granules dark violet to black violet reddish Nuclei Lymphocytes Monocytes Granulocytes: neutrophilic eosinophilic basophilic Thrombocytes violet Erythrocytes red-purple to blueviolet plasma blue plasma grey-blue granules light violet granules brick-red to dark violet granules dark violet to black reddish grey Giemsa Pappenheim dilution 1:20 May-Grünwald dilution 30 ml solution add 150 ml dh 2 O and 20 ml buffer solution

6 Giemsa Wright Staining rack Methanol 3 minutes Giemsa's solution minutes Rinse with buffer solution Coplin jar Blood smears Air dried Methanol 3 5 minutes Giemsa solution minutes Wash in buffer solution 2 x 1 minutes Staining automat Methanol 3 minutes Giemsa solution minutes Wash in buffer solution 1 minutes Tap water 2 minutes 3 minutes Staining rack Wright's solution 1 minutes Buffer solution (1ml) add, mix, stain 4 minutes Rinse with buffer solution Coplin jar Blood smears Air dried Wright's solution 3 minutes Dilute Wright's solution 6 minutes Wash in buffer solution 2 x 1minutes Staining automat Wright's solution 3 minutes Dilute Wright's solution 6 minutes Wash in buffer solution 1 minutes Tap water 2 minutes 3 minutes Nuclei Lymphocytes Monocytes Granulocytes: neutrophilic eosinophilic basophilic Thrombocytes Erythrocytes Blood parasites Giemsa dilution 1:20 purple to violet plasma light blue plasma dove blue granules delicate violet granules red to greyblue granules strong violet violet weak red nuclei bright red Nuclei Lymphocytes red violet to blue-violet plasma light blue plasma dove-blue Monocytes Granulocytes: - neutrophilic: granules light violet - eosinophilic: granules brick-red to red-brown - basophilic: granules dark violet to black Thrombocytes Erythrocytes violet reddish-grey

7 Giemsa (paraffin sections of bone marrow) Deparaffinise and rehydrate the sections Distilled water 10 second Undiluted, Giemsa minutes 0.1 % acetic acid 10 second Distilled water 10 sec. Isopropyl 10 second Isopropyl 10 second Isopropyl 10 second Xylene 5 minutes Xylene 5 minutes Mount with DdMount Cell nuclei, cells blue, dark blue Collagen, osteoid pale blue Eosinophilic grain red Acidophilic mucopolysaccharides, mastocytes, cartilage matrix reddish violet Acidophilic materials orange red Leishman Staining rack Leishman solution 1 minutes Buffer solution (2 ml) add, mix, stain 5 minutes. Dilute 30 ml of Leishman with 150 ml distilled water and add 20 ml buffer solution. Rinse with buffer solution 1 minutes Dry Coplin jar Leishman solution Dilute Leishman solution Rinse with buffer solution Dry Staining automat Leishman solution Dilute Leishman solution Buffer solution Running water (rinse) 3 minutes 3 minutes 6 minutes 2 x 1 minutes 3 minutes 6 minutes 1 minutes 2 minutes Nuclei Lymphocytes Monocytes Neutrophilic granulocytes Eosinophilic granules Basophilic granules Erythrocytes red to violet plasma blue plasma gray-blue granules light violet granules brick-red to red-brown granules dark violet reddish

8 Fast color kit Application. Rapid method for differentiates stain of blood figured elements. The method can be used for staining slides of: cytological, fine needle, cytocentrifuge, malaria, and supplying a chromatic picture of the traditional method May Grünwald Giemsa. Principle. Fast color is a fast-acting variation of May Grünwald Giemsa stain. In hematology, it allows differential staining of blood smears (white blood cell count, morphological erythrocyte analysis and testing of parasites). It is ideal for emergencies and for studying smears in specimens showing as positive on cell counter machine. In cytology, it allows cytological examinations of fluid and of fine-needle punctures. In pathology, it allows emergency examinations of frozen tissue section completed during surgical operation. Stains present in this kit are the same utilized in traditional May Grünwald Giemsa. In this case, the rapidity with which the coloration process is completed is due to the fast dissociation of the tyazinic and others stain contained that they rented extremely rapid the absorption of cellular structures. Auto-Fast color Fixation, staining and rinsing of blood smears can be accomplished with these solutions. The methanolic dye solution also fixes the smears. Staining is achieved by the alkaline dye (azure) binding to the acidic builders of the cells, e.g. chromatin, spongioplasm, and the acidic dye (eosin) binding to the alkaline constituents, e.g. cytoplasm. Sample material Air-dried blood and bone marrow smears. Place the glass slides with the material downwards to the staining strip. Air-dried blood smears are transported in a fully automatic flow system (no immersion system) over the staining strip. Pass a carefully measured, fresh quantity of dye, buffer and rinse solutions, in this order, into the capillary space between the slides and the staining strip. The solutions are transferred to the staining strip by adjustable peristaltic pumps. Method Blood smear Dip the slides 5 times for 1 second into solution 1. Dip the slides 5 times for 1 second into solution 2. Dip the slides 5 times for 1 second into solution 3. Briefly wash in running water to remove excess and dip into alcohol 5-6 time. Malaria (Plasmodium) Dip the slides 3 times for 1 second into solution 1. Dip the slides 2 times for 1 second into solution 2. Dip the slides 2 times for 1 second into solution 3. Briefly wash in running water to remove excess and dip into alcohol 5-6 time. Cytological of fluid and fine-needle punctures Dip the slides 5 times for 1 second into solution 1. Dip the slides 5 times for 1 second into solution 2. Dip the slides 10 times for 1 second into solution 3. Wash carefully in running water to remove excess and dip into alcohol 5-6 time. Histological slides Dip the slides 10 times for 1 second into solution 1. Dip the slides 15 times for 1 second into solution 2. Dip the slides 15 times for 1 second into solution 3. Dehydrate and mount with DdMount. Pneumocystis carinii Cryptosporidia Leishmania Cryptococcus Microfilaria Toxoplasma Dip the slides 10 times for 1 second into solution 1. Dip the slides 10 times for 1 second into solution 2. Dip the slides 10 times for 1 second into solution 3. Briefly wash in running water to remove excess and dip into alcohol 5-6 time.

9 Frozen tissue section Dip the slides 10 second in solution 1. Drain the surplus. Dip the slides 15 second in solution 2. Drain the surplus. Dip the slides 20 second in solution 3. Drain the surplus. Dehydrate and mount with DdMount. Blood cells result: Nuclei: chromatin: purple Leucocytes: cytoplasm with out ARN: light pink eosinophilic granules: orange brown basophilic granules: dark purplish blue neutrophilic granules: purple ± intense Lymphocytes: cytoplasm with ARN: blue cytoplasm with out ARN: light blue azurophilic granules: red Monocytes: cytoplasm: grey-blue Erythrocytes: light red Platelets: chromomer: purplish red hyalomere: bluish Blood parasites: nucleus red (Malaria) cytoplasm blue Please note Staining time may changer according to thickness, and dryness of slides.

10 Blood cell counting Staining of reticulocytes in blood with blu cresy brilliant l Method for the visualization of erythrocyte regeneration by counting of reticulocytes Use for the measurement of substantia granulofilamentosa (ribo nucleo proteins) with fresh, non-fixed, young erythrocytes (supravital staining). Four stages of substantia granulo filamentosa maturation can be distinguished depending on the stage of reticulocyte development: coiled skein (I), incomplete network (II), complete network (III) and granular form (IV). In peripheral blood the development stages III and IV are found most commonly. When stained with brilliant Cresyl blue they display a bluish black network or bluish black dots. Sample material Venous blood, in exceptional cases capillary blood Counting under the microscope Count the reticulocytes per 1,000 erythrocytes with oil immersion under the microscope following a meandering pattern. In order to avoid confusion when counting it is advisable to place a reticulocyte counting grid subdivided into small squares (or a square paper diaphragm) in one of the two eyepieces. In peripheral blood the development stages III and IV are found most commonly. When stained with blue cresyl brilliant they display a dark blue network and dark blue dots. The reticulocyte count is expressed in relation to 1,000 counted erythrocytes (i.e. as 0/00). If the erythrocyte count is low, then the absolute reticulocyte count /μl is used. Single tests Draw 20 μl blood and 20 μl blue cresyl brilliant solution into a haemoglobin pipette and fill into a small sealable container. Mix thoroughly and after about 30 min prepare a thin smear. Tests in series Prepare thin smears of blue cresyl brilliant solution on microscope slides using a glass rod. Air-dried slides prepared in this way can be stored for 2 3 weeks. For reticulocyte counts smear a small drop of blood quickly over the stain layer, and immediately place the still wet preparation in a moist chamber (Petri dish with damp filter paper). Leave for 5 10 minutes and then allow to dry in air. Calculation Reticulocyte count = E/μl x R (0/00) [cells/μl] 1,000 E = erythrocyte count - R = reticulocyte count Normal range Patient 0/00 reticulocyte count/μl Adults ,000 75,000 Newborn babies , ,000

11 Türk solution Reagent for manual leukocyte counting Leukocyte counting is a routine method. The basis of all counting methods is the dilution and preparation of a blood sample of known volume. The erythrocytes are hemolyzed by the acetic acid of Türk solution and the leukocytes are stained by the dye contained. The required cell type in a defined volume is counted and the number of cells per microliter of blood is then calculated. Sample material Anticoagulant venous blood, in exceptional cases capillary blood Preparation Filling the pipette Draw blood into the leukocyte pipette up to the 1.0 mark, and then draw Türk solution up to the 11 mark. The dilution is 1:10. A dilution of 1:20 can also be used (draw blood up to the 0.5 mark and Türk solution up to the 11 mark). Mix blood and Türk solution carefully, leave for maximally 1 h. Filling the counting chamber Discard the first 3 drops and fill the counting chamber. Counting under the microscope Counting is performed with a x 10 objective, in older microscope models the condenser should be lowered and its front lens swung out. Count the leukocytes in the 4 large corner squares, the sides of which each measure 1 mm. It is advisable to determine the counts in duplicate; the results should not differ by more than 15 %. The results are expressed as the means of the duplicate determinations. Calculation Leukocyte count = x (dilution 1:10) 4 Leukocyte count = x 25 [cells/μl] x = total number of cells counted in the 4 corner squares Normal range Patient Leukocyte count/μl Adults 4,000 9,000 School children 5,000 12,000 Small children 6,000 15,000 Infants 7,000 17,000 Newborn babies 10,000 30,000

12 Water quality for wash, rinse and dilution steps. Especially hematological staining methods react very sensitive and visible of changes in the quality and ph range of used rinsing solution. Tap water or distilled water is used normally for wash/rinse steps and for dilution of the staining solutions. Tap water has a ph of around 7 and distilled water has a ph < 7, distilled water which is stored for a while react more acid, the ph is lower. Acid water Eosin Y, an acid dye, reacts more intense when the used water has an acid ph. The staining result is more reddish. Alkaline water methylene blue and the oxidation products give a reinforcement and more intense azure metachromasy and simultaneously an extenuation of the eosin effect, the bluish-grey shade prevails. Phosphate buffer solution. The ph of the water should be in the range of 6.8 to 7.2. Optimal and reproducible staining results are accomplished by the use of buffered solution. The phosphate buffer are especially prepared for thebuffering of water for wash, rinse and dilution steps in hematological staining methods. According to the desired staining result, the ph of the used buffer solution is to choose. Preparation of buffer solution Dissolve the dry buffer pack in 1 L distilled water. It take some time to dissolve. The buffer solution is stable for 4 weeks and should be replaced by a fresh buffer solution after that time. Sample material Fresh and if possible native sample material should be used for the preparation of blood and bone marrow smears. That is the starting material for all types of staining. The use of anticoagulant as EDTA should be reduced to a minimum. Anticoagulants can reduce the stainability of blood and bone marrow samples and it could be critical especially when the material is used for enzyme cytochemical methods. Blood and bone marrow smears must be dried in the air for at least 30 min and fixed then with the relevant fixative and according to the instruction. Hematological staining methods are applicable for clinical specimens in cytology as well. Specimen as urine sediment, sputum, fine needle aspiration biopsies (FNAB), imprints, lavages can be processed with these methods in a very good way and belong to the standard application. Fixation Methanol is the standard fixative for blood and bone marrow samples. Methanol is a solvent with a long tradition as fixative in hematology. It reacts fast and is inert e.g. no changes of fine structures will be recognized when the fixation is done with sufficient air dried material. The used methanol should have a concentration of 100 %. A sufficient quality grade should be used to prevent problems with the stainability and the quality of the sample material. Methanol is the gold standard as hematological fixative but it is under discussion because of the strong hazardous classification of the solvent. Methanol has to be used in well ventilated and air-conditioned working places. Safety protection for the staff and the working place must be organized according to the safety instructions.