Stains. for blood and bone marrow. Overview of the classic staining methods, fast stain and foil staining. Focussing your hematology targets.

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1 Stains for blood and bone marrow Overview of the classic staining methods, fast stain and foil staining Focussing your hematology targets.

2 Advantages of the hematological staining methods from Merck 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 For the staining of blood and bone marrow smears are 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, the Hemacolor fast stain as well as the foil staining without dye solutions with Sangodiff. The water quality at the blood smear 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. Reddish shades dominates. If the water is alkaline, it comes to one reinforcement of the azure-metachromasie 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 the mounting medium Entellan new is highly recommended. As soon as the slide has dried, few drops of Entellan new 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.

4 Pappenheim May-Grünwald Giemsa 2. Dilute Giemsa's solution (1:20): Add 10 ml Giemsa's solution to 190 ml buffer solution (ph 6.8 or 7.2), mix well, allow to stand 10 minutes and filter if necessary. Add 15 ml Giemsa's solution to 220 ml distilled water and 65 ml buffer solution (ph 6.8 or 7.2) mix well, allow to stand for 10 minutes and filter if necessary. 2. Dilute : Dilute 30 ml with 150 ml distilled water and 20 ml buffer solution. Add 30ml buffer solution and 220 ml distilled water to 50 ml May-Grünwald's solution, mix and allow to stand for 10 minutes before using. 2. Dilute Giemsa's solution (1:20): Add 10 ml Giemsa's solution to 190 ml buffer solution (ph 6.8 or 7.2), mix well, allow to stand 10 minutes and filter if necessary. Slowly add 25 ml Giemsa solution to 275 ml buffer (ph 6.8 or 7.2) mix well, allow to stand for 10 minutes and filter if necessary. Methanol Buffer solution (1ml) add, mix, stain Dilute Giemsa's solution 3 5 min min Buffer solution (1ml) add, mix, stain Dilute Giemsa's solution Methanol Dilute Giemsa solution min 3 5 min min Dilute Giemsa's solution 3 5 min min Dilute May-Grünwald's solution 2 x 2 x 2 x Methanol 4 min Dilute Giemsa solution min Dilute Giemsa solution 20 min Dilute May-Grünwald's solution Lymphocytes: plasma light blue Monocytes: plasma dove- - neutrophilic: granules light to red-brown, - basophilic: granules dark to black Erythrocytes: reddish Lymphocytes: Monocytes: red-purple to blue- plasma blue plasma grey-blue - neutrophilic: granules light to dark to black Thrombocytes: Erythrocytes: reddish grey Lymphocytes: plasma light blue Monocytes: plasma dove blue - neutrophilic: granules delicate - eosinophilic: granules red to grey-blue - basophilic: granules strong Erythrocytes: weak red Blood parasites: nulei bright red

5 Wright Leishman Hemacolor 2. Dilute Wright's solution: Dilute 30 ml Wright's solution with 150 ml distilled water and 20 ml buffer solution. Add 30 ml buffer solution and 220 ml distilled water to 50 ml Wright's solution. 2. Dilute Leishman's solution: Dilute 30 ml Leishman's solution with 150 ml distilled water and 20 ml buffer solution. Add 30 ml buffer solution and 220 ml distilled water to 50 ml Leishman's solution. Buffer solution ph 7.2: Wright's solution Leishman's solution 1min Buffer solution (1ml) add, mix, stain 4 min Buffer solution (1ml) add, mix, stain 5 min Hemacolor solution 1 (fixing solution) 5 x 1 sec Hemacolor solution 2 (colour reagent red) Hemacolor solution 3 (colour reagent blue) 3 x 1 sec 6 x 1 sec Wright's solution Leishman's solution 2x10 sec Dilute Wright's solution Wright's solution 2 x 1min Dilute Leishman's solution Leishman's solution 2 x 1min Hemacolor solution 1 Hemacolor solution 2 Hemacolor solution 3 30 sec 3 sec 6 sec Dilute Wright's solution Dilute Leishmany's solution 10 sec 1min 10 sec Lymphocytes: Monocytes: red to blue- plasma light blue plasma dove-blue - neutrophilic: granules light to red-brown to black Erythrocytes: reddish-grey Lymphocytes: plasma blue Monocytes: plasma grey-blue - neutrophilic: granules Erythrocytes: red Lymphocytes: plasma light blue Monocytes: plasma dove blue - neutrophilic: granules light to red-brown Erythrocytes: reddish

6 Sangodiff Auto-Hemacolor Buffer solution ph 6.8 (7.2): The kit is for automatic blood smear staining on Hema-Tek * Slide Stainer. Use the solutions contained in the set to fix, stain and wash blood smears. * Trademark of Ames Company, Division Miles Laboratories Inc., Elkat, Indiana, USA Staining foils Methanol Add 1 drops buffer solution and cover with stain foil Remove foil Wash with buffer solution 10 sec 10 min 1- Lymphocytes: plasma light blue Monocytes: plasma dove blue - neutrophilic: granules light to red-brown Erythrocytes: reddish Lymphocytes: plasma blue Monocytes: plasma grey-blue - neutrophilic: granules Erythrocytes: red

7 Ordering information Method Product Merck Cat. No. Package size Pappenheim May-Grünwald's ml, 500 ml, 1 l, 2.5 l eosin methylene blue solution Giemsa's ml, 500 ml, 1 l, 2.5 l azure eosin methylene blue solution May-Grünwald May-Grünwald's ml, 500 ml, 1 l, 2.5 l eosin methylene blue solution Giemsa Giemsa's ml, 500 ml, 1 l, 2.5 l azure eosin methylene blue solution Wright Wright's ml, 500 ml, 2.5 l eosin methylene blue solution Leishman Leishman's ml eosin methylene blue solution Hemacolor Hemacolor staining set ml of each solution 1, 2 and 3 for blood smears 3 buffer tablets ph 7.2 Hemacolor staining set ml of each solution 1, 2 and 3 for blood smears 6 buffer tablets ph 7.2 Hemacolor -solution l fixing solution Hemacolor -solution l colour reagent red Hemacolor -solution l colour reagent blue Sangodiff Sangodiff staining foils staining foils, for manual blood smear staining buffer solution ph 6.0 Auto-Hemacolor Auto-Hemacolor staining set ml stains, for automatic blood smear staining 500 ml buffer solution and 1 l wash solution Additional reagents Application Product Merck Cat. No. Package size Waterquality Weise buffer tablets ph tablets for preparing buffer solution acc. to Weise Weise buffer tablets ph tablets for preparing buffer solution acc. to Weise Weise buffer tablets ph tablets for preparing buffer solution acc. to Weise Fixation Methanol GR f.a. ACS, ISO, Reag. Ph Eur l, 2.5 l Mounting media Entellan new ml, 500 ml Entellan ml Neo-Mount ml Immersion oil Immersion oil ml, 500 ml Immersion oil acc. to ISO 8036 mod ml

8 Further information: Merck KGaA Darmstadt, Germany Phone +49 (0) 6151/72-0 Fax +49 (0) 6151/ W /07 We provide information and advice to our customers on application technologies and regulatory matters to the best of our knowledge and ability, but without obligation or liability. Existing laws and regulations are to be observed in all cases by our customers. This also applies in respect to any rights of third parties. Our information and advice do not relieve our customers of their own responsibility for checking the suitability of our products for the envisaged purpose.