Blood and Bone Marrow Staining PDF

Blood and the bone marrow Laura Ahmed The blood film Made using a drop of blood Slide is air dried Blood is fixed using methanol Stained to allow features o...

Blood and the bone marrow Laura Ahmed The blood film Made using a drop of blood Slide is air dried Blood is fixed using methanol Stained to allow features of cells to be seen most common stain in UK is May- Grunwald Giemsa  Dacie and Lewis, Practical haematology 12th edition Dacie and Lewis, Practical haematology 12th edition Staining technique Fix in methanol for 5 minutes For bone marrow- fix for 15-20 minutes Stain in May-Grunwald diluted 50:50 in buffered water pH 6.8 for 5 minutes Stain in Giemsa stain diluted in 1:10 buffered water pH 6.8 for 15 minutes Stand in buffered water (pH 6.8) for 5 minutes Examination of the blood smear Oil immersion microscopy Examine area of film where red blood cells are just touching Work along the slide in battlement formation either side to side or to thick part and back Right area Red blood cells just touching – allows detail to be seen Too thick Can’t see detail in the cells Too thin Red blood cells become circles (spherocytic) Red cell morphology Hypochromic cells Polychromasia Anisocytosis White cell morphology Lymphocytes Neutrophil Monocyte Eosinophil Basophil Metamyelocyte Myelocyte Promyelocyte Myeloblast Bone marrow Normal bone marrow Myeloid to erythroid ratio 3:1 (varies from 2:1 to 4:1) Cellular and fat component Even distribution All cell types present, mature and immature  95% range Mean Mean Myeloblasts 0-3 1.4 0.4 Promyelocytes 3-12 7.8 13.7 Myelocytes (neutrophil) 2-13 7.6 - Metamyelocytes 2-6 4.1 - Neutrophils 22-46 32.1M , 37.4w 35.5 Myelocytes (eosinophil) 0-3 1.3 1.6 Eosinophils 0.3-4 2.2 1.7 Basophils 0-0.5 0.1 0.2 Lymphocytes 5-20 13.1 16.1 Monocytes 0-3 1.3 2.5 Plasma cells 0-3.5 0.6 1.9 Erythroblasts 5-35 28.1M, 22.5W 23.5 Megakaryocytes 0-2 0.5 Macrophages 0-2 0.4 2.0 Dacie and Lewis Practical Haematology, 4th edition, pp122 Required samples FBC at same time Trephine biopsy not always required Aspirate Number of slides depends on clinician, usually at least 4 – one or two for MGG, spares for other staining i.e. cytochemistry EDTA sample for cytogenetics (usually 2x5 ml) The bone marrow – trephine biopsies Fixation – 10% formal saline up to 48 hours Decalcification Method laboratory dependent EDTA Staining H+E Iron stain – Perls’ reaction Immunohistochemistry Reticulin staining (silver impregnation) Bone marrow collection  http://www.pathpedia.com/education/eatlas/histology/bone_marro w/Images.aspx?13 http://www.pathpedia.com/education/eatlas/histopathology/bone_ marrow.aspx