Bone Marrow Examination PDF

17 Bone Marrow Examination Q. Enumerate the indications and describe the findings in bone marrow aspiration and biopsy. (PA17.2) ===================================================================================================== **Ans.** Bone marrow examination is the pathologic examination of...

17 Bone Marrow Examination Q. Enumerate the indications and describe the findings in bone marrow aspiration and biopsy. (PA17.2) ===================================================================================================== **Ans.** Bone marrow examination is the pathologic examination of a sample of bone marrow, which is indicated when a disease cannot be diagnosed and classified on the basis of the blood counts and peripheral blood smear examination alone. In some cases, examination of the bone marrow is required for monitoring the course of therapy, for example in leukaemia; performing special studies, such as cytogenetics and microbiological analysis or flow cytometry. Bone marrow can be obtained through: i. Bone marrow aspiration i. Bone biopsy **Indications for bone marrow aspiration** - Evaluation of unexplained leukocytopenia, thrombocytopenia, and/or anaemia. - Diagnosis of aplastic anaemia, myelodysplastic syndrome, chronic myelogenous leukaemia, myelofibrosis and essential thrombocythaemia. - Evaluation of monoclonal hypergammaglobulinaemia. - Diagnosis and evaluation of idiopathic thrombocytopenic purpura. - Diagnosis and management of acute leukaemia and sub-leukaemic leukaemia. - Staging of lymphomas. - Evaluation of pyrexia of unknown origin, unexplained lymphadenopathy and hepatosplenomegaly. - Diagnosis of infectious diseases, such as leishmaniasis, disseminated fungal disease or HIV. A bone marrow biopsy and aspiration may also be ordered at intervals when a person is being treated for a cancer, to evaluate whether marrow function is being suppressed and, if it is, when its function begins to recover. **Contraindications for bone marrow aspiration:** Absolute contraindications to bone marrow aspiration or biopsy include haemophilia and related coagulation disorders. If there is a skin or soft tissue infection over the hip, a different site should be chosen for bone marrow examination. Bone marrow aspiration and biopsy can be safely performed even in the setting of thrombocytopenia. **Sites for bone marrow aspiration** i. The **sternum** is considered a good site for aspiration because in this site the cortical bone is thin and the marrow contains abundant cells and little fat. However, one has to be careful that the needle is correctly inserted in the sternum, or else, there is a danger of perforating the inner cortical layer and damaging the underlying large blood vessels and right atrium, with serious consequences. Penetration of the underlying mediastinal organs can result in mediastinitis, pulmonary embolism, pneumothorax, cardiac tamponade and cardiac tissue injury, and for these reasons, biopsies are not to be performed from the sternum. Also, this site should not be used in children as the bone is very thin and the marrow cavities are small. i. The **posterior iliac crest** is optimal for reasons of safety and ease of performance. The **iliac spines** have the advantage that if no material is aspirated, a trephine biopsy can be performed immediately. These sites, however, are difficult to aspirate from in patients with morbid obesity, in which case puncture of the sternum may be necessary. Performance of bilateral iliac biopsies is thought to increase the probability of detecting focal lesions when there is patchy bone involvement, as in carcinoma and lymphoma staging. With the availability of positron emission tomography (PET) for staging of lymphomas and the current inclusion of PET-positive bone disease as indicative of bone involvement in lymphoma, bilateral bone marrow biopsies are rarely done. i. **Spinous processes of the lumbar vertebrae** may also occasionally be used for aspiration. i. In infants and children younger than 2 years the medial side of the upper end of **tibia** is considered the site of choice. **Aspiration technique** - Consent for the procedure is obtained. - The patient is positioned as follows: - Lateral decubitus position, knees flexed, and pillow under head, for posterior iliac crest aspiration. - Supine position hips and knees flexed, for anterior iliac crest aspiration. - Supine position, for a sternal puncture. - The skin over the chosen site is sterilized with 70% alcohol or 0.5% chlorhexidine. - The skin, subcutaneous tissue and periosteum overlying the selected site is then infiltrated with a local anaesthetic such as 2-5 mL 2% lignocaine. - A skin incision is made with a small surgical blade, through which the bone marrow aspiration needle, with a stylet locked in place, is inserted. The most common reusable needles are the **Salah** and **Klima** needles (Fig. 17.1). The needle is passed perpendicularly by slowly rotating clockwise and counter clockwise into the cavity of the ilium at the centre of the oval posterior superior iliac spine or 2 cm posterior and 2 cm inferior to the anterior superior iliac spine or second intercostal space in the midline for sternal aspiration. When the needle is firmly in place and a slight give in pressure is felt, the cavity has been entered. - After penetrating the bone, the stylet is removed, a 1 or 2 mL syringe is attached, and marrow contents are sucked up for making films. - If a larger sample is needed (e.g. for cytogenetic or immunophenotypic analysis), a second 5 or 10 mL syringe is attached and a second sample is aspirated. **Preparation and interpretation of smears** - The marrow is poured onto slides placed at an angle of 30 degree so that the blood present in the marrow can be drained off. Films should be made very quickly from the aspirated material, as bone marrow tends to clot faster than blood. - By using a smooth-edged glass spreader, smears are made of the aspirated marrow, by dragging the marrow fragments behind the spreader. Most of the blood is quickly sucked off from the edge of the drop with the marrow syringe or a fine plastic pipette. - The remaining material can be stored in EDTA and used later to make more films. Heparin should be used for **immunophenotyping** or **cytogenetic studies**. Formalin for a Cytoblock preparation (clot). - The smears can be fixed in absolute methanol as soon as they are dry, for subsequent staining by **Romanowsky, Perls** or **cytochemical stains.** - The preparation is considered satisfactory when marrow particles and free marrow cells can be seen in stained films. The differential counts should be made in the cellular trails, commencing from the marrow fragment and working back towards the head of the film. - A well-spread film with easily identifiable particles is picked. Several particles are examined with a low-power (×10) objective to estimate the cellularity of the marrow (whether the marrow is hypocellular, normocellular or hypercellular). A cellular area of the film where the nucleated cells are well stained and well spread is chosen and the cells in this area are then examined under higher power (×40). - At least 500 cells are counted and percentages of different cells are calculated. The **myeloid:erythroid** (M:E) ratio (proportion of myeloid and erythroid cells) is determined; normal M:E ratio varies between 2:1 and 4:1. - **Maturation of erythroid cells** is classified as normoblastic, megaloblastic or dyserythropoietic. - **Leukocytic maturation** is looked at and **megakaryocytic number, maturation sequence** and **morphology** are noted. - Attempt is made to identify **abnormal cells** (as seen in metastatic carcinomas, lymphomas and metabolic disorders). - **Macrophages** are examined at higher power for evidence of infection (presence of malarial pigment, parasites, bacteria or fungi). - Areas of **bone marrow necrosis** are looked for. Bone marrow necrosis may occur in sickle cell disease, lymphomas, leukaemia, myeloproliferative diseases, metastatic carcinoma, septicaemia and tuberculosis. - **Iron content** of macrophages is assessed by Perls stain. At least seven particles should be examined to optimally assess a bone marrow aspirate for iron stores. - A sample for **bone marrow histology** should ideally be obtained at the same time as bone marrow aspiration, particularly in the case of a \'dry tap\'. A dry tap or failure to aspirate marrow suggests bone marrow fibrosis or infiltration. **Cell counts on aspirated bone marrow** - For practical purposes, the degree of marrow cellularity can be assessed as increased, normal, or reduced by inspection of a stained film containing marrow particles. As a rough guide, if less than 25% of the particle is occupied by haemopoietic cells, it is hypocellular, and if more than 75%-80% is occupied, it is hypercellular. - A 200-500 cell differential can be undertaken and the cells can be classified as erythroid, myeloid, lymphoid and plasma cells. - Sometimes it may be important to specifically count one cell type (e.g. blasts in acute leukaemia for assessing response to chemotherapy). **Sources of error in cell counts** - **Age of the patient:** The cellularity of the marrow is affected by age. In adults, a smaller proportion of the marrow cavity is occupied by haemopoietic marrow compared with children, and the proportion of fat cells to cellular marrow is increased. - **Distribution of the marrow cells:** Irregular distribution of the marrow cells in films is an established source of error. For example, megakaryocytes are irregularly distributed in marrow preparations and tend to be carried to the tail of the film. Rarely, aspiration of a lymphoid follicle can result in an abnormally high percentage of lymphocytes in the differential count. - **Dilution with blood:** This can be overcome by aspirating only a small volume and counting cells in the trails left behind marrow particles as they are spread on the slide. - **Marrow fibrosis:** In the presence of increased reticulin, some cell types resist aspiration or remain embedded in marrow fragments and may not be adequately represented in the differential count. **Complications of bone marrow aspiration** Albeit extremely rare, complications from bone marrow aspiration and biopsy include: i. Infection and bleeding. i. Even rarer complications include iliac bone perforation and haemorrhage, usually due to poor technique and positioning of the patient. i. Cardiac tamponade can occur during aspiration of sternum. **Bone marrow trephine biopsy** Bone marrow biopsy provides a core of bone with marrow spaces and is usually obtained after marrow aspiration. The sites are the same as those for marrow aspiration (except the sternum). Needles used for this purpose include **Jamshidi** (most common)**, Westerman Jensen** or **Islam**. The tissue obtained is rolled between two slides to make **imprint smears**. The biopsy is then decalcified and processed. Sections of the marrow are cut and stained with **haematoxylin** and **eosin, reticulin** and **cytochemical** stains. **Trephine biopsies of the bone marrow are indicated in the following conditions:** i. Conditions that yield a \'dry tap\' on bone marrow aspiration (e.g. myelofibrosis, infiltrations). i. In conditions in which analysis of the architecture of the marrow is important (e.g. Hodgkin disease, lymphoma). i. To detect micrometastasis in disseminated cancer. i. To diagnose metabolic bone disease. Fig. 17.1 Bone marrow aspiration needles.