Bone Marrow Examination PDF

CHAPTER 7 PREPARATION, STAINING AND EXAMINATION OF BONE MARROW SMEARS 1 Objectives At the end of this chapter the student would be able to:  Indicate the conditions for which bone marrow examination is indicated  Indicate the sites of bone marrow aspiration...

CHAPTER 7 PREPARATION, STAINING AND EXAMINATION OF BONE MARROW SMEARS 1 Objectives At the end of this chapter the student would be able to:  Indicate the conditions for which bone marrow examination is indicated  Indicate the sites of bone marrow aspiration in the different age groups  Discuss the techniques for preparation and examination of bone marrow smears  Discuss bone marrow cellularity  Discuss myeloid to erythroid ratio 2  Introduction  Bone marrow examination – an indispensable aspect of the hematological investigation and diagnosis  Marrow examination is necessary for: Discovering or confirming a variety of diagnoses The monitoring of Hematologic and non hematologic diseases (solid tumors and leukemic patients undergoing intensive chemotherapy  Cytological and histological examination are the major aspects of bone marrow investigation although in recent years: Bone marrow culture for cytogenetic and kinetic studies Isotopic labeling Processing for electron microscopy Clonal studies Culture and other methods have become established  Bone Marrow Specimens  Samples of bone marrow can be obtained by: Aspiration using a special needle and syringe, e.g., Salah, Klima, and Islam’s aspiration needles Percutaneous trephine in which a section of bone is taken for examination Open surgical biopsy or open trephine that requires full operating theatre practice  Most bone marrow samples for hematological purposes are obtained by aspiration often combined with needle or trephine biopsy The aspiration procedure is simple, safe and relatively painless 4 Aspiration Biopsy  The site selected for the aspiration depends on: The age of the patient Whether or not a needle or trephine biopsy is required  Adults - active marrow is normally confined to the central skeleton and the convenient sites are: The sternum The best site when aspiration only is needed The easiest to puncture Considered to yield the most cellular samples  A disadvantage is that the patient has a clear view of the procedure which may cause distress Anterior or posterior iliac spines Have the advantage that if no material is aspirated, a micro trephine biopsy can be performed immediately 5 Bone Marrow Specimens cont’d A needle used for bone marrow aspiration, with removable stylet 6 Aspiration Biopsy cont’d Bone marrow biopsy from the superior part of the posterior iliac spine (back of the hipbone) 7  Aspiration Biopsy cont’d  Infants and children: the sternum is naturally thin and an alternative site is preferred Under 12 years – iliac crest Under 2 years – the presence of active marrow in the long bones makes the proximal anterior portion of the tibia a possible site  In disorders associated with replacement of hemopoietic marrow by other tissues or cells (e.g., malignancies in the bone marrow) Marrow aspiration may be difficult or impossible, the so-called ‘dry tap’  In such cases, a needle or trephine biopsy is essential 8  Aspiration Biopsy cont’d  A minimum amount of marrow should be aspirated  Volumes over 0.5ml will almost certainly be diluted with blood making processing and interpretation more difficult  Careful preparation is essential  It is desirable, if possible, to concentrate the marrow cells at the expense of the blood in which they are diluted 9 Bone marrow Films Method 1. Deliver single drops of aspirate on to slides about 1cm from one end and then quickly suck off most of the blood with a fine Pasteur pipette applied to the edge of each drop.  Alternatively, place the slides on a slop to allow the blood to drain away  The irregularly shaped marrow fragments tend to adhere to the slide and most of them will be left behind 10  Bone marrow Films cont’d 2. Make films 3-5 cm in length, of the marrow fragments and the remaining blood using a smooth-edged glass spreader of not more than 2cm in width  The marrow fragments are dragged behind the spreader and leave a trail of cells behind them  It is in these cellular trails that the differential counts be made commencing from the marrow fragments and working back towards the head of the film; in this way, smaller numbers of cells from the peripheral blood become incorporated in the differential count The preparation can be considered satisfactory only when marrow particles as well as free marrow cells can be seen in stained films. 11 Bone marrow Films cont’d 3. Fix the films of bone marrow and stain them with Romanowsky dyes as for peripheral films  However, a longer fixation time (at least 20 minutes in methanol) is essential for high quality staining  The staining time should also be increased if the marrow is hyper cellular 12 Bone marrow Films cont’d Film of aspirated bone marrow. The marrow particles are easily visible, mostly at the tail of the film 13 "Particle/Crush Smears"  Aspirated marrow particles are isolated crush preparations made by gentle pressure of a second slide combined with the sliding apart of the two slides either in one movement or by a series of interrupted movements  Technique gives preparations of authentic marrow cells  Squashing and smearing out the particles causes disruption and distortion of cells The resultant thick preparations are difficult to stain well 14 "Particle/Crush Smears" cont’d Squash preparation and meandering smear for the cytological analysis of bone marrow 15 Examination and Assessment of Stained Bone marrow Preparations  The first thing to do is to look with the naked eye at a selection of slides and to choose from them the best spread films containing easily visible marrow particles  The particles should then be examined with a low power objective with particular reference to their: cellularity and an estimate of whether the marrow is:  Hypoplastic/hypocellular  Normoplastic/normocellular Hyperplastic/hypercellular 16  Cellularity of Bone marrow  It is expressed as the ratio of the volume of hemopoietic cells to the total volume of marrow  It is judged by comparing the areas occupied by fat spaces and by nucleated cells in the particles  Normal marrow is normocellular or normoplastic  Cellularity varies with the age of the subject and the site from which the bone marrow is taken E.g., Age 50 years: vertebrae = 75% sternum = 60% iliac crest = 50% 17 Cellularity of Bone marrow cont’d  If the percentage is increased for the age of the patient  The marrow is said to be hypercellular or hyperplasic  Such hyper cellular marrow is seen in:  Myeloproliferative disorders (e.g., CGL, AML)  Lymphoproliferative disorders (e.g., ALL, CLL)  Infections Polycythemia 18 Cellularity of Bone marrow cont’d NORMAL/NORMOCELLULAR MARROW BIOPSY 19 Cellularity of Bone marrow cont’d Hypocellular marrow Aplastic marrow 20 Cellularity of Bone marrow cont’d Hypercellular marrow 21 Cellularity of Bone marrow cont’d  If the percentage is decreased for the age of the patient  The marrow is said to be hypo cellular or hypo plastic  It is a finding in conditions associated with marrow failure  Aplastic anemia  Toxicity (drugs, chemicals) 22 Myeloid to Erythroid Ratio (M:E)  It is an expression of the myeloid and erythroid compartments relative to each other  It is calculated after classifying at least 200 cells (leucocytes of all types and stages of maturation are counted together)  In normal adult bone marrow, the myeloid cells always outnumber the erythroid cells with a mean value of 4:1  An increased M:E ratio shows:  An increase in the number of leucocytes, and  Depression of the erythroid series  A decrease in the ratio shows: The presence of erythroid hyperplasia and suppression of granulocytes 23  Differential Count on Aspirated Bone marrow: The Myelogram  Expression of the incidence of the various cell types as percentages is not a mandatory part of bone marrow examination because of: The relatively long time required to perform the count Little clinical usefulness of such an effort  The count is also unreliable due to: Irregular distribution of the marrow cells, and Inclusion of cells from the peripheral blood for which there is no compensation  If at all to be attempted, a minimum of 200 cells should be studied 24 Differential Count on… cont’d  Because of the naturally variegated pattern of the bone marrow and the irregular distribution of the marrow cells: Differential counts on marrow from normal subjects vary widely Minor degrees of deviation from the normal occurring in disease are difficult to establish 25 Marrow Differential Counts in Adults Myeloblasts: 0.0-3.5% Promyelocytes: 0.0- 6.0% Myelocytes: 8.0-15.0% Metamyelocytes: 9.0-25.0% Band and Segmented: 15.0-27% Neutrophilic: 7.0-25.0% Eosinophilic: 0.0-4.0% Basophlic: 0.0-1.0% Pronormoblasts: 0.0-3.0% Basophilic normoblasts: 1.0-5.0% Polychromatophilc normoblasts: 5.0-20.0% Orthochromatic normoblasts: 1.0-15.0% Lymphocytes + Precurssors: 3.0-20.0% Plasmacytes + Precurssors: 0.0-3.5% Monocytes + Precurssors:0.0-2.0% M:E Ratio: 1.5-3.5% 26 Review Questions 1. Indicate the sites of bone marrow aspiration in adults, children under 12 years of age and children under 2 years of age. 2. What elements of the stained bone marrow architecture are mainly assessed in bone marrow examination? 27