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Manual of Bone Marrow Examination
By Anwarul Islam Trafford Publishing
Copyright © 2013 Anwarul Islam, MD, PhD. FRCPath, FACP
All rights reserved.
ISBN: 978-1-4669-1615-9
CHAPTER 1
INTRODUCTION
INTRODUCTION TO BONE MARROW AND ITS EXAMINATION
Bone marrow is housed within the inner fixed confines of bone and is the hematopoietic organ responsible for the production of the blood cellular elements that perform vital functions of oxygen transport, protection against bacterial and viral pathogens, control of inflammatory responses, and participation in endothelial repair as well as clot formation. The importance of bone marrow examination in any hematological disorder cannot be overemphasized. In its absence the investigation of any hematopoietic disorder, unless otherwise well defined, documented and prognostically evaluated, is incomplete. Even in many cases where the diagnosis is clinically and pathologically established an examination of the bone marrow remains an integral part of the practice of effective, scientific, hematologic medicine.
CYTOLOGIC AND HISTOLOGIC ANALYSIS
There are two methods available for diagnostic access to the bone marrow, cytologic and histologic. In the former approach, i.e. bone marrow aspiration, a sample of marrow is withdrawn from a bone via an aspirating needle and a syringe delivering a mixture of free hematopoietic cells, small aggregates or clusters of marrow cells and fat (often termed bone marrow particles, fragments or units), and a variable amount of sinusoidal blood. This material is utilized to prepare dry film smears which are typically stained with a Romanowsky type dye (Leishman, Giemsa etc.). Conversely in the histologic analysis of bone marrow, a biopsy is obtained which provides an undisturbed segment of marrow tissue with its cellular, vascular and osseous in situ relationships intact. This tissue is then fixed in a suitable fixative and prepared for paraffin or plastic embedding, sectioning, staining, and subsequent analysis.
ACCESS TO BONE MARROW
Various sites are available for the access of hematopoietic bone marrow in man. Satisfactory samples can be routinely aspirated from the sternum, the iliac crest(s) in the region of the anterior or posterior iliac spines and the spinous processes of the lumbar vertebrae (Figure 1). A bone marrow aspirate sample can also be obtained from ribs, vertebral bodies or any other bones which show radiologic or other evidence of osseous lesion. The region of the tibial proximal epiphysis is an excellent safe site for sampling bone marrow in children but is not appropriate for adults because of the replacement of its red marrow with adipose cells (inactive yellow marrow). Sternal puncture has historically been the most commonly used technique of aspirating bone marrow. In recent years, however, due to the availability of improved, more durable bone marrow aspiration instruments the region of the posterior iliac spine has become more frequently the site of choice. Bone marrow histologic (solid tissue) biopsies, often termed needle or core biopsies, are usually performed at the anterior or posterior iliac crests. Again, as a result of technological improvements as well as ease of access the posterior locus is becoming more frequently utilized.
QUANTITATIVE REQUIREMENTS FOR ASPIRATION AND SECTIONED TISSUE SAMPLING
For routine bone marrow cytology the amount of marrow required to be aspirated is minimal. Usually 0.3-0.5 ml is sufficient to prepare several dry film smears. Small volumes are also cited as advantageous because this prevents a dilution of the hematopoietic cells with circulating blood and its cellular contents. Larger volumes may be necessary when cytogenetic and and/or fluorescent flow cytometric analyses are required. For solid tissue (needle) biopsies a 15-20 millimeter-long core of marrow tissue should be obtained to insure the retrieval of adequate, histlogically representative, undisturbed specimens. Sections of variable, limited usefulness are also sometimes obtainable by collecting excess bone marrow units from an aspirate, allowing them to aggregate in a plasma/thromboplastin clot and submitting this mass to the embedding and sectioning process.
CHAPTER 2
BONE MARROW ASPIRATION
STERNAL PUNCTURE
Introduction
In 1929, Mikhail Innokent'evich Arinkin (Figure 2) a Russian physician first introduced the technique of bone marrow aspiration from the sternum when he used a lumbar puncture (spinal tap) needle to obtain a marrow aspirate from this site. Since then sternal puncture has become one of the most common intraosseous diagnostic procedures used in the field of hematology. Although the structure in adult humans which yields the largest quantity of bone marrow is the posterior ilium, the sternum has remained one of the most commonly used sites for obtaining aspirates for hematological diagnosis. This preferential selection may be due to the fact that this locus of hematopoietic marrow is near the surface of the body and is easily accessible.
Site
Sternal puncture is normally performed in the upper part of the body of the sternum, below the sternal angle of Louis and opposite the second intercostal space, midway between the midsternal line and the right or left sternal border (Figure 3). This particular site is chosen because the needle enters one of the previous centers of ossification where the red marrow is usually more abundant than in the midsternal line. The sternum is most stable at this level and least likely to move or fracture under applied pressure. This area is also separated from the underlying great vessels by a distance of 2-3 cm, while at the levels of third and fourth intercostal spaces, the sternum lies in close proximity to the pericardium and heart.
The manubrium of the sternum can also be used as a site for obtaining bone marrow aspirates and is perhaps the safest area of this bone to puncture. But as a rule, particularly in elderly subjects, the manubrium contains more adipose cells than the sternal body and as a result an aspiration at this site may yield an inadequate or non-representative marrow sample. However, completely satisfactory samples are obtained more often than not from the manubrium. If the manubrium is selected for aspiration, the appropriate site for the puncture is about 1 cm above the sterno-manubrial angle and slightly lateral to the mid-line.
Instrumentation
The needles most commonly used for obtaining bone marrow aspirate samples from the sternum are the Salah and Klima needles (Figure 4) or their modifications.
They were designed in the 1930's and except for the introduction of various kinds of stops and guards there has been very little change over the years in their basic construction and design. These instruments are small, do not conveniently fit the operator's hand, and the lack of a T-bar handle in most instances often makes them difficult to maneuver during the sternal puncture procedure. The recently introduced Islam sternal puncture needle (Figure 5) overcomes these disadvantages. The domed handle of its stilette rests comfortably in the operator's hand while the T-bar handle provides a firm grip and precise control of the needle movement. An important feature of the Islam sternal puncture needle is the short length of the penetrating segment which is almost half the length of a conventional sternal puncture needle (Figure 6).
This reduces the possibility of accidental penetration of the inner cortex (posterior wall) of the sternum and injuring the great blood vessels that lie underneath (Figure 7). Further it also has an adjustable guard to prevent accidental over penetration and a sloped stop at its expanded proximal end for easy fitting and withdrawal of the stilette.
The Islam Sternal Puncture Needle
The instrument
The instrument (Figure 8) consists of three parts. The standard size needle has an overall length of 45 mm, a uniform external diameter of 2.0 mm and a constant internal diameter of 1.25 mm except for the 2-3 mm distal portion where it is beveled to produce a tip (cutting edge) very similar to hypodermic needles but much shorter in configuration. The proximal end of the needle has been fitted with a large metal bar specifically shaped to insure a firm grip and a standard female luer-lock to receive the nozzle of a syringe and to fit the male luer-lock of the stilette.
The female luer-lock of the needle also has a sloped stop for easy positioning and resting of the metal stud attached to the male luer-lock of the stilette. This arrangement makes it possible to automatically position the cutting edge of the stilette and needle in the same plane. This design also facilitates the easy unlocking and withdrawal of the stilette (by gentle anti-clockwise rotary motion) during the sternal puncture procedure. As has been mentioned the tip of the needle has been specially designed to make it short (Figure 6) considering the narrowness of the space between the inner and outer plates of the sternum but sharp enough to easily penetrate the overlying soft tissue and bony cortex. The overall length (from the tip to the base, Figure 6) of the penetrating portion of the needle is approximately one half of a conventional needle, thus reducing the fear of accidental penetration of the inner plate of the sternum and injuring the pericardium or heart. The stilette is a solid shaft of 1.24 mm in diameter except for the distal portion where it ends in a 2-3 mm beveled tip to fit the beveled tip of the needle to provide means of easy penetration of the soft tissue and bony cortex. The proximal end of the stilette has been fitted with a male luer-lock mounted on the inner side of the dome handle to fit the female luer-lock of the needle. It also has a metal stud which fits the sloped stop at the proximal end of the needle and help automatically align the penetrating end of the stilette and needle in the same plane. The proximal end of the stilette is capped with a smooth dome-shaped solid nylon handle 25 mm in diameter and 15 mm deep with 5 mm lightly milled edge. It rests snugly in the operator's hand and the two together (the dome and the T-bar handle) provides a uniquely designed instrument to carry out the sternal puncture procedure with efficiency. The metal guard is adjustable and can be fixed at any point over the needle by tightening the screw. The adjustable guard is provided mainly to control the depth of penetration during the sternal puncture and also as a precaution and protection against accidents. In obese patients it may be necessary to adjust the guard to a higher level before attempting to enter the sternum. In such circumstances the positioning of the index finger over the shaft (Figure 9) of the needle helps stabilize the needle and permits adequate control during the sternal puncture procedure. If the sternal puncture needle is used for bone marrow aspiration from sites other than the sternum the adjustable guard may be removed before attempting to penetrate the bone and aspirating the marrow.
Procedure:
1. Position of the patient: Place the patient on his/her back with the head and neck comfortably resting on a soft low-lying pillow. In men it may be necessary to shave the skin over the sternum prior to puncture.
2. Site: In adult the sternal puncture is usually performed in the proximal region of the body of the sternum, at the level of the second intercostal space, half way between the midsternal line and the left or right sternal border.
3. Identify the area of sternal puncture by palpating the angle of Louis at the junction of the manubrium and body of the sternum. Mark the location with an indelible marker or digital pressure. Surgically prepare the area down to the fourth intercostal space with alcohol and iodine and then drape the site.
4. First withdraw 3-5 ml of anesthetic (2% lignocane) through a 21 gauge 1 1/2" needle into a 5 ml syringe. Then substitute the 21 gauge needle for a 25 gauge 5/8" hypodermic needle and make an intradermal injection producing a 5 mm papule. Replace the 25 gauge with a 21 gauge needle and pass it through the papule infiltrating the subcutaneous tissue with the local anesthetic. Then with the needle still in place also inject about 1.0 ml directly into the periosteum. Give ample time for the anesthetic to take effect.
5. It is useful to probe the site with a 21 gauge, one and a half inch needle (with or without an attached syringe) to roughly assess the effect of the anesthetic and the depth at which the sternum will be struck. Using this as a guide, adjust and firmly fix the guard on to the shaft of the bone marrow aspiration needle.
6. No skin incision is necessary for this procedure.
7. Hold the needle assembly with the domed handle in the palm, the middle and fourth fingers over the transverse handle, and the index finger against the shaft of the needle. The position of the index finger against the shaft (arrow) helps stabilize the needle and controls it during the sternal puncture procedure.
8. After skin sterilization and local anesthesia of the skin, subcutaneous tissue and periosteum the needle with its stilette in place is slowly and gently advanced towards the sternum so as to hit the bone at a right angle (or at 45°). When the sternum is reached it is then penetrated by gentle rotary (clockwise/ counterclockwise) motion of the needle.
9. Entrance into the sternal marrow cavity is recognized by a "sudden" give of the needle which indicates that the outer table of the sternum has been perforated. Once the cortex is penetrated advance the needle only few millimeters into the marrow cavity with gentle, reciprocal clockwise/counterclockwise rotary motion.
10. Once the needle is in place — hold it in position with the left hand, (as shown in the illustration), unlock the stilette and domed handle by anticlockwise twist and gently remove it.
11. Attach a syringe to the needle mount.
12. Apply a negative pressure and aspirate the required amount of marrow sample from the sternum.
13. Following aspiration of the required amount marrow remove the syringe from the needle mount and using sterile technique, close the proximal opening of the needle (arrow) to prevent any loss of marrow or blood from the wound while the marrow specimen collected in the syringe is being delivered into a bottle containing EDTA (or other anticoagulant) for processing and examination.
14 Replace and lock the stilette with domed handle by a twisting clockwise motion.
15. Slowly withdraw the needle with the right hand with a gentle alternating rotary motion while fixing the sternum with the index and middle fingers of the left hand.
16. After withdrawal of the needle, apply firm pressure for one or two minutes over the site of the puncture to prevent any bleeding and then cover the wound with a small dressing.
Note: To protect the marrow aspiration needle and obtain the desired results the needle should never be introduced or withdrawn without the stilette in place.
Schematic Representation of the Sternal Puncture Procedure Using the Islam Sternal Puncture Needle
1. The sternal puncture needle, with the stilette and handle in place and the guard firmly secured at a proper distance from the distal end of the needle, is slowly advanced through the skin and subcutaneous tissue towards the sternum so as to hit the bone at 90°. When the sternum is reached, it is gently penetrated by clockwise and counterclockwise rotary motion of the needle (Figure 10, upper).
2. Once the outer table of the sternum is penetrated the needle is advanced slowly a few millimeters in the marrow cavity by the same rotary motion of the needle (Figure 10, lower).
3. Once the needle is in place within the marrow cavity, the stilette is removed with an anticlockwise twisting action, a syringe is attached, and the aspiration performed.
4. Following aspiration of the desired amount of bone marrow, the stilette is promptly replaced and adequately secured and the whole needle assembly is withdrawn with a rotary motion.
(Continues...)
Excerpted from Manual of Bone Marrow Examination by Anwarul Islam. Copyright © 2013 Anwarul Islam, MD, PhD. FRCPath, FACP. Excerpted by permission of Trafford Publishing.
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