Joanne B. Messick Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA Bone marrow, the primary site of hematopoiesis, is one of the most widely distributed organs in the equine body. While hematopoiesis in the horse is similar to that described in humans, it is much less well studied and we rely heavily on extrapolation to understand this process in the horse. It is well established that hematopoietic stems cells (HSC), found mainly in the bone marrow, give rise to all the cells in the peripheral blood [1, 2]. In addition, the bone marrow serves as an important component of the reticuloendothelial system. Clusters of HSCs and their progeny in the marrow are nurtured and develop in the extravascular spaces between a vast network of vascular channels known as sinusoids. A support lattice for the developing hematopoietic cells is formed by cytoplasmic extensions from reticular cells that cover the surface of the sinusoids. Additional components of the bone marrow stroma include vascular endothelial cells, macrophages, and adipocytes, as well as associated extracellular matrix molecules that regulate and control the process of hematopoiesis and provide an adhesive framework for the developing precursors. These components are collectively referred to as the microenvironment of the bone marrow. The regenerative capacity and plasticity of bone marrow is extraordinary, with millions of red cells, platelets, and leukocytes produced every second of life. In times of hematopoietic stress or challenge, such as with blood loss, hemolysis, or infections, the marrow has tremendous reserve capacity and can increase production to 5–10 times normal [3]. The skeleton of the horse is mainly composed of two types of bony tissue that are important to our understanding of the bone marrow and collection of marrow for cytological or histological evaluation. Cortical or compact bone, which forms a dense, hard osseous shell around the bone, makes up about 80% of bone mass in the adult animal. Its outer surface is covered by a fibroelastic tissue known as periosteum, while a thin vascular membrane of connective tissue, the endosteum, lines its inner surface. Cavities within the cortical bone (medullary cavities) contain a meshwork of bony plates and rods, known as trabecular or cancellous bone, which are lined by endosteal cells, a continuum of their cortical counterpart [4]. The thickness and strength of the cortical bone that surrounds the trabecular meshwork vary depending on the location, as well as inherent stresses applied to that area [5]. Trabecular bone makes up much of the enlarged ends of long bones and it is within the spaces formed in this trabecular meshwork that hematopoietically active bone marrow tissue is found. A large portion of the medullary cavity of bones is filled with active blood‐forming tissue at the time of birth. However, in the adult horse, active marrow is found only in the axial skeleton, including the sternum, ribs, vertebrae, pelvis, and proximal ends of the femur and humerus. The most accessible of these sites for collection of bone marrow in the horse are sternum, dorsal ribs, and tuber coxae of the ileum [6]. The various bone marrow sites, it is assumed, have relatively uniform cellularity and proportions of hematopoietic cells under normal physiological conditions. Thus, generalization about hematopoiesis can be made from collection of bone marrow from a single site. The diagnostic accuracy for most hematological disorders does not appear to be improved by collecting samples from multiple bone marrow sites. It is essential that bone marrow evaluation, whether an aspiration or core biopsy, should be preceded by an extensive review of all pertinent historical information and clinical features. Meticulous assessment of the blood film, complete blood count (CBC) data, and other laboratory tests, as well as any imaging studies, are essential to making an accurate diagnosis. When a routine blood smear evaluation and other diagnostic testing fail to uncover the reason for an observed abnormality on the CBC, a bone marrow evaluation is indicated. The most common indication for this procedure is the presence of peripheral cytopenia in one or several hematopoietic cell lines for which an etiology cannot be found. However, unexplained elevations in blood cell numbers, abnormal cellular morphology, suspicion of marrow malignancy, and other clinical or laboratory abnormal findings, such as fever of unknown origin, hypercalcemia, hyperproteinemia, and presence of a lytic bony lesion, may also warrant a bone marrow evaluation [7]. There are few contraindications for obtaining a bone marrow sample. Marrow aspiration or core biopsies can be safely obtained even in horses with thrombocytopenia. However, in horses with an abnormal coagulation profile or platelet dysfunction [8, 9], severe thrombocytopenia (<30 × 103/μL), and when there is concomitant use of anticoagulants, these procedures should be approached with great caution. The most frequent adverse events reported in humans are hemorrhage, persistent pain at the marrow site, and infection [10]. It was concluded in one study that bone marrow aspiration from the sternum of the horse did not elicit a measurable pain response during, or in the two hours following, the procedure [11]. While the collection of a bone marrow sample from the sternum is considered an innocuous procedure, there have been case reports of fatal cardiac puncture and nonfatal pneumopericardium in the horse [12]. This emphasizes the need for a thorough understanding of the anatomy of the equine sternum as well as the exact site and depth of needle placement before attempting the collection of bone marrow samples from this site. The two most commonly used sites for bone marrow collection in horses are the sternum and the tuber coxae. While the sternum is not covered by thick muscle and is often the preferred site, the awkward and somewhat unsafe position the person must assume when performing this procedure, and the possibility of severe complications, cannot be dismissed. These sites have been compared relative to the adequacy of samples obtained for harvesting the mononuclear cell fraction to be used for in vitro culture expansion of mesenchymal stem cells (MSC). MSC are shown to have high osteogenic capacity and may be used to treat tendon, ligament, and meniscal injuries, as well as enhancing the healing of cartilage defects and subchondral bone cystic lesions in the horse. For collection of multipotent mononuclear cells, the amount of bone marrow sample obtained and MSC yield were greatest from the sternal site [13]. The tuber coxae of the ilium may be used for collection of a bone marrow sample in younger horses, but it is doubtful whether an adequate sample can be obtained from this site in the adult horse, particularly as it relates to the collection of cells for therapeutic purposes. A bone marrow sample can also be obtained from the iliac crest in adult horses, but the needle must penetrate deeply through the overlaying layer of muscle to reach this site and needle placement appears to be more critical [14]. The bone marrow aspiration is typically done on the standing horse that has been sedated. Recent evidence suggests that the fifth sternebra is the most consistent and safe site for aspiration of bone marrow in horses. It is usually located just caudal to the point of the elbow, but identification of the correct site and accurate placement of the bone marrow needle into its medullary cavity can be greatly facilitated by the use of ultrasonography [12, 15]. The fourth sternebra may also be used for collection of bone marrow. Both the fourth and fifth sternebrae are somewhat spherical in shape and possess a dorsoventral extension of at least 2 inches [15]. Care must be taken to avoid moving more caudally as the sixth and seventh sternebrae are thinner and closer to the apex of the heart. It is this author’s experience that moving too far caudally and penetrating too deeply are major mistakes that may result in a catastrophic outcome. The bone marrow needles most commonly used for sample collection in the horse include the Illinois sternal/iliac bone marrow needle for aspirates and Jamshidi bone marrow biopsy needle for core bone marrow samples. It is suggested the bone marrow needle should be positioned substantially cranial to the most caudal aspect of bony structures of the sternum, which are also identified by this imaging modality; while this is a rather gross indication of “where not to tread,” it may help to avoid a catastrophic outcome. The reader is referred to Kasashima et al. [12] for photographs, as well as ultrasonographic and radiographic images that delineate the topographical anatomy of the sternum and the appropriate sternebrae for obtaining a bone marrow aspiration or core biopsy. A wide band of hair overlying the sternal site that is approximately 8 cm wide and 10 cm long and centered on the mid‐line is clipped. The skin is over the fifth sternebra is aseptically prepared, cleaning the intended biopsy site outward in increasingly large concentric circles with surgical scrub and alcohol. Sterile gloves should be worn and aseptic technique maintained throughout the procedure to avoid the possibility of infection. Several 12 mL syringes should be preloaded with the appropriate anticoagulant. For therapeutic bone marrow harvesting, syringes are preloaded with 0.5 mL of 5000 IU/mL heparin solution, which gives a final concentration of 250 IU/mL of heparin after the addition of 9.5 mL of bone marrow [12]. Dipotassium ethylenediamine tetraacetic acid (K2 EDTA) is the anticoagulant of choice for bone marrow to be used for morphological evaluations. Syringes should be preloaded with 20 mg of K2 EDTA per each 10 mL of bone marrow volume collected; thus, three drops of a 15% K2 EDTA solution should be added to each 12 mL syringe. The skin at the entry point for a bone marrow aspiration is infiltrated with local anesthetic, extending the injection of anesthetic to all tissues down to and including the periosteum of the bone. Following a second surgical scrub, a small stab incision is made through the skin and the bone marrow needle with its stylet in place is inserted to the level of the bone. The ventral surface of the sternebra is usually contacted at a depth of about 1.5–2 inches; the needle is advanced an additional 0.5–0.75 inches through the cortical bone into the marrow cavity using steady pressure and a gentle forward and backward rotation of the needle. For an aspiration sample, the stylet is removed once the needle is in place, a 12 mL syringe preloaded with K2 EDTA is attached, and rapid, strong negative pressure is applied to obtain approximately 7–10 mL of bone marrow. If necessary, additional syringes may be attached to the needle hub and the aspiration repeated to obtain more bone marrow. A sterile cap is placed on the syringe and marrow samples are immediately mixed by gentle inversion for, at the least, several minutes. The needle is removed after checking to make certain that an adequate bone marrow aspiration sample has been obtained, and pressure is applied to the site to control any bleeding that may occur following the procedure. Smears from bone marrow aspirates for microscopic examination may be prepared using either the wedge‐spread or crush techniques. There are advantages to each of these techniques; since particles are collected after the well‐mixed aspiration sample is poured into a Petri dish, slides prepared by the crush technique of the transferred particles have less blood dilution and assessment of megakaryopoiesis appears to be more accurate. On the other hand, the wedge‐spread may be more beneficial for the assessment of total cellularity. Whilst this author prefers the crush technique, there may be merit to routinely making slides using both of these techniques [16]. The prepared slides are air‐dried and at least 2–3 slides, representing each of these techniques, are stained with a Romanowsky type stain such as Wright–Giemsa for cytological evaluation. Any remaining marrow sample in K2 EDTA and unstained slides should also be sent to the laboratory. This sample may be useful for additional studies such as cytochemistry, immunocytochemistry, or polymerase chain reaction evaluation, as well as automated hematology analysis. The core biopsy sample can be collected through the same stab incision in the skin, but the needle should be directed in a slightly different direction to avoid the disrupted marrow spaces resulting from the aspiration procedure. As previously mentioned, the ventral surface of the sternebra is typically contacted at a depth of about 1.5–2 inches; the needle is then advanced through the cortical bone using firm pressure with an alternating clockwise and counterclockwise motion. It is suggested to position the forefinger on the Jamshidi needle about 0.5 inches from the skin surface to limit the depth of penetration of the needle through the cortex into the marrow space [6]. Once in the marrow space, the stylet is removed and the Jamshidi needle is advanced about 1–1.25 inches before rotating it several times in place to ensure that the core sample is detached. The needle is withdrawn and the core sample removed from its shaft and placed into 10% formalin for fixation. Notice the total depth of the needle penetration should not exceed 0.5 inches through cortical bone and 1.25 inches into the marrow space or 1.75 inches total, which is slightly less than the dorsoventral extension of this bone. Bone marrow aspiration and core biopsy samples must be interpreted in the context of a current CBC and should be evaluated by an experienced veterinary pathologist (see Cases 1 and 2). The CBC should be collected at the same time or within 24 hours of bone marrow collection. A careful review of the peripheral blood smear may reveal important clues that are directly related to an underlying hematological problem or provide information to help direct marrow evaluation and other diagnostics. In general, the bone marrow aspirate examination includes an overall assessment of cellularity and iron stores, as well as evaluation of the myeloid to erythroid ratio and judging whether maturation of the myeloid, erythroid, and megakaryocytic lineages is orderly and their morphology is normal. Further, other cell types such as lymphocytes, plasma cells, histiocytes (macrophages and dendritic cells), mast cells, stroma cells, and putative neoplastic cells must be characterized in terms of their abundance, distribution, and morphology. Bone marrow cellularity can be estimated on preparations of an aspirated sample, but should be verified by a determination made from a good‐quality core biopsy section. The assessment of cellularity on slides prepared from an aspirated sample is subjective and can be influenced by adequacy of specimen collection, as well as hemodilution of the aspirated sample. The overall cellularity in multiple bone marrow fragments or particles is evaluated. Cellularity is expressed in terms of the hematopoietic tissue as a percentage of total tissue within the particle, including hematopoietic and adipose tissue. The percentage of hematopoietic tissue within several particles should be estimated and this should be done on several slides before calculating an average. If the cellularity is highly variable amongst the particles, more particles should be assessed to provide as accurate an estimate as possible. An average particle cellularity varies from about 25% to 75%, but the age of the horse must be taken into consideration. The younger the animal, the higher the cellularity; it is not unusual in animals less than 6 months of age to have greater than 75% cellularity within particles and for the neonate, this may approach 100%. Cellularity decreases with age and senior horses (>25 years of age) may have as little as 20% cellularity within particles. This is considered normal cellularity given the young or advanced age of the animal, respectively. In an adult horse (~2–20 years), bone marrow cellularity is considered hypocellular when the percentage of hematopoietic cells within particles is on average less than 25%, whereas hypercellularity is defined when marrow particles have greater than 75% cellularity (see Case 1) (Figure 3.1a,b). The orderliness and completeness of erythroid maturation in the bone marrow, determination of a myeloid to erythroid (M:E) ratio, and percentage of marrow reticulocytes are critical components for assessing the erythroid lineage in horses. To calculate the M:E ratio, a minimum of 500 nucleated cells should be classified as belonging to either the myeloid or erythroid lineage; it should be evaluated in several areas of the bone marrow slide and on several different slide preparations and should be correlated to the overall marrow cellularity. The range for the M:E ratio in healthy adult horses is 0.5–2.4 whereas the marrow reticulocyte count, which is a reflection of erythroid activity, is typically less than 3% [6]. In the horse, these parameters should be assessed concurrently in order to classify an anemia as regenerative or nonregenerative. The anemia is regenerative when the M:E ratio is <0.5 and the reticulocyte count >3%, whereas a low reticulocyte percentage in the bone marrow and a normal or increased M:E ratio support a nonregenerative erythroid response. In an intensely regenerative erythroid response, reticulocytosis in the bone marrow may increase dramatically and values as high as 60% have been reported [17, 18]. The presence of a peripheral regenerative response is difficult to assess in horses, since they do not tend to release reticulocytes into the blood but rather the vast majority of these cells mature in the marrow or spleen [19]. In addition to assessing the bone marrow components as discussed above, an increase in the red cell distribution width (RDW) and mean corpuscular volume (MCV) of circulating red cells may be subtle indicators of a regenerative response [6]. A clearer indication that the horse has mounted a regenerative response is sequential packed cell volume (PCV) measurements over time that show a steady increase, provided the hydration status of the horse is unchanged. It has been suggested that the reticulocyte count using an automated hematology analyzer, where thousands of the horse’s erythrocytes are counted in assessing this response, may be a sensitive way to establish the presence of a regenerative response in some hemolytic anemias [18, 20]. An assessment of the morphology of erythroid precursors in the marrow is also essential. The nucleus and cytoplasm should be individually evaluated for any abnormalities and for the synchronicity of their maturation. Morphological irregularities or dysplastic changes of erythroid cells may present as abnormally large erythroid precursors, bizarre nuclear shapes, nuclear fragments, multiple nuclei, and asynchronous maturation of nucleus relative to its cytoplasm. While these changes are uncommon in horses, erythroid hyperplasia with a left shift in maturation and concurrent dysplasia eventually progressing to severe erythroid hypoplasia in the bone marrow of foals with fell pony syndrome or foal immunodeficiency syndrome (FIS) has been reported [21]. Iron stores should be easy to find in the bone marrow of horses (see Case 1) (Figure 3.1c), and a tendency for stores to be more prominent in older animals is reported. In some instances, Prussian blue staining of the bone marrow may be indicated to assess the presence or absence of iron stores. This procedure may be particularly useful if an anemia due to iron deficiency is suspected, although this is an extremely rare condition in horses [22].
3
Bone Marrow Evaluation
3.1 Indications, Technique, and Evaluation of Equine Bone Marrow Cytology
3.2 Indications
3.3 Technique for Bone Marrow Collection
3.4 Bone Marrow Evaluation
3.4.1 Bone Marrow Aspirate
3.4.1.1 Cellularity
3.4.1.2 Myeloid to Erythroid Ratio and Marrow Reticulocyte Count
3.4.1.3 Erythroid Lineage, Iron, and Abnormalities
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