Bone marrow collection in the dog and cat (Proceedings)

Hematopoiesis

Hematopoiesis is the production of new blood cells such as white blood cells (WBC), red blood cells (RBC) and platelets.  Although in utero a number of organs are hematopoietically active, including the liver and the spleen, from birth onwards hematopoiesis is centered almost exclusively in the bone marrow.  Although the need to produce new blood cells continues throughout life, the magnitude of hematopoiesis declines with maturity and, in the adult animal, hematopoietically active (red) marrow is limited predominantly to the flat bones and the epiphyses of the long bones.  Active marrow contains mature blood cells and their various precursors, reticular cells and reticulin fibres, adipocytes (fat cells) and vascular sinuses.  Less active (yellow) marrow has a higher proportion of adipocytes.

All of the blood cell lines derived from the marrow appear to originate from a common progenitor cell, known as the hematopoietic stem cell.  The hematopoietic stem cell, which is capable of self-renewal, is a pluripotential cell: that is, it is capable of differentiating into the stem cells (also known as colony-forming units or CFUs) that eventually develop into erythroid, lymphoid, granulocyte-monocyte and megakaryocytic cell lines.  As pluripotential stem cells divide and develop, they become unipotential stem cells (cells committed to a single cell line), and with further maturation and development eventually lose their ability to self-renew.  This process occurs under the influence of various growth factors, including interleukins, colony-stimulating factors, and poietins such as erythropoietin.  Stem cells are present in relatively small numbers within the bone marrow, and are not recognisable using standard microscopic techniques.

The normal canine or feline marrow contains very few primitive or immature stem cells, and the vast bulk of cells (80-90%) within the marrow are either mature blood cells ready for release into the circulation, or very recognisable and well-differentiated blood cell precursors.  In conditions of increased demand for circulating blood cells, however, the stem cells are capable of rapidly dividing and maturing into well-differentiated cells, and of filling the marrow cavity with the precursor cells that are needed.  For example, in an animal with anemia due to blood loss or hemolysis, marked marrow erythroid hyperplasia can develop within one week of the onset of anemia.

Bone marrow collection and analysis is sometimes required in order to accurately evaluate hematopoietic function.  In the clinical setting, however, the function of the marrow can often be accurately estimated based on peripheral blood responses, without a need for marrow collection.  For example, in an animal with a strongly regenerative RBC response secondary to hemolytic anemia can safely be presumed to have marrow erythroid hyperplasia, and a patient with a marked inflammatory leukogram in response to a pyometra can be reliably presumed to have marrow myeloid hyperplasia.  In many other circumstances, however, marrow collection and analysis are necessary in order to accurately define marrow function, and to determine the cause of marrow dysfunction.

Bone marrow collection/interpretation

Collection of bone marrow is a relatively simple technique that is readily achievable in general practice.  The procedure does, however, require some degree of practice to perfect, and can be frustratingly difficult and time-consuming in the live, unwell patient.  Prior perfection of marrow collection techniques on cadavers, when available, is therefore strongly recommended.

Indications for Bone Marrow Analysis

Diagnosis or staging of cancer

·         Diagnosis:               Multiple myeloma

                                Leukemia

·         Staging:                   Lymphosarcoma

                                Mast cell tumor

Diagnosis of various hematological disorders

Marrow analysis is often indicated in patients with:

·         Persistent unexplained decreases in blood cells:

o    Unexplained pancytopenia

o    Unexplained severe neutropenia

o    Impaired erythropoiesis (non-regenerative anemia)

o    Impaired thrombopoiesis (thrombocytopenia without shift platelets)

·         Persistent unexplained increases in blood cells:

o    Polycythemia

o    Marked thrombocytosis

o    Marked neutrophilia, lymphocytosis or eosinophilia

·         Abnormal cells in the circulation:

o    Unexplained rubricytosis (increased nucleated RBCs)

o    Inappropriate preponderance of immature cells of any cell line

o    Suspected neoplastic cells (e.g. mast cells)

Investigation of Specific Medical Problems

Marrow analysis can, on occasion, become part of the standard work-up investigating:

Fever of unknown origin

·         Unexplained hypercalcemia

·         Unexplained hyperglobulinemia (especially monoclonal gammopathies)

Search for Infectious Agents

·         Deep mycoses, especially histoplasmosis in cats

·         Protozoal infections, such as leishmaniasis in dogs

·         Testing for feline leukemia virus (immunofluorescent assay)

Specialized diagnostic testing

·         Immunologic staining for antibodies to RBC, WBC or platelet precursors           

Monitoring Response to Therapy

Follow-up marrow analysis is very useful at monitoring response to therapy, particularly as such responses will be evident in the bone marrow well before causing an improving peripheral blood cell count.  Can monitor:

·         Response to immunosuppression (immune-mediated marrow disorders)

·         Response to chemotherapy (leukemia)

Since marrow collection can seem difficult to the inexperienced, it is tempting to avoid marrow aspiration and/or biopsy and ‘best guess' diagnostic/therapeutic approaches.  Regular marrow collection whenever indicated, however, is the best means of developing confidence in the technique.  Once accepted as a routine procedure, the results of marrow analysis often significantly improve the quality of patient care.

Sites for Marrow Collection

Many sites are potentially available for marrow collection in the dog and cat, including:

·         Dorsal iliac crest

·         Femoral shaft (via the intertrochanteric fossa)

·         Humeral shaft (via the lateral aspect of the greater tubercle)

·         Costochondral junction (through the cartilage and into the rib marrow cavity)

Most of the preceding sites will yield good marrow samples, although marrow can be rather sparse in the iliac crest or rib of small or old patients.  In most instances (but not invariably), marrow from any location is reasonably representative of the overall status of all of the patient's bone marrow.  My personal preference is to use the dorsal iliac crest for most dogs over about 5 kg (10 lb) and the proximal humerus for cats and smaller dogs.  Marrow can usually be collected from most co-operative or depressed dogs using local anesthesia with or without sedation (acepromazine/buprenorphine, for example), while most cats require either heavy sedation (ketamine/valium, for example) or general anesthesia.  Marrow collection is undoubtedly a moderately painful procedure.  Even with periosteal application of local anesthetic agents, some human patients report excruciating pain associated with marrow collection.  This is hardly surprising, since the endosteal lining of the marrow cavity contains pain receptors which cannot feasibly be pre-treated with local anesthetic.  Dogs and cats often seem to exhibit the same pain response upon disruption of the marrow cavity (particularly during aspiration). The procedure can therefore be somewhat nerve-wracking for the inexperienced, who may prefer to perform marrow collection under general anesthesia.

Methods of Bone Marrow Evaluation

Bone marrow can be evaluated either by the cytological examination of marrow aspirates, or the histopathologic evaluation of core biopsies.  Each method has certain advantages and disadvantages.

Marrow aspiration cytology

Bone marrow is aspirated via a marrow aspiration needle, smeared onto a microscope slide, air-dried, stained using standard hematologic stains and evaluated cytologically.

Advantages

·         Rapid turn-around time from collection to examination.  Theoretically, marrow aspirates can be evaluated in hospital, although in reality marrow cytology is complex and difficult to interpret, and is therefore usually best performed by an experienced clinical pathologist.

·         Cytological examination of collected cells often provides superior evaluation of individual cell cytoplasmic and nuclear detail.

·         Preferred technique for immunoflurescent staining.

Disadvantages

·         Many marrow diseases tend to provide poorly cellular or acellular aspirates, or provide unrepresentative samples.  Marrow diseases that often aspirate poorly include aplastic anemia, myelofibrosis and some leukemias.  In contrast, good samples are relatively easy to obtain from healthy marrow.

·         Provides little useful information regarding overall marrow cellular architecture.

·         Inexperienced practitioners can easily disrupt cells during smear preparation.

Marrow core biopsy histopathology

A bony core of marrow material is collected using a specialized core biopsy needle, fixed (usually in formalin), decalcified and examined histopathologically.

           Advantages

·         With experience, perseverance and the right tools, a representative core sample should be obtainable from every patient, regardless of the severity or type of marrow disease.

·         Overall marrow architecture and cell-to-cell relationships can be evaluated.

·         Immunoperoxidase staining can be performed on formalin fixed marrow samples.

           Disadvantages

·         Samples must be sent to a pathology laboratory, and results will invariably be delayed for 3-4 days, and can take longer if decalcification is particularly slow (samples from older patients).

·         Cytoplasmic and nuclear detail of individual cells can be sub-optimal.

Whether aspiration or core biopsy is performed depends on multiple factors, including the suspected disease process, the type of needle available, and the personal preferences of the practitioner.  Many clinicians will initially attempt marrow aspiration and then, if a diagnosis is not obtained, collect a core biopsy.  Evaluation of bone marrow is a specialized art, and it can be difficult to find a pathologist or clinical pathologist who is confident at handling marrow specimens.  Sample collection may therefore depend upon who is available:  obviously, clinical pathologists prefer aspirates, and pathologists prefer cores.  Ideally, send specimens to a specialist who is experienced at evaluating both types of sample.

Since both marrow aspirates and core biopsies can be collected from the same location, since both require the same level of local anesthesia, sedation and/or general anesthesia, and since each technique can provide different (and often complimentary) information, I prefer to collect both aspirates and cores at the same time.  When in doubt regarding which samples to collect, and how to handle them, contact your laboratory for advice prior to sample collection.  Most specialists will prefer that blood be submitted for hematologic analysis along with marrow samples.

Specific techniques

The following two protocols outline collection of bone marrow aspirates and cores from the iliac crest, my preferred site in most dogs.  Collection of marrow via the iliac crest is usually associated with few problems (occasionally a little bruising is observed) in larger dogs.  In cats and smaller dogs, however, the marrow content of the iliac crest may be insufficient to allow collection of representative samples, especially in older animals.  Fracture of the ileum, although rare, is also a potential complication of using this site in smaller patients.  With minor modifications, the techniques used for the iliac crest can be applied to collect marrow via the trochanteric fossa of the femur or the greater tubercle of the humerus.  Marrow collection from these latter two sites usually requires general anesthesia.

Marrow aspiration (canine iliac crest)

Bone marrow aspiration may be performed with either multi-use or single use (disposable) aspiration needles.  Whatever type is used, aspiration is relatively easy with a sharp needle, and practically impossible (and painful) with a blunt needle.  In my experience, practitioners tend to sharpen multi-use needles inadequately and too infrequently, and therefore become disillusioned at their lack of success whenever attempting bone marrow collection.  For this reason, I prefer to use so-called single use needles.  Although these needles are relatively expensive, and cannot be autoclaved, they can be cold sterilized.  In the hands of an experienced operator, such needles can be re-used approximately 5 to 10 times before becoming appreciably blunted.

Procedure

1.        Sedate or anesthetise patient if needed, and position either standing or (my preference) in sternal recumbency.  Take care to keep the patient as square and straight as possible in order to maximize preservation of landmarks.

2.        Locate the left or right iliac crest by palpating firmly backwards along the lumbar musculature on either side of mid-line.  The first bony prominence met in the pelvic region is the iliac crest.  As long as care is taken to avoid mid-line (that is, the vertebral column) and to stay well forward of the hip joint and nearby sciatic nerve, no delicate structures will be damaged by marrow collection, however crudely performed.

3.        Clip the dorsal aspect of one iliac crest, and infiltrate the skin, subcutaneous tissue and periosteum of the ileum with 1-2 mls of local anesthetic.

4.        Surgically prepare the clipped area (some people even apply drapes) and, using a scalpel blade, make a small stab incision through the skin overlying the iliac crest.

5.        Prepare saline mixed with EDTA anticoagulant.  Probably the easiest method of accomplishing this in practice is to add 2-3 mls of saline to a 10 ml EDTA-containing vacutainer tube, mix thoroughly, and then withdraw into a 10 ml syringe.  Since EDTA solution is affected by prolonged exposure to light, it is simplest to make up a new solution for each procedure.

6.        Remove the stylet from a marrow aspiration needle, flush through with a small amount of EDTA solution, and re-insert the stylet.

7.        Wearing sterile gloves, and using a firm, downward twisting motion, insert the marrow needle into the iliac crest until firmly embedded.  Considerable amounts of exertion may be necessary, and if the needle slides off the medial or lateral aspect of the iliac crest, repeat from a slightly different angle.  Gentle hammering is sometimes needed in older dogs, particularly if a blunt needle is used.  When properly inserted, the needle should be so firmly embedded that the dog can almost be lifted off the table via the needle.

8.        Remove the stylet, and firmly attach the 10 ml syringe containing a few mls of EDTA solution to the marrow needle.  Vigorously and repeatedly aspirate until a small amount of marrow (which looks like thick venous blood) enters the syringe:  as little as 0.5 ml will suffice, and greater than 2 mls is superfluous to requirements.  Initial aspiration often elicits a pain response from the patient.

9.        Failure to obtain marrow indicates either marrow disease or poor location of the needle tip.  Re-insert the stylet, and redirect the needle tip (usually by pushing in a little further) before attempting repeat aspiration.  Two or three attempts without success from a well embedded needle usually indicates significant marrow disease although, since blood clots can occasionally occlude the needle lumen, it is worth removing the needle and flushing through again with EDTA solution.  Core biopsies should be collected if aspirates are persistently unsuccessful.

10.     Produce smears of the marrow aspirates using several different techniques:

a.        Cover several microscope slides with the marrow/EDTA mix, let sit for 30 seconds or so, then gently tilt the slide to run off the excess fluid.  Marrow granules will have settled down on to the slide, and are visible to the naked eye as numerous slightly fatty white or cream raised speckles. Then, drag another angled microscope slide  (using the same technique as used when dragging a slide back into a blood droplet for blood smears) backwards through a section of the slide that is covered with granules, in order to pick up some granules on the underside of the angled slide.  Then lift the angled slide, place it on a fresh clean slide, and make a smear on the new slide using exactly the same technique as is used for a blood smear.  The granules picked up on the underside of angled slide will be non-traumatically spread out across the smear.  Repeat this smearing technique several more times.  Then, use another slide to scrape all of the remaining granules into a clump at one end of the first slide.  Distribute small aliquots of the granules to fresh slides, and prepare multiple slides using a standard squash smear technique (lie a second slide across the granules, and rapidly draw the two slides apart)

b.        Stand several clean slides propped up at a 45° angle.  Place a drop of marrow/anticoagulant mix near the top of the slide.  Gravity will run the drop down to the bottom of the slide, leaving a trail of dispersed granules.  Then perform squash smear slides as described previously.

11.     Slides may be submitted to the laboratory once air-dried, or stained for in-practice examination.

12.     Remove the marrow needle.

More than one smear preparation technique is recommended because marrow aspirates are very prone to handling problems that may hinder diagnosis, and different types of marrow disease do better with different preparation techniques.  Blood smear-type techniques protect fragile cells from crushing damage, and are therefore ideally suited for round cell tumors affecting the marrow (such as lymphosarcoma and mast cell tumors) since round cells tend to rupture easily when handled roughly.  However, with blood smear techniques, marrow granules can be so densely clumped that it is impossible to see cytological detail.  The use of a squash smear technique helps to spread out these granules, allowing better cytological examination of granule contents.

Marrow core biopsy (canine iliac crest)

As with aspiration, bone marrow core biopsy may be performed with either multi-use or single use (disposable) biopsy needles.  Core biopsy needles are extremely difficult to use unless sharp, very easily blunted, and practically impossible to sharpen.  Therefore, as with aspiration needles, I prefer to use single use biopsy needles.  Again, these needles are expensive and cannot be autoclaved, although they can be cold sterilized and re-used 5 to 10 times before becoming blunted.

Procedure

1.        Prepare patient as for marrow aspiration (steps 1 through 4 above).

2.        Wearing sterile gloves, and using a firm, downward twisting motion, insert the marrow core biopsy needle, with stylet in place, into the iliac crest until firmly embedded.  As for insertion of the aspiration needle, considerable amounts of exertion may be necessary to insert the biopsy needle, and re-direction may be necessary if the needle slides off the crest.  Since the biopsy needle is much longer and thicker than the aspiration needle, deep tissues can potentially be damaged if the needle is accidentally plunged off the top of the crest.  For this reason, it is advisable to rest one index finger along the barrel of the biopsy needle, so that the needle cannot be accidentally inserted by more than a few centimeters.

3.        Once the needle is embedded, remove the central stylet.  Since, for this procedure, the needle needs only to be through the cortex of the iliac crest, the stylet may be removed once the needle is embedded into the crest to an estimated depth of one quarter inch or so. 

4.        Using a firm rotating motion, continue to insert the hollow biopsy needle much deeper into the marrow cavity (at least one-half to one inch deeper, and even deeper if possible).  This will, on most occasions, fill the cavity of the needle with a core of marrow.

5.        Break the marrow core off into the needle.  This can be done either by more vigorously rotating the needle (take care not to bend or break it), or by removing the needle slightly and redirecting (thereby cutting off the base of the core).  Some biopsy needle have a cap that may be screwed over the hub of the needle.  Attach the cap at this point, since a firmly applied cap will create a vacuum that will encourage removal of the core.

6.        Remove the needle by rotating as it is pulled out, and remove the screw-on cap.

7.        Apply a stylet through the needle (from the tip directed towards the hub is preferable).  Hopefully, a solid core of bone and marrow material will be pushed out of the needle lumen.  Repeat several more times if an adequate core is not obtained.

8.        Place the core in formalin.  Since some pathologists prefer fixatives other than formalin, it is advisable to contact the laboratory prior to sample collection.  Prior to fixation, the core can be rolled along a microscope slide, which is subsequently air-dried for cytology if aspiration was previously unsuccessful.  This procedure will sometimes (but not always) provide adequate samples for immediate cytology: unfortunately, the same diseases that make aspiration unsuccessful often prevent adequate exfoliation of cells from a rolled core.

Examination of a Marrow Smear

Clinical pathologists examine marrow smears using a systematic approach.  Smears are looked at under low magnification, and then at higher magnification

Low magnification

·         Determine adequacy of the sample.

·         Estimate degree of cellularity of marrow granules

·         Estimate megakaryocyte numbers

·         Evaluate iron stores

Adequate samples have at least several granules, AND areas where the marrow elements are spread out enough to allow evaluation of the cytoplasmic detail of individual cells.  Cellularity varies with age, with as little as 25% fat within granules in young animals, and as high as 90% fat in older animals.  Megakaryocytes are giant multinucleated cells: typically each granule in a smear contains approximately 5 to 10 megakaryocytes, although a precise estimation of megakaryocyte numbers can not be derived from smear examination because of variable cellularity of aspirates.  Clinical pathologists evaluate iron stores, which are typically visible as brown-black densities on standard Wright's stained marrow smears, because in most species an absence of stainable iron is consistent with iron deficiency.  In cats, however, the absence of stainable iron has little diagnostic value, since many normal cats have no detectable marrow iron stores.  The presence of stainable iron in a cat, when observed, indicates the presence of adequate iron stores.

High magnification

1.              Evaluate erythroid and myeloid numbers and morphology, and whether all stages of maturation within each series are present.

2.              Determine myeloid to erythroid ratio.

3.              Identify and estimate numbers of round cells such as plasma cells, mast cells, and lymphocytes.

4.              Identify malignant or abnormal cells.

5.              Identify infectious agents (Histoplasma, for example).

Normally maturing RBC and WBC series within the bone marrow contain a preponderance of the later stages of cell maturation.  For example, metamyelocytes, band and segmented neutrophils comprise up to 80% of the myeloid series, and rubricytes and metarubricytes comprise up to 90% of the erythroid series.  The recognizable granulocytic series begins with poorly differentiated round blast cells (myeloblasts), and then progresses through promyelocytes, myelocytes, metamyelocytes, and band neutrophils until eventually the mature cell form (segmented neutrophil) develops.  For the inexperienced cytologist, probably the metamyelocyte, with its indented nucleus that eventually becomes band-shaped, is the first cell in this series that can be readily recognized as a neutrophil precursor. 

The recognizable erythroid series begins with rubriblasts, and progresses through prorubricytes to rubricytes to metarubricytes (nucleated RBCs), before the RBC nucleus is extruded and the immature cell is released into the circulation as a polychromatic RBC.  For the less experienced observer, probably the rubricyte, which can be identified as the immediate precursor to the readily apparent metarubricytes, is the first recognizable RBC precursor.  The myeloid to erythroid ratio in normal dogs and cats ranges from 1:1 to 5:1.

Interpretation of Marrow Smears

While clinicians with an interest in cytology should definitely look at marrow aspirates and develop a preliminary diagnostic impression, marrow smears should always also be sent to an experienced clinical pathologist for a definitive interpretation.  While some marrow diseases may be readily diagnosed by the 'enthusiastic amateur' (for example, histoplasmosis, aplastic anemia, pure red cell aplasia, and advanced forms of marrow neoplasms like multiple myeloma, lymphosarcoma, mastocytosis or leukemia), most marrow disorders have subtleties of interpretation that require the input of an expert.

Although marrow cytology and histopathology should be evaluated by an expert, the clinician's diagnostic role is still far more than that of a non-thinking collector and submitter of samples.  Interpretation of marrow is not just an exercise in cytology: interpretations are also based on assimilating extra information like history (prior drug exposure, duration of disease, treatments), hematology, and the results of other diagnostic tests.  For example, a marrow that has an overwhelming preponderance of immature RBC precursors and relatively few late stages may be a marrow that is in the early stages of recovering from an acute blood loss or hemolytic anemia, or a marrow with severe erythroid hyperplasia that is just now beginning to respond to appropriate and specific therapy (like immunosuppressive therapy for pure red cell aplasia, erythropoietin for chronic renal failure, or iron supplementation for iron deficiency), or a marrow that has myelodysplasia and associated RBC 'maturation arrest', or a marrow that has an immune disease directed against a later stage of RBC precursor. 

In this instance, the clinician responsible for the case may be better equipped than the clinical pathologist to differentiate each of these possibilities, because the clinician can answer the key questions that will fill in the gaps in the diagnostic puzzle: 'is the anemia acute or chronic?', 'has appropriate therapy been commenced recently?', and 'is there evidence of peripheral hemolysis or hemorrhage?'.  When in doubt, repeat marrow sampling at one to two week intervals will often enable differentiation of each of these possibilities.  Effective communication between the clinician and the clinical pathologist is essential.

Marrow diseases: specific conditions

Many primary and secondary marrow diseases can often be diagnosed by marrow analysis, including:

·         Pure red cell aplasia

·         Aplastic anemia

·         Myelofibrosis

·         Amyloidosis

·         Myelodysplasia

·         Hematopoietic cell neoplasms:

o    Acute leukemia

o    Chronic leukemia

o    Erythroid neoplasms (erythemic myelosis and polycythemia vera)

o    Essential thrombocytosis

·         Other neoplasms:

o    Multiple myeloma

o    Lymphosarcoma

o    Mast cell tumors

o    Metastatic carcinomas

·         Infectious diseases:

o    Histoplasmosis and other fungal infections

o    Leishmaniasis

o    Ehrlichiosis

o    Feline leukemia virus