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When Do I Need to Collect Bone Marrow, and HOW?!?!
You may have seen it written at the bottom of a pathologist’s comment on their CBC path review, cytology, or biopsy report:
“Consider bone marrow evaluation”
Many practitioners are wary of sampling bone marrow due to uncertainty over when and how to collect it. In this article, I discuss the indications for bone marrow cytology and/or core biopsy, the basics of collection technique, and how to assess sample quality.
Indications for Bone Marrow Assessment
Bone marrow, particularly within the long bones, is the site of hematopoietic tissue that produces all the blood cells in the body. The most basic reasons for evaluating bone marrow are to look for the cause of a hematologic problem. Here are some of the main indications:
- Unexplained, persistent, and/or worsening cytopenia(s)
- If a patient has a non-regenerative anemia, thrombocytopenia and/or neutropenia that lasts more than a week or so (enough time for a regenerative response to kick in) without improvement, and no cause is apparent, a bone marrow may reveal the cause
- Pancytopenia is frequently an indication for bone marrow sampling
- Unexplained, persistent, and/or worsening increases in one or more cell lines
- This is particularly the case for moderate to marked lymphocytosis or other white blood cells to differentiate reactive/inflammatory causes versus acute or chronic lymphoid or myeloid leukemia
- (Marked) increases in erythrocytes or platelets may also be an indication for bone marrow, although the clonal disorders polycythemia vera (increased RBCs) and essential thrombocythemia (increased PLTs) are diagnoses of exclusion
- Presence of abnormal cells in blood
- Staging or diagnosing occult neoplasia
- Sampling the bone marrow is part of the full staging process for lymphoma, and may also be indicated for mast cell tumors, histiocytic sarcoma, and occasionally others
- Unexplained hypercalcemia
- T cell lymphoma is a common cause of hypercalcemia of malignancy, and it can lurk in the bone marrow (in some cases that may be the only location where it’s found!)
- Unexplained hyperglobulinemia
- Similar to the previous category, neoplastic plasma cells infiltrate the marrow in multiple myeloma
- Search for infectious agents
- Some organisms have a predilection for the marrow, and you may find various bacterial, fungal, rickettsial, or protozoal agents hiding there, especially Histoplasma, Leishmania, and Mycobacteria
- Fever of unknown origin
- Similar to the previous entry, a number of diseases that cause fever may be found within the bone marrow
- In one study of 101 dogs with FUO, the single largest disease category (22% of case) was dogs with primary bone marrow pathology1
- These can include organisms, although numerous sterile causes like inflammatory, immune-mediated and neoplastic diseases may also produce FUO
- BM for FUO should be performed after 1st tier (“minimum database” CBC/Chem/UA/serology and imaging) and 2nd tier (i.e. joint taps, CSF, pathology of any masses/lesions) fail to identify a cause
- Evaluate body iron stores for suspected iron deficiency anemia
Sample Collection Technique
Bone marrow can be collected by both aspiration (for cytology) and core biopsy (for histopathology). Which type of sample should you collect? Ideally both, because they provide different pieces of information. BM aspirate cytology generally provides better cellular detail to assess the morphology and can be critical to identify dysplasia, neoplastic cells, and small organisms. Core biopsy histopathology is better at assessing overall cellularity of hematopoietic tissue, can look for architecture changes that aren’t evident on cytology like myelofibrosis, and you can apply special stains for things ranging from iron to IHC for specific cell lines. If money or logistical concerns preclude doing both, proceeding with a BM aspirate first is a logical step, as you can often get a diagnosis from that, and it is (relatively) less invasive.
The main anatomic sites for collection of either sample type are the proximal humerus, the iliac crest, and proximal femur, although others have been described (including the sternum2). These procedures are painful in human medicine and are assumed to be in our animal patients too. Therefore, appropriate anesthesia is paramount. Bone marrow aspiration requires at least mild to moderate sedation, while core biopsy is recommended under heavy sedation or general anesthesia.
Image: Common anatomic landmarks for bone marrow collection (aspirate and core)
Whatever the procedure and anatomic site, you should approach it with aseptic technique like any other minor surgical procedure (i.e. joint taps, CSF collection, placing chest or feeding tubes). This includes clipping (a 2x2 - 3x3 inch area usually sufficient), surgical scrub prep, and open gloving into sterile surgical gloves.
An Illinois bone marrow needle is generally used for bone marrow aspirates (smaller syringes and needles can be used for sternal marrow collection) whereas a Jamshidi bone marrow biopsy tool is used for core samples. Both needles come in a variety of gauges and your selection depends on patient size, technique and preferences. A small stab incision can be made with a scalpel blade (I preferred a #11 blade) to facilitate entry into the deeper tissues and to avoid premature dulling of your biopsy instrument.
Bone marrow can be aspirated with or without 5% EDTA, but the inclusion of that anti-coagulant will prevent premature clotting and can lead to better samples.
For a detailed, step-by-step description of the technique for BM aspiration and biopsy, readers are referred to the following resources3,4
Preparing the sample
Preparing high quality slides is essential for diagnostic bone marrow assessment. Here are some tips by modality:
Bone marrow aspirate cytology
Even if you submit the liquid bone marrow-EDTA sample, it is highly recommended that you prepare slides in your clinic immediately after collection. This is both to assess sample quality in case re-aspiration is needed while the patient is sedated/anesthetized as well as to provide the pathologist with the best representation of what the marrow cells looked like at “time zero” before any artifacts from shipping, storage, and handling at the referral lab. Hematopoietic cells are fragile and may degenerate quickly.
There are several ways you can prepare BMA slides. First, you can place a drop on a glass slide and prepare it via the “horizonal pull-apart” technique like any other effusion or FNA sample. Using the Illinois needle to aspirate back excess blood can improve the slide by reducing hemodilution. Alternatively, you can expel the material into a petri dish and select several grossly visible particles with forceps and place them onto slides before smearing, but in the author’s experience, this technique is more cumbersome yet not superior to the simpler method.
Bone marrow core biopsy
Collecting at least 2 core fragments is advisable in case one of them is non-diagnostic. Expel the core by inserting the stylet into the smaller diameter opening of the Jamshidi (the portion that entered the bone) and gently push the fragment into the cassette for 10% neutral buffered formalin. Look for a mix of red and white tissue along with bone fragments. If the whole thing is white or appears fatty or like muscle/connective tissue, that fragment may be non-viable.
Important note: Even trace amounts of formalin fumes can damage cytology samples, so make sure to keep the jar of formalin separated if you are preparing aspirate slides at the same time.
Sample Quality Assessment
Cytologic evaluation of bone marrow is challenging and takes years to master, so that is out of scope of this article. I’d encourage you to send bone marrow to a board-certified pathologist, ideally one with interest and expertise in hematopathology. What is reasonable for folks in practice is staining a smear in clinic to check and look for cellularity and sample quality.
You will certainly want to see a sample with lots of nucleated cells that are intact and well-spread. Bone marrow samples are loaded with “particles” or “spicules” that may be seen grossly if some of the aspirate is expelled into a petri dish with EDTA. A highly cellular BM aspirate will have dense, “chunky” streaks of blue-stained material in the middle of an oval smear:
Source: eClinPath, https://eclinpath.com/cytology/bone-marrow/indications-methods/bone-marrow-slides/
Under the microscope, a highly cellular bone marrow aspirate will look like this:
Source: eClinPath, https://eclinpath.com/atlas/bone-marrow/normal-marrow/nggallery/page/1
At higher power, finding large, multilobulated megakaryocytes is a good indicator that you sampled bone marrow (as opposed to just blood contamination):
Source: eClinPath, https://eclinpath.com/atlas/bone-marrow/bone-marrow-non-neoplastic/nggallery/page/1
Summary
- There are multiple indications for bone marrow sampling, particularly related to hematologic and neoplastic disease
- Bone marrow can be sampled by aspirate cytology and core biopsy histopathology (ideally both)
- Common locations are the proximal humerus, proximal femur, iliac crest, and sternum
- Appropriate anesthesia and sterile prep are important aspects of collection
- Proper sample handling and quality checking is critical and improves the likelihood of getting a diagnosis the first time
Further Reading:
- Dunn, K.J. and Dunn, J.K. (1998), Diagnostic investigations in 101 dogs with pyrexia of unknown origin. Journal of Small Animal Practice, 39: 574-580. https://doi.org/10.1111/j.1748-5827.1998.tb03711.x
- Defarges, A., Abrams-Ogg, A., Foster, R.A. and Bienzle, D. (2013), Comparison of sternal, iliac, and humeral bone marrow aspiration in Beagle dogs. Vet Clin Pathol, 42: 170-176. https://doi.org/10.1111/vcp.12036
- Rudloff, E. Bone marrow sampling. Clinicians Brief, May 2013. https://www.cliniciansbrief.com/article/how-collect-bone-marrow-samples
- Harvey, J.W. Bone marrow core biopsy. Clinicians Brief, October 2004. https://www.cliniciansbrief.com/article/bone-marrow-core-biopsy
About the Guide: Eric J. Fish, DVM, PhD, DACVP (Clinical Pathology)
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Dr. Eric Fish has over a decade of experience in veterinary diagnostics, ranging from academia to the private sector. He was an early adopter of digital cytology, having started a static telepathology service called CytoVetStat and co-founding Lacuna Diagnostics. His interests include hematology, oncologic pathology, iron metabolism and biomarkers, bone marrow disorders, and digital pathology.
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