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Keywords:

  • Cat;
  • erythropoiesis;
  • granulopoiesis;
  • hematopoietic tissue;
  • histopathology

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Background

Commonly used 11ga or 13ga biopsy needles are relatively large for cats and often preclude successful collection of bone marrow (BM) core biopsies.

Objectives

The objective was to compare 15ga to 13ga BM core biopsy ease of collection and specimen quality.

Methods

In 10 cats, humeral biopsies obtained with 15ga EZ-IO needles were compared with iliac biopsies obtained with 13ga Jamshidi needles. Body condition, ease of collection, section quality, postprocedure pain, and swelling at biopsy sites were scored. Specimen length on mounted slides was measured and specimens with quality scores of 3–5 out of a maximum value of 5 were considered to be of acceptable diagnostic quality. The distribution of all parameters was assessed by Shapiro–Wilk tests, and differences in parameters were assessed by ANCOVA.

Results

There were no significant differences between 15ga and 13ga biopsies, except that the 15ga humeral biopsy was judged to be easier to perform than 13ga iliac biopsy, and there was more severe postbiopsy swelling with 13ga biopsies. Facility score (mean ± SD), section quality score (median ± SD) and specimen length (mm, mean ± SD) were 12.7 ± 2.3, 2.0 ± 1.4, and 6.0 ± 2.1 for 15ga biopsies, respectively, and 8.9 ± 2.4, 1.0 ± 1.8, and 7.5 ± 2.5 for 13ga biopsies, respectively. Three specimens of acceptable quality were obtained with each 15ga and 13ga biopsies.

Conclusions

In cats, BM biopsy of the humerus with a 15ga needle is easier and causes less postbiopsy swelling than biopsy of the ilium with a 13ga needle. Sites and needles are equivalent with respect to yielding specimens of acceptable quality. Neither technique consistently captured high-quality specimens.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Bone marrow (BM) biopsy is indicated in animals with hematologic abnormalities that are not explainable based on clinical findings and examination of blood.[1] Aspiration biopsy of BM is performed more often than core biopsy, but the latter is necessary when aspiration yields an inadequate sample because of myelofibrosis or poor technique. Furthermore, core biopsy of the BM allows better assessment of focal lesions, cell density, cell distribution and marrow architecture. Core biopsy was superior to aspiration biopsy for assessment of cellularity and detection of metastases in people, and for detection of lymphoma in dogs.[2-4] However, ideally, both types of specimens should be collected as they provide complementary information.[1-5]

An impediment to BM core biopsy in cats may be the size of typical biopsy needles, which are designed for use in people. The smallest available needle, 13ga × 51 mm, may be awkward to use in cats and may increase the risk of fracture and needle slippage off the bone into the surrounding soft tissue. In addition, the pressure required to penetrate the osseous cortex may result in lameness due to soft tissue joint injury at the humeral head or femoral trochanteric fossa sites. To the authors' knowledge, there are no reports on the frequency of complications of BM core biopsy in feline patients, but the above difficulties have anecdotally been identified.

In studies in people, 58% of BM core biopsies from adults and up to 50% from children were of inadequate quality.[6, 7] Specimen quality varied by operator.[6, 7] Similar information has not been reported for cats, but, for our teaching institution with variable levels of operator experience, it is estimated that < 30% of core biopsies from cats are of diagnostic quality. This may reflect difficulty encountered when performing BM core biopsy with 13ga or larger needles.

In an effort to both encourage BM core biopsy in small dogs and cats, and improve sample quality, we have investigated BM core biopsy using smaller needles. We previously reported the feasibility of obtaining BM core biopsies in small dogs using 15ga needles intended for intraosseous fluid therapy (EZ-IO Intraosseus Infusion System needles; Vidacare, San Antonio, TX, USA).[5] In the study reported here, these same needles were used to obtain BM core biopsies in cats, and the biopsies were compared with those obtained with conventional 13ga needles.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Cats

Ten clinically healthy neutered male (5) and spayed female (5) Domestic Shorthair or Longhair cats ranging in age from 2 - 7 years (mean 4.7 and median 5.0 years) were studied. All cats had unremarkable hemograms (Advia 120 Hematology System, Siemens Healthcare Diagnostics; Mississauga, ON, Canada), blood smear evaluation and serum biochemistry profiles (Cobas C 501; Roche Diagnostics Canada, Laval, QC, Canada), and were seronegative for feline leukemia and immunodeficiency virus infections (SNAP FIV/FeLV Combo Test; IDEXX Laboratories, Markham, ON, Canada). Cats ranged in weight from 3.4 - 8.4 kg (mean 5.8 and median 6.1 kg). Body condition scores on a scale of 1–9 (Nestlé Purina Body Condition System, www.purina.com/cat/weight-control/bodycondition.aspx) were independently ascribed by 2 investigators with 90% agreement, and in the one case of disagreement, the mean of the 2 scores was used. The body condition scores ranged from 5 - 9 (mean and median = 7).

Anesthesia for BM biopsy was induced with an intramuscular injection of 0.08 mg/kg medetomidine (Cepetor; Modern Veterinary Therapeutics, Coral Gables, FL, USA) combined with 5.8 mg/kg ketamine (Vetalar; Bioniche Animal Health Canada Inc., Belleville, ON, Canada) and 0.4 mg/kg butorphanol (Torbugesic; Wyeth Animal Health, Guelph, ON, Canada). Cats were then intubated and anesthesia maintained with isoflurane (Aerrane; Baxter Corporation, Mississauga, ON, Canada). Following recovery from anesthesia, analgesia was provided with 4–5 mg/kg tramadol per os every 12 hours for 3 days (Chiron Compounding Pharmacy Inc., Guelph, ON, Canada) and cats were assessed for postbiopsy pain (Colorado State University Veterinary Medical Center Feline Acute Pain Scale, range 0 [no pain] to 4 [maximal pain] at 24, 48, and 72 hours by 2 investigators [AAO and AD]).[8] Postprocedural swelling of biopsy sites was also scored at each time point as 0 (none), one (mild), 2 (moderate), or 3 (marked). All procedures met guidelines set by the Canadian Council on Animal Care.[9, 10] The study was approved by the Animal Care Committee at the University of Guelph and was performed in accordance with the Animals for Research Act (Ontario, 1980).

Bone marrow biopsy and evaluation

Humeral and iliac biopsies (Figure 1) were obtained from each cat with the order of biopsy randomly assigned. Humeral biopsies were obtained with disposable 15ga × 25 mm EZ-IO needles (Figures 1A to G). The cats were positioned in right lateral recumbency, and the needle was inserted into the greater tubercle of the left humerus and advanced at an approximately 30° angle from the long axis of the bone. Each needle was used for 2 cats with re-sterilization between procedures (STERRAD, Advanced Sterilization Products; J & J Medical Products, Markham, ON, Canada). Iliac biopsies were obtained with disposable 51 mm Jamshidi Bone Marrow Biopsy/Aspiration Needles (Cardinal Health Canada Inc, Vaughan, ON, Canada) (Figure 1H), using standard technique.[1, 5] The cats were positioned in sternal recumbency, and the needle was advanced from the medio-dorsal aspect to the latero-ventral aspect of the ilium (Figure 1I). Each needle was used for one cat. Biopsies were placed directly into Davidson's fixative (consisting of 2 parts 37% formalin, 3 parts isopropanol, one part glacial acetic acid, 3 parts water, and eosin to color, prepared by the Animal Health Laboratory, University of Guelph, Guelph, ON, Canada) and after 15 minutes wrapped in fixative-soaked tissue paper (Bio-Wrap; Leica Microsystems, Winnipeg, MB, Canada), placed into a biopsy cassette, and then transferred to 10% buffered neutral formalin for 24 hours. Specimens were decalcified for 15 minutes in an acidic chelating solution (Fisher Scientific, Nepean, ON, Canada), embedded in paraffin and mounted as 3 μm sections using standard histotechnology methods (Animal Health Laboratory).

image

Figure 1. Types of needle and anatomical location for bone marrow biopsy in cats. (A) 15ga × 25 mm EZ-IO needle showing trochar tip of stylet and trephine-like tip of cannula. (B) Hub without stylet for manual maneuvers (the stylet had been cut off with a metal-cutting saw). (C) The assembled cannula and stylet were inserted into the humeral head cortex. (D) The stylet was removed. (E) Hub without stylet was attached to the cannula. (F) The cannula was advanced with manual effort clockwise into the marrow to full depth, and then withdrawn with clockwise action. (G) The probe (15ga in diameter) from the 13ga Jamshidi needle was used to extrude the specimen in an anterograde direction into Davidson's fixative. The specimen was wrapped in tissue paper and after 15 minutes transferred to formalin. (H) 13ga biopsies were obtained from the left ilium using a 51 mm Jamshidi needle. (I) Biopsy paths for 15ga biopsy of the humerus (left) and 13ga biopsy of the ilium (right).

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All biopsies were performed by one investigator (AAO) assisted by a second investigator (AD). Each biopsy procedure was repeated up to a maximum of 3 times until a specimen with an estimated length ≥ 5 mm was obtained. Site localization, needle insertion, and needle advancement were individually scored on a scale of one (most difficult) to 5 (most easy) with respect to ease, and summed for a facility score, as previously described.[5] The lengths of stained biopsy sections on the mounted slides were measured with a ruler as previously described.[5]

All biopsies were coded to blind the evaluator (DB) to cat and biopsy site. For fragmented biopsies, if fragments were not displaced and clearly represented a single cylindrical piece broken into several fragments, the lengths of the individual fragments were summed.[5] For disjointed biopsy fragments, the longest fragment was recorded. Biopsy quality was scored from one (low) to 5 (high) according to the number of intertrabecular spaces free of artifacts, as previously described.[5] A score of 0 was given if no hematopoietic tissue was present. Hematopoietic tissue was also quantitatively and qualitatively assessed.[5]

Statistical analysis

Data were assessed with statistical analysis software (SAS Institute, Cary, NA, USA). The normality of data for each variable was assessed with a Shapiro–Wilk test and examination of residuals after processing the data through a general linear model. Variables with nonnormal distributions were log-transformed to improve normality, and then parallel analyses were performed with transformed variables. Nontransformed and transformed variables were evaluated for differences between biopsy collection methods by ANCOVA for repeated measures accounting for effects of weight and body condition score. These variables included biopsy time, number of attempts, facility score, biopsy quality score, biopsy section length, and 24 hours and 48 hours postprocedure pain scores and swelling at biopsy sites. Relationships between biopsy quality and length were examined with Spearman's correlation coefficients. The number of biopsy attempts was compared between collection methods with Friedman's test. For all tests  .05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Comparison of 15ga humeral and 13ga iliac biopsies

There were no significant differences between biopsy methods with respect to time or number of attempts, but facility scores for 15ga humeral biopsies were significantly higher than for 13ga iliac biopsies (Table 1). Greater facility of obtaining humeral biopsies was due to easier needle insertion (= .03) and advancement (< .001), but not due to easier localization of biopsy site (= .18). There were no significant differences in specimen quality and length. With respect to postprocedure assessment of pain, all cats were assessed to be normal for psychological and behavioral parameters, and for body tension. No painful reactions to palpation were observed at humeral sites, whereas 2 cats had mild reactions to palpation of iliac sites. No postprocedure swelling was observed at humeral sites, but 7 cats had mild swelling (score = 1) at iliac sites at 24 hours, which was less apparent at 48 hours in all but one cat. All cats were clinically normal at 72 hours. There were no significant effects of body weight or body condition on any variable.

Table 1. Facility of acquisition, specimen quality, and postprocedural clinical findings for 15ga humeral and 13ga iliac bone marrow core biopsies obtained from 10 clinically healthy cats.
Site, Type of NeedleTime (Minutes)*Attempts (Number)Facility (Score)*, Specimen Quality (Score)Specimen Length (mm)*Painful Reaction to Palpation at 24 Hours§ (Score)*Swelling at 24 Hours (Score)*Painful Reaction to Palpation at 48 Hours§ (Score)*Swelling at 48 Hours (Score)*
  1. Significantly different: a= .003, b,c< .001.

  2. *Mean ± SD (minimum-maximum).

  3. Median ± CI (minimum-maximum).

  4. Facility score calculated for the last (ie, successful) attempt.

  5. §Colorado State University Veterinary Medical Center Feline Acute Pain Scale: Response to Palpation.

Humerus, 15ga EZ-IO5.1 ± 2.6 (2.2–9.3)2.0 ± 1.1 (1–3)12.7 ± 2.3a (9–15)2.0 ± 1.4 (0–4)6.0 ± 2.1 (3–10)0000
Ilium, 13ga Jamshidi6.1 ± 1.7 (4.5–9.5)2.0 ± 0.6 (1–3)8.9 ± 2.4a (6–12)1.0 ± 1.8 (0–5)7.5 ± 2.5 (5–12)0.2 ± 0.4 (0–1)0.9 ± 0.3b (0–1)0.3 ± 0.5 (0–1)0.7 ± 0.5c (0–1)

Nine of the 15ga and 7 of the 13ga biopsies captured hematopoietic tissue; the other specimens (one 15ga and 3 13ga specimens) contained only blood and cortical bone. Examples of specimens with high quality score and/or length for each collection method are shown in Figures 2A–C. Only one specimen was given a quality score of 5 (Figure 2B). Poor quality of 15ga specimens was most often due to fragmentation and crush artifact (eg Figures 2D–E). Fragmentation was apparent in some 13ga specimens (eg Figure 2C); and poor quality was also assigned due to presence of cortical bone rather than trabecular bone with interspersed hematopoietic tissue (eg Figure 2F–G). Other artifacts were not noted. Four of the 15ga biopsies were of higher quality than the corresponding 13ga biopsies from the same cat, and 3 were of equivalent quality. Three of the 13ga biopsies were of higher quality than the corresponding 15ga biopsies. Considering all 15ga and 13ga biopsies combined, hematopoietic tissue was obtained from all cats, but specimens with a quality score of ≥ 3 were obtained from only 5 cats (3 humeral and 3 iliac biopsies; one cat had biopsies with a quality of score of 4 from both sites). There was no significant effect of the number of attempts on specimen quality or length. Specimen quality and length were significantly correlated for humeral (ρ = 0.80, = .02), but not iliac (ρ = .6, = 0.06) biopsies.

image

Figure 2. Images of sections of 7 humeral or iliac bone marrow biopsies obtained with 13 and 15ga needles from cats. H&E, bar = 1 mm. (A) 15ga humeral biopsy with highest quality score and length; quality = 4; length = 10 mm. (B) 13ga iliac biopsy with highest quality score; quality = 5; length = 7 mm. (C) 13ga iliac biopsy with the greatest length; quality = 4; length = 12 mm. (D) Poor quality 15ga humeral biopsy; quality = 2; length = 5 mm. (E) Poor quality 15ga humeral biopsy; quality = 1; length = 3 mm. (F) Acceptable quality 13ga iliac biopsy; quality = 3; length = 10 mm. (G) Poor quality 13ga iliac biopsy; quality = 1; length = 11 mm.

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Six of the 15ga and 5 of the 13ga biopsies were considered adequate specimens for assessment of bone marrow architecture and morphology; all intact spaces in these biopsies had normal hematopoietic tissue and cell morphology. Comparison of adequate biopsy specimens from 4 cats showed no significant differences for cell density and presence of lymphocytes/plasma cells. However, cellularity, assessed as high in 2 of the 13ga biopsies, was assessed as medium in the corresponding 15ga biopsies; and megakaryocytes, assessed as 4–9/low-power field in one 13ga biopsy, were assessed as ≤ 3/low-power field in the 15ga biopsy. Granulocytic:erythrocytic ratio was greater in 15ga biopsies compared with 13ga biopsies in 2 cats, and lower in the 15ga biopsy of one cat. All granulocytic:erythrocytic ratios were within the expected range for hematologically normal cats. Discordant results between biopsies were not related to differences in quality.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In this study, we investigated 15ga BM core biopsies from clinically healthy cats using EZ-IO intraosseous infusion needles. The procedure used to obtain biopsies from the humerus was similar as previously reported for dogs.[5] The cannulae were advanced manually rather than with the use of a power driver because the latter had neither increased facility scores in dogs[5] nor in preliminary studies with feline cadavers (data not shown), and there appeared to be more directional control of the advancing cannula with manual effort.

Results demonstrated that 15ga BM core biopsy was feasible in cats and that EZ-IO needles were more easily inserted and advanced in the humeral head compared with 13ga Jamshidi needles in the ilium. Furthermore, 15ga humeral biopsies were associated with less pain and swelling postoperatively. However, the different biopsy needles are not entirely comparable as the biopsy site probably also affected procedural facility and postprocedural clinical signs. Iliac 13ga biopsy was selected as the gold standard against which to compare the novel technique as an informal institutional review revealed that this site had yielded the highest quality biopsies in cats compared with humeral and femoral biopsies. The humeral head was chosen for 15ga biopsy as it is anecdotally a common site for BM biopsy in cats and the site was previously used successfully in small dogs.[5] Comparative 15ga biopsy of the ilium was not performed because of the low quality of such biopsies in small dogs.[5]

There were no significant differences in specimen quality and length, but the main factors reducing biopsy quality were fragmentation and crush artifact in 15ga biopsies, and presence of cortical rather than trabecular bone in 13ga biopsies. The former finding was also observed in dog BM biopsies,[5] and emphasized the importance of careful specimen handling. Biopsy quality and length were strongly correlated for 15ga humeral biopsies in this study, and previously in dogs,[5] likely attributable to more intertrabecular spaces containing hematopoietic tissue free of artifact. Davidson's fixative was used in this study in an attempt to minimize cell loss. This fixative is alcohol-based and causes rapid coagulant fixation, while formalin more slowly fixes tissue by cross-linking methylene bridges.[11] Presence of cortical rather than trabecular bone along the length of 13ga biopsies probably resulted from cutting along the margins of the narrow marrow cavity of the ilium. As 13ga Jamshidi needles have tips tapered to 15ga, the actual biopsy diameter is 15ga, but hematopoietic tissue and short pieces of trabecular bone probably expand once within the cannular cavity. However, extensive cortical bone in biopsies may interfere with such expansion, leading to a relatively narrow appearance and probably lower quality of such specimens.

The average quality score for 15ga humeral biopsies in cats was 2.0 ± 1.4, which was similar to results with this needle in dogs, although the biopsies from dogs were generally longer.[5] The longer specimens captured in dogs may reflect their larger body size (6–13.4 kg). Assessment of biopsy quality as “acceptable” was by strict guidelines necessitating presence of at least 3 intertrabecular spaces free of artifact.[5] This amount of hematopoietic tissue was considered essential to adequately interpret cellularity and architecture, but may be a more stringent criterion than often applied to BM biopsies from small animals. Although this study was insufficiently powered to measure agreement on cellular composition in different biopsies, there appeared to be similar assessment of hematopoietic tissue morphology. Lower cellularity and lower megakaryocyte counts in 2 and one of 15ga biopsies, respectively, suggest that some caution should be exercised in diagnosing hypocellularity in such samples, especially if they are of limited or poor quality.

A limitation of this study was that the pain scoring system has not (to the authors' knowledge) been validated. Since data acquisition, a scoring system for acute postoperative pain in cats with initial validation has been reported.[12] Post-hoc application of this system to the data reported herein would yield similar results.

In conclusion, this study demonstrated that 15ga BM biopsy is feasible in cats, that the humerus is a suitable site for such biopsy, that good quality specimens may be obtained, and that the probability of obtaining a good quality specimen with 15ga needles from the humerus is similar to 13ga biopsy of the ilium. However, obtaining adequate BM biopsies from cats remains challenging with either type of needle and site, and overall, only approximately half of all attempts yielded an acceptable specimen with ≥ 3 intertrabecular spaces free of artifact. Further investigation of methods to improve specimen quality in feline BM core biopsy is warranted.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This project was funded by the Ontario Veterinary College Pet Trust. The authors thank Jennifer Brandow and Michelle Visnesky for technical assistance; Vicky Abrams-Ogg for photographs in Figure 1; technicians of the Histopathology Laboratory, University of Guelph for section preparation; and Gabrielle Monteith for assistance with statistical analysis.

Disclosure: The authors have indicated that they have no affiliations or financial involvement with any organization or entity with a financial interest in, or in financial competition with, the subject matter or materials discussed in this article.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
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  • 2
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    Available at: www.ivapm.org. Accessed October 5, 2011.
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    Olfert ED, Cross BM, McWilliam AA, eds. Guide to the care and use of experimental animals. Vol 1. 2nd ed. Ottawa, Canada: Canadian Council on Animal Care; 1993.
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    Olfert ED, Cross BM, McWilliam AA, eds. Guide to the care and use of experimental animals. Vol 2. Ottawa, Canada: Canadian Council on Animal Care; 1984.
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    Nowacek JM, Kiernan JA. Fixation and tissue processing. In: Kumar GL, Kiernan JA, eds. Education Guide: Special Stains and H & E, 2nd ed. Carpinteria, CA: Dako North America; 2010:141152.
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    Brondani JT, Luna SP, Padovani CR. Refinement and initial validation of a multidimensional composite scale for use in assessing acute postoperative pain in cats. Am J Vet Res. 2011;72:174183.