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Keywords:

  • Cytology;
  • fine-needle aspiration;
  • sample collection;
  • spleen

Abstract

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

Background: Ultrasound-guided fine needle aspiration of the spleen is commonly used in the diagnostic evaluation of veterinary patients. Techniques using suction delivered through a 6–20-cm3 syringe are the most commonly described means of obtaining cytologic samples of the spleen. Comparison studies of various human lesions have shown nonaspiration techniques to produce equal or superior cytologic specimens with less blood than specimens obtained using aspiration techniques.

Objective: The purpose of this study was to compare the quality of splenic cytology specimens obtained using aspiration and nonaspiration techniques.

Methods: Client-owned dogs (n=24) and cats (n=7) receiving an abdominal ultrasound at the University of Tennessee College of Veterinary Medicine were enrolled in the study between January and June 2005. Samples were obtained from patients with and without sonographic splenic abnormalities. Two clinical pathologists, working independently and blinded to the method of sample collection, graded the cytologic specimens using a subjective scoring system for cellularity, amount of blood, and preservation of cellular morphology.

Results: Agreement between the 2 independent observers was good. Direct comparison of the 2 techniques showed that samples obtained by the nonaspiration method had higher cellularity (P=.0002), less blood (P=.0023), and similar cell morphology (P=1.0000) compared with samples obtained by the aspiration method.

Conclusion: These results suggest the nonaspiration technique is a superior method for obtaining a high-quality cytologic specimen from the canine and feline spleen.


Introduction

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

Cytologic evaluation of the spleen is a commonly used tool in the diagnostic work-up of veterinary patients. Samples are often obtained with ultrasound guidance. Indications for obtaining cytologic samples from the spleen include evaluation of nodular or focal masses, evaluation of diffuse splenomegaly or abnormal ultrasonographic echotexture, and staging or characterization of systemic or multicentric hemic disorders.1 The most commonly reported method for acquiring cytologic specimens of the spleen of veterinary patients is through the use of fine-needle aspiration.2–5 In this technique, a 22–25-G needle and a 6–20-cm3 syringe typically are used to apply suction and obtain negative pressure for harvesting cells. A second technique, referred to as the nonaspiration, capillary, or pincushion technique, does not require a syringe and no suction is applied.6,7 In the nonaspiration technique, a 22–25-G needle is guided into the target organ and incompletely retracted and redirected several times in an attempt to shear and displace cells into the cylinder of the needle. Studies in human patients have shown that the nonaspiration technique produces equal or superior cytologic specimens with less blood than specimens obtained using the traditional aspiration technique.8–11 Although the nonaspiration technique also has been described for use in animals12–15 and has been used in a recent investigation in dogs and cats,16 to the authors' knowledge, no studies have been carried out to objectively evaluate this method in dogs and cats.

The objective of this study was to compare the quality of splenic cytology specimens obtained using aspiration and nonaspiration techniques. Comparison of the 2 techniques was based on the overall cellularity, amount of blood, and preservation of cell morphology in the cytologic specimens.

Materials and Methods

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

Animals

Dogs and cats in this prospective study were client-owned pets that received an abdominal ultrasound examination at the University of Tennessee College of Veterinary Medicine Veterinary Teaching Hospital. If the spleen was found to be sonographically abnormal, and fine-needle aspiration was requested, the patient was entered into the study. Patients with sonographically normal spleens also were entered into the study if sampling was requested for staging metastatic neoplasia or if the owner consented to the procedure. The experimental protocol was approved by the University of Tennessee Institutional Animal Care and Use Committee.

Samples

Ultrasound-guided cytology specimens were obtained by 1 of 5 on-duty sonographers using 2 techniques. For the aspiration technique, a 22-G needle was attached to a 6- or 12-cm3 syringe; the needle was guided into the spleen and negative pressure was applied to the syringe. For the nonaspiration technique, a 22-G needle was guided into the spleen and then gently moved up and down along the needle tract in an attempt to harvest cells; no negative pressure was applied to the syringe. Once a sample was obtained, it was deposited onto a glass slide(s) and a smear was prepared using a slide-over-slide technique; no attempt was made to control the manner in which the material was deposited onto the slide. All slides were stained with Wright's stain using an automated stainer (Aerospray 7120, Wescor, Logan, UT, USA). A random numbers table was used to code the slides “A” or “B” for the 2 techniques (aspiration, nonaspiration).

Before being evaluated specifically for this study, the slides were evaluated by the on-duty clinical pathologist for routine diagnostic purposes. Diagnostic interpretations included no cytologic abnormalities (n=9), lymphoid hyperplasia (n=4), extramedullary hematopoiesis (n=9), neoplasia (n=4), or a combination of these abnormalities (n=5). An interpretation of “no cytologic abnormalities” was based on the absence of lymphoid hyperplasia, no or mild extramedullary hematopoiesis, and no evidence of neoplasia.

Cytologic evaluation

Between 4 and 12 slides per case were prepared. No attempt was made to determine which slides were of diagnostic value; rather, 1 of the investigators (M.M.F.) scanned all of the slides, and for each case selected the 2 subjectively considered to be the best representatives of each sample collection technique (ie, 4 slides per case). Two board-certified veterinary clinical pathologists (C.J.L., M.M.F.), working independently and blinded to the method of sample collection, then graded the slides using a semiquantitative scoring system (Table 1). If the 2 observers' scores agreed within 1 point, the mean was assigned as the value for statistical analysis. If the scores did not agree within 1 point, the observers examined the slide together and determined a consensus value for statistical analysis.

Table 1.   Scoring system for evaluating splenic cytology samples obtained by aspiration and nonaspiration techniques.
CharacteristicScoreInterpretationDescription
Cellularity3HighVery high numbers of nucleated cells; includes both aggregates of stroma and individual cells
2ModerateMany nucleated cells in addition to those expected based on the amount of blood
1LowFew nucleated cells in addition to those expected based on the amount of blood; no or very few small aggregates of stromal cells
 0AcellularNo to rare nucleated cells in addition to those expected based on the amount of blood
Blood3MarkedMany RBCs, including areas of densely packed RBCs, impairing evaluation of nucleated cells
2ModerateMany RBCs, but not densely packed
1MildFew RBCs
 0NoneRare to no RBCs
Morphology3ExcellentEstimated > 90% nucleated cells intact
2GoodEstimated > 50% nucleated cells intact
1FairEstimated > 50% nucleated cells disrupted
0PoorEstimated > 90% nucleated cells disrupted

Statistical analysis

Agreement between observers was described as complete agreement (same score), partial agreement (score difference=1), or disagreement (score difference > 1). For each case, evaluation scores for cellularity, blood, and cell morphology for both slides obtained using the same technique were combined into a final mean score. Results were expressed as mean±SD, median, and minimum–maximum values for canine and feline samples separately and combined. Mean scores for dogs and cats combined were compared using a 2-tailed Wilcoxon's test for paired samples (MEDCALC Version 8.2.1.0, http://medcalc.be). Mean scores for dogs and cats were not statistically compared separately because of low sample numbers.

Results

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

Twenty-four dogs and 7 cats were enrolled in the study. The data set thus included 124 slides (31 cases × 2 techniques/case × 2 slides/technique). Agreement between observers was complete for > 70% of scores, partial for > 20% of scores, and in disagreement (on cellularity) for 1% of scores (Table 2).

Table 2.   Agreement between 2 clinical pathologists in the independent, blinded evaluation of cellularity, blood, and cell morphology in splenic cytology specimens from dogs and cats.
AgreementCellularityBloodMorphology
  1. Data are the number (percentage) of slides in each category. The total number of slides under Morphology is < 124 because some slides were acellular and therefore preservation of cell morphology could not be evaluated.

Complete agreement89 (72%)97 (78%)86 (77%)
Partial agreement34 (27%)27 (22%)26 (23%)
Disagreement1 (1%)0 (0%)0 (0%)

For mean evaluation scores from dogs and cats combined, samples obtained with the nonaspiration method had higher cellularity (P=.0002) and less blood (P=.0023) than samples obtained with the aspiration method (Table 3). The 2 methods resulted in no significant difference in cell morphology (P=1.0000). In 4 cases, the aspiration method produced higher cellularity than the nonaspiration method, but in each of these instances the difference in mean score was <1. Direct comparison of morphology included only 24 cases, because 7 cases had at least 1 slide that was judged to be acellular (data not shown) and mean scores therefore could not be determined.

Table 3.   Summary of evaluation scores for cellularity, blood, and cell morphology in splenic cytology specimens obtained using aspiration and nonaspiration techniques.*
SpeciesParameterCellularityBloodMorphology
AspirationNonaspirationAspirationNonaspirationAspirationNonaspiration
  • *

    Specimens were graded on a scale from 0 to 3 based on the criteria outlined in Table 1.

  • †Does not include 3 cases from dogs and 4 cases from cats because the samples were acellular and preservation of morphology could not be evaluated.

  • ‡Significantly different from aspiration sample (2-tailed Wilcoxon's test).

Dogs (n=24)Mean ± SD1.66 ± 0.62.17 ± 0.62.88 ± 0.262.68 ± 0.392.05 ± 0.152.07 ± 0.21
Median1.752.2532.8822
Minimum–maximum0.5–2.51–32–32–31.75–2.51.75–2.75
Cats (n=7)Mean ± SD1.11 ± 0.811.57 ± 0.792.29 ± 0.221.93 ± 0.372.17 ± 0.292.08 ± 0.29
Median11.52.25222.25
Minimum–maximum0–20.5–2.52–2.51.5–2.52–2.51.75–2.25
Combined (n=31)Mean ± SD1.53 ± 0.682.03 ± 0.682.74 ± 0.352.51 ± 0.492.06 ± 0.172.07 ± 0.21
Median1.752.2532.522
Minimum–maximum0–2.50.5–32–31.5–31.75–2.51.75–2.75

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 demonstrated that cytologic specimens from the canine or feline spleen obtained using a nonaspiration technique were more cellular and had less blood and similar cell morphology compared with specimens obtained using an aspiration technique.

Zajdela et al8 first described the use of a nonaspiration technique to obtain a cytologic sample of human mammary tumors in 1987. Although a statistically significant difference was not found with comparison with samples obtained by aspiration, the uncontrolled trial reported a lower percentage of nondiagnostic samples using the nonaspiration technique. The authors of this study also felt the nonaspiration technique caused less trauma to the tumor and surrounding tissues, reduced the amount of peripheral blood contamination in samples, and allowed for a more sensitive assessment of tumor consistency during sampling. In a study comparing the 2 techniques concurrently, dual sampling of 100 superficial mass lesions found the aspiration method to be diagnostic in more cases, but when diagnostic, the nonaspiration method more frequently produced superior quality specimens.10 A study evaluating ultrasound-guided sampling of malignant lymph nodes in humans found the aspiration technique produced more cellular samples but did not increase diagnostic yield. Furthermore, aspiration technique samples were associated with more peripheral blood contamination.11

Previous studies and review articles describing cytologic evaluation of the spleen in dogs and cats discuss only aspiration techniques as a means of obtaining a cytologic specimen.2–5 Recent veterinary textbooks include descriptions of nonaspiration methods for obtaining cytologic specimens from vascular tissues.12–14 The nonaspiration technique for acquiring ultrasound-guided fine-needle samples of various tissues in veterinary patients was first described in 1995.15 Similar to other reports,8–10 these authors advocated the nonaspiration technique as a means of obtaining samples with less hemodilution and higher relative cellularity. To the authors' knowledge, the study reported here is the first to directly compare aspiration and nonaspiration techniques in any animal species. The spleen was evaluated in this study because it is a highly vascular organ that is commonly sampled using a fine-needle technique.

Although previous reports advocate the nonaspiration technique as a means of obtaining less hemodiluted samples, the degree of cellularity appeared to be more variable.8–11 Therefore, the finding of higher cellularity in the nonaspiration specimens in this study was noteworthy. The spleen normally contains highly concentrated blood—a function of the splenic microvasculature that enables the increase in peripheral PCV following splenic contraction—so it is inappropriate to consider blood in a cytologic specimen from the spleen to be a contaminant.17,18 However, a cytologic diagnosis typically is based on the nucleated cell population(s), and a dense background of blood can sometimes interfere with microscopic evaluation. The results of this study suggest that the nonaspiration technique is a way to increase the yield of nucleated cells in splenic cytologic samples relative to the amount of blood. Although diagnostic utility of the 2 techniques was not specifically evaluated in this study, and the differences in cellularity, though significant, are small, it seems likely, based on the dual advantage of maximizing the yield of nucleated cells and minimizing the amount of blood, that the nonaspiration method would have a diagnostic yield equal to or greater than that obtained with the aspiration method.

A limitation of this study was that all specimens were not obtained by the same sonographer. Although this adds another potential variable to data interpretation, the fact that statistically significant differences were found in 2 of 3 variables, despite the relatively low number of samples, suggests it had a minor effect on the results. An additional limitation was that data from dogs and cats were combined before statistical analysis. The number of feline cases was too low to analyze statistically, and it is possible that our conclusions may not apply in equal measure to both species. However, inspection of the data suggests the feline results were consistent with the overall findings.

Although the results of this study would likely hold true for sampling of superficial lesions and other internal organs, future comparison studies need to be conducted to confirm the superiority of the nonaspiration technique in tissues other than the spleen. In conclusion, the nonaspiration technique is recommended for obtaining more cellular, less hemodiluted cytologic specimens of the canine or feline spleen.

Acknowledgments

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

This work was supported by a grant from the University of Tennessee, College of Veterinary Medicine Companion Animal Fund. The authors wish to thank the radiologists who collected samples, and the laboratory staff who helped with sample preparation.

References

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