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Research Papers

Improved Method for the ASTM Platelet and Leukocyte Assay: Use of Minimal Heparinization in a Screening Test for Hemocompatibility of Blood-Contacting Medical Devices

[+] Author and Article Information
Tim Schatz

American Preclinical Services,
8945 Evergreen Boulevard,
Coon Rapids, MN 55433
e-mails: tschatz@apsemail.com;
tschatz@sjm.com

Sarah Howard

American Preclinical Services,
8945 Evergreen Boulevard,
Coon Rapids, MN 55433
e-mail: showard@apsemail.com

Deanna Porter

Abbott Laboratories,
177 East County Road B,
St. Paul, MN 55117
e-mail: DPorter@sjm.com

Kent Grove

Abbott Laboratories,
177 East County Road B,
St. Paul, MN 55117
e-mail: KGrove@sjm.com

Mark E. Smith

American Preclinical Services,
8945 Evergreen Boulevard,
Coon Rapids, MN 55433
e-mail: msmith@apsemail.com

Yan Chen

American Preclinical Services,
8945 Evergreen Boulevard,
Coon Rapids, MN 55433
e-mail: ychen@apsemail.com

1Present address: Abbott Laboratories, 177 East County Road B, St. Paul, MN 55117.

2Corresponding author.

Manuscript received April 16, 2018; final manuscript received August 3, 2018; published online November 19, 2018. Assoc. Editor: Matthew R. Myers.

J. Med. Devices 13(1), 011004 (Nov 19, 2018) (7 pages) Paper No: MED-18-1077; doi: 10.1115/1.4041805 History: Received April 16, 2018; Revised August 03, 2018

Most blood-contacting medical devices must be assessed for potential thrombogenicity prior to regulatory approval. A common assay for screening and qualifying devices involves monitoring the reduction of platelet and leukocyte (P&L) counts in whole blood exposed to the device. We have validated an improved method for assessing a device's effect on platelet activation and surface adhesion, offering significant improvement over the current ASTM F2888-13 method, which uses blood fully anticoagulated by acidified citrate (known to significantly inhibit platelet responsiveness). Our method uses minimal heparinization (final concentration 1 IU/mL) to optimize the response to commonly used control materials: latex, black rubber, and high-density polyethylene (HDPE). We also have shown the assay's capacity to appropriately assess a legally marketed comparator device (LMCD) with a documented clinical history. The test materials were prepared for incubation and allowed to remain in contact with the citrated or heparinized blood for ∼1 h at 37 °C. A complete blood count was performed prior to exposure, and at the end of the incubation period, reductions in P&L counts were recorded. Results from citrate-anticoagulated assay showed only a marginal response to the positive control, black rubber. Using heparinized blood, the assay generated a robust response to the positive controls, the “intermediate scoring” controls, and also assessed a legally marketed and approved device as clearly nonthrombogenic. This modification adds robustness and sensitivity to this quick and inexpensive thrombogenicity assay and should be incorporated into the next ASTM standards.

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References

ISO, 2017, “ Biological Evaluation of Medical Devices—Part 4: Selection of Tests for Interactions With Blood,” International Standards Organization, Geneva, Switzerland, Standard No. ISO 10993-4.
Wolf, M. F. , and Anderson, J. M. , 2012, “ Practical Approach to Blood Compatibility Assessments: General Considerations and Standards,” Biocompatibility and Performance of Medical Devices, J.-P. Boutrand , ed., Woodhead Publishing, Oxford, UK.
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ASTM, 2013, “ Standard Test Method for Platelet Leukocyte Count—An In-Vitro Measure for Hemocompatibility Assessment of Cardiovascular Materials,” American Society for Testing and Materials, ASTM International, West Conshohocken, PA, Standard No. F2888-13.
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Lu, Q. , Nehrer, J. , and Malinauskas, R. , 2015, “ Platelet and Leukocyte Counts as Hemocompatibility Indicators for Biomaterials: An Evaluation of ASTM Standard Test Method F2888-13,” Society for Biomaterials Annual Meeting and Exposition 2015: Driving Biomaterial Innvoation and the Race to Translation, Charlotte, NC, Apr. 15–18, p. 60.
Schatz, T. , Beltrame, A. , Howard, S. , and Smith, M. , 2017, “ Modification of the ASTM Platelet and Leukocyte Assay—Use of an Alternative and Clinically Relevant Anticoagulant: An In-Vitro Hemocompatibility Assessment of Blood Contacting Devices,” ASME Paper No. DMD2017-3345.
Grove, K. , Deline, S. , Schatz, T. , Howard, S. , Porter, D. , and Smith, M. E. , 2017, “ Thrombogenicity Testing of Medical Devices in a Minimally Heparinized Ovine Blood Loop,” ASME J. Med. Devices, 11(2), p. 021008. [CrossRef]
St. Jude Medical, 2016, “ St. Jude Medical Complaint Handling Database; Search Performed for Thromboembolic Events for the St. Jude MedicalTM ResponseTM Electrophysiology Catheter, May 2011–April 2016,” St. Jude Medical Data Analytics, St. Paul, MN.
van Oeveren, W. , Tielliu, I. F. , and de Hart, J. , 2012, “ Comparison of Modified Chandler, Roller Pump, and Ball Valve Circulation Models for In Vivo Testing in High Blood Flow Conditions: Application in Thrombogenicity Testing of Different Materials for Vascular Applications,” Int. J. Biomater., 2012, p. 673163. [CrossRef] [PubMed]
Sefton, M. V. , Sawyer, A. , Gorbet, M. , Black, J. P. , Cheng, E. , Gemmell, C. , and Pottinger-Cooper, E. , 2001, “ Does Surface Chemistry Affect Thrombogenicity of Surface Modified Polymers?,” J. Biomed. Mater. Res., 55(4), pp. 447–459. [CrossRef] [PubMed]
Wendel, H. , 2014, “ Hemocompatibility Testing in the 21st Century: Options and Pitfalls,” University of Tubingen, Tübingen, Germany, accessed Apr. 14, 2014, https://www.fda.gov/downloads/medicaldevices/newsevents/workshopsconferences/ucm397150.pdf

Figures

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Fig. 1

Summary of platelet count data from heparinized blood and results of Tukey analyses of normalized platelet counts as percent of blank control. Blank = negative blank control, HPDE = negative reference material, RC = Response™ Electrophysiology Catheter, APU = abraded polyurethane, L = latex, N = nitrile, BR = black rubber. Letters on the graph represent significantly different mean responses for calculated percent of negative blank control. Columns sharing a common letter are not statistically different. Materials not sharing a common letter were significantly different from all the other materials. Figure 1(a) shows normalized platelet counts for the initial assay and Fig. 1(b) shows normalized platelet counts for the secondary assay, each with statistical comparisons depicted. Figure 1(c) shows the combined mean normalized platelet counts (per cent of blank) for positive controls, HDPE negative reference material, and Response Catheter (RC) are not different from each other. Stratification of significant differences in mean platelet responses were observed among the three additional materials tested, all showing significant differences from the blank, with the nitrile showing a greater response not significantly different from the black rubber positive control.

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Fig. 2

Summary of leukocyte count data from heparinized blood and results of Tukey analyses of normalized leukocyte counts as percent of blank control. Blank = negative blank control, HDPE = negative reference material, RC = Response™ Electrophysiology Catheter, APU = abraded polyurethane, L = latex, N = nitrile, BR = black rubber. Figure 2(a) shows normalized leukocyte counts for the initial assay, Fig. 2(b) shows normalized leukocyte counts for the secondary assay and Fig. 2(c) shows normalized leukocyte counts for the combined assays, each with statistical comparisons depicted. The results of this Tukey analysis appear to be consistent with those observed in the analysis of platelet depletion. Letters on the graph represent significantly different mean responses in calculated percent of negative control (as detailed in the legend for Fig. 1).

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Fig. 3

Means and standard deviations of cell count results from the initial assay comparing performance of citrated blood to heparinized blood; see panel a for platelet counts and panel b for leukocyte counts. Each column represents the mean and standard deviation from a total of 15 replicates (five donors prepared in triplicate). A two-way analysis of variance was conducted to compare the main effects of test material and anticoagulation and the interaction with platelet and leukocyte cell counts. Only black rubber produced results significantly different from the mean response of the blank negative control for both platelet and leukocyte counts and only in the heparinized blood (*post hoc Tukey Kramer analysis p < 0.05).

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Fig. 4

Visible clots formed on black rubber (a) latex (b) and the abraded polypropylene (c). There was a lack of a clot presence formed on the negative control, HDPE (d).

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Fig. 5

Representative examples of the LMCD Response™ Catheters are shown, post exposure in the in vitro platelet and leukocyte assay incubated in either heparinized or citrated blood. Note the deposition of thrombus only on the distal tip of the catheter in the heparinized-blood assay (panels (a) and (b)) with no evidence of formation elsewhere and no obvious evidence of thrombus formation in the citrated-blood assay (panels (c) and (d)).

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