Research Article | Open Access

¹Department of Periodontics, Army Postgraduate Dental School, Postgraduate Dental College, Uniformed Services University, Fort Eisenhower, GA
²Department of Periodontics, United States Army Dental Activity, Fort Leonard Wood, MO
³Department of Periodontics, United States Army Dental Activity, Fort Riley, KS
⁴Department of Periodontics, United States Army Dental Activity, Fort Wainwright, AK
⁵Department of Oral Pathology, Army Postgraduate Dental School, Tripler Army Medical Center, HI

*Corresponding author: Alan R George, Department of Periodontics, Army Postgraduate Dental School, Postgraduate Dental College, Uniformed Services University, Fort Eisenhower, GA

Abstract

---

Background: In periodontics, interest in use of autologous blood-derived products (ABPs) has increased due to demonstrated safety, enhanced initial healing, and for some applications, superior clinical outcomes. Periodontists commonly place peripheral intravenous catheters for moderate sedation, thus encountering opportunities to utilize ABPs routinely. The purpose of this report is to describe a safe blood collection technique for ABP preparation and define typical blood volume requirements for periodontal procedures.
Methods: Five cases requiring various amounts of liquid and membrane-formed platelet-rich fibrin are presented. Case 1 involves treatment of an infrabony periodontal defect. Case 2 illustrates alveolar ridge preservation at a maxillary right second premolar site. Case 3 demonstrates repair of a defect related to nasopalatine duct cyst removal. Cases 4 and 5 illustrate the use of PRF in sinus elevation and root coverage, respectively.
Results: Use of PRF in the presented cases added minimal procedural time and expense. Blood samples varied from 20 to 60 ml in volume. There were no complications related to blood collection or use of PRF. Each patient reported minimal discomfort limited to the first few postoperative days. Favorable early healing was observed in each case.
Conclusions: The blood collection method described in this report, which is consistent with published standards of practice, necessitates few additional steps and supplies for practitioners already placing peripheral intravenous catheters. Blood volumes necessary for typical procedures in periodontics are safe and well below maximum attainable samples.
Key words: Clinical Protocols; Biological Products; Blood; Blood Platelets; Platelet-Rich Fibrin; Treatment Outcome
Abbreviations: ABP: Autologous Blood-Derived Products, DFDBA: Demineralized Freeze-Dried Bone Allografts, PRP: Platelet-Rich Plasma, PRF: Platelet-Rich Fibrin, ARP/ARR: Alveolar Ridge Preservation/Reconstruction, IV: Intravenous,APDS: Army Postgraduate Dental School, FDBA: Freeze-Dried Bone Allograft, NDC: Nasopalatine Duct Cyst, SCTG: Subepithelial Connective Tissue Graft, TBV: Total Blood Volume, ED: Emergency Department, ANTT: Aseptic Non-Touch Technique

Introduction

---

It has been more than a half-century since Marshall Urist demonstrated the capacity of demineralized bone and dentin to induce bone formation when implanted into animal skin and muscle [1,2]. Dr. Gerald Bowers later presented histologic evidence of new bone, cementum, and functionally-oriented periodontal ligament fibers following application of demineralized freeze-dried bone allografts (DFDBAs) in human infrabony periodontal defects (IBDs) [3]. Since these landmark studies, multiple biologically active therapeutic agents have been developed to manipulate innate wound healing processes and induce bone and periodontal regeneration [4,5]. Most of these agents, known collectively as “biologics,” are commercially available proteins that stimulate recruitment, growth, differentiation, and metabolic activity of cells involved in regeneration [5]. Among biologics currently in widespread use, only autologous blood-derived products (ABPs) are patient derived [5,6].

The first ABP with significant dental application was platelet-rich plasma (PRP) [7-9]. This first-generation platelet concentrate was hampered by a complicated, time-consuming, and expensive chairside preparation involving the addition of an anticoagulant, two centrifugation cycles, and multiple containers [10]. Moreover, PRP delivered inconsistent efficacy in controlled clinical investigations [6,9]. In 2006, Choukroun introduced a simplified preparation protocol in which a blood sample without anticoagulant was centrifuged in a single cycle for 10 minutes at 3000 rpm [10]. The modifications initiated the second generation of plate concentrates—platelet-rich fibrin (PRF)—which offered superior handling properties and clinical performance [6,10]. Notably, ABP preparation protocols, including centrifugation parameters, continue to evolve. A recent review identified ten unique ABP preparations [11]. The temperature, magnitude and duration of force applied to the blood, addition of anticoagulant, and centrifugation tube material have the potential to alter the biological properties and clinical handling of the product [6,11].

Despite technological advances, questions regarding the clinical efficacy of biologics remain. Performance of each biologic must be assessed in the context of specific procedure types, and the available data are incomplete [5,6]. For example, evidence that a particular biologic accelerates soft tissue healing does not necessarily imply long-term clinical benefit or a favorable influence across a spectrum of procedures. Despite limitations in available research, a recent American Academy of Periodontology best evidence consensus statement concluded that ABPs significantly enhance clinical and radiographic outcomes following surgical treatment of IBDs, PRF producing superior outcomes in this context compared with enamel matrix derivative and PRP [6]. Furthermore, ABPs improve handling properties of bone biomaterials and possibly enhance mineralized tissue formation in alveolar ridge preservation/reconstruction (ARP/ARR) [6]. Multiple biologics, including ABPs, enhance initial soft tissue healing and may diminish complication risk following some surgical procedures [5,6].

Intuitively, ABPs, which are patient derived, should be safe for clinical use, and indeed, no ABP-related risks or detrimental effects have been reported [5,6]. Considering the consensus that ABPs add clinical benefit to conventional periodontal procedures and the common practice of intravenous (IV) access for moderate sedation in periodontics, routine use of ABPs appears attractive. However, in the periodontics literature, best practices for obtaining blood samples from peripheral lines have received minimal attention. The purpose of this report is to describe an armamentarium and protocol for safely obtaining blood samples for ABP preparation and demonstrate sample volumes typically needed in periodontics.

Materials And Methods

---

Patients in this report presented to the Department of Periodontics, Army Postgraduate Dental School (APDS), Postgraduate Dental College, Fort Eisenhower, Georgia, and completed an informed consent process involving verbal and written components. All patients received intravenous cannulation for moderate sedation and elected to provide blood samples for PRF preparation using the described protocol (Figures 1 and 2, Tables 1 and 2) [12-16]. In all cases, a single 20-gauge intravenous catheter was used for both blood collection and fluid/medication delivery [17-20]. In each case the blood was centrifuged at 1300 RPM for 14 minutes in sterile vacuum tubes.

Case 1
In June of 2020, a healthy male aged 41 years presented for evaluation of bone loss at the distal surface of the right mandibular second molar. Probing depths measured 1-3 mm generally, with bleeding 8-mm pockets distal to tooth #31. Radiographic findings included vertical bone loss to the apical third of the distal root. The patient elected a bone replacement graft with guided tissue regeneration using PRF. A 60-ml blood sample was collected for ABP preparation. Liquid PRF was combined with a particulate freeze-dried bone allograft (FDBA) and autogenous bone scrapings, then placed in the defect. Two PRF membranes were layered over the FDBA. Wound closure for primary intention healing was achieved with dense polytetrafluoroethylene sutures. Early healing was uneventful. After 2 years, partial radiographic bone fill was noted, residual probing depths measuring 4-5 mm (Figures 3 and 4).

Case 2
In September of 2020, a healthy male aged 37 years was referred for extraction of non-restorable tooth #4, ARP, and implant placement. A 20-ml blood sample was drawn using the described protocol. Minimal buccal and palatal mucoperiosteal flaps were reflected to permit membrane stabilization, and tooth #4 was extracted. A PRF membrane was trimmed and condensed into the apical third of the socket, and a particulate FDBA filled the remainder of the alveolus. A second PRF membrane was placed over the socket orifice and stabilized under the buccal and palatal flaps using a crossed horizontal mattress suture# (Figures 5 and 6). After 4 months of healing, a 4 x 10 mm implant** was installed with insertion torque of 40 N-cm. An implant-supported crown was delivered 6 months following implant placement.

Case 3
In July of 2022, a healthy male aged 57 years presented for implant evaluation. A 7.5-mm heart-shaped, low-density lesion near the midline, apical to the maxillary central incisors, was an incidental finding on the cone-beam computed tomography volume (Figure 7). An oral and maxillofacial radiology consult confirmed the lesion was consistent with nasopalatine duct cyst (NDC), and enucleation was recommended. A 60-ml blood sample was collected using the described technique. Aspiration of the lesion was negative. A full thickness palatal flap was reflected from tooth #5 to tooth #11. The thin, superficial bone partially covering the NDC was removed. The cyst was then enucleated and stored in formalin. The biopsy was interpreted by an oral pathologist who confirmed the NDC diagnosis. A particulate FDBA (1.2 ml) was mixed with liquid PRF. The PRF-allograft mixture was applied within the defect and covered with a PRF membrane (Figures 8 and 9). The palatal flap was approximated and stabilized with simple interrupted sutures.# The patient reported minimal discomfort limited to the first few postoperative days. At the 1-year follow-up assessment, the maxillary anterior appeared normal. No adverse effect of the surgery was appreciated.

Case 4
In July of 2020, a healthy male aged 22 years missing tooth #4 presented for implant evaluation. A CBCT scan revealed sinus pneumatization and mucosal thickening in the tooth #4 area. The patient elected sinus elevation with delayed implant placement. A 60-ml blood sample was collected for ABP preparation as described. A full thickness buccal flap was reflected, and the maxillary sinus was accessed. The Schneiderian membrane was reflected to the medial wall, and no perforation was detected. A 2.4-ml particulate FDBA was hydrated in a tetracycline hydrochloride solution (100 mg/ml), rinsed thoroughly, combined with liquid PRF, and applied in the sinus. Wound closure for primary intention healing was achieved using PTFE sutures, and healing was uneventful. At postoperative month 6, a CBCT volume was acquired to permit virtual implant placement and surgical template fabrication. Seven months following sinus elevation, a 4 x 10 mm implant** was stabilized with insertion torque > 35 N-cm. A bone core biopsy was obtained at implant surgery (Figures 10 and 11) and processed for histological evaluation. The alveolar ridge appeared clinically and histologically favorable for implant placement (Figure 12). Healing was uneventful, and an implant-supported crown was delivered 6 months after implant placement.

Case 5
In January of 2021, a healthy female aged 22 years complained of cold sensitivity from the left maxillary canine area. Tooth #11 exhibited 3 mm of gingival recession and a width of attached gingiva < 2 mm. The treatment plan included coronally advanced flap with subepithelial connective tissue graft (SCTG) and PRF. A 40-ml sample of blood was drawn for ABP preparation. Following full thickness facial flap reflection, the root was debrided with rotary, ultrasonic, and hand instruments. A periosteal releasing incision facilitated the coronal advancement of the flap, and the interproximal papillae were de-epithelialized. A SCTG was harvested from the left side of the palate using a single incision technique. A PRF membrane was placed in the palatal wound, and the margins were approximated with 5-0 chromic gut sutures.# The SCTG was submerged in liquid PRF, then stabilized at the cementoenamel junction of tooth #11. The coronally advanced flap was secured with sling†† and simple interrupted sutures.# Early healing was uneventful. Through one year of follow-up, no residual root exposure was noted. Esthetics were acceptable to the patient, and her chief complaint of hypersensitivity was adequately addressed (Figure 13).

Discussion

---

Specific guidelines on blood collection for ABP preparation do not exist. However, literature on blood collection for various medical purposes offers insight into best practices for periodontists. Blood sampling for screening and diagnostic testing ranks among the most common procedures in medicine, [21,22] and in hospitals, IV catheters are the most frequently used invasive medical devices [23-25]. Moreover, blood donation/banking for transfusion has been in widespread use since the 1940s. [26]. Admittedly, blood volumes safely obtainable during donation may have minimal relevance for patients undergoing surgical procedures. Typical blood donors are not subject to surgery-related stress and do not experience blood loss from a surgical wound. Nevertheless, the World Health Organization recommends limiting donations to 13% of total blood volume (TBV), which is known to correlate with body mass [27,28]. A typical 70-kg adult maintains about 5600 ml (5.9 quarts) of blood [27,28]. In perspective, routine blood donations range between 350 and 500 ml in volume [27]—7- to 10-fold higher than the average volume utilized in cases presented here.

Recommended total blood draw volume limits for screening, diagnostic testing, and other purposes have been defined for healthy and unhealthy individuals. For healthy patients, the maximum recommended sample volume within a 24-hour period is 3% of TBV (≈ 168 ml for a 70-kg adult) [27]. This limit is reduced to 2.5% of TBV (≈ 140 ml for a 70-kg adult) for compromised individuals [27]. Blood volumes collected for ABP preparation described in this report were substantially less than half of this maximum. At most institutions, maximum allowable blood sample volumes for research purposes mirror these clinical limitations [29,30].

Applying a saline lock (Figure 2) allows clinicians flexibility in the timing of blood collection. Preparation time required to produce an ABP varies by product type. Accordingly, clinicians may choose to collect blood prior to initiating surgery or pause intraoperatively to obtain the sample. In addition to deciding upon the blood draw timing, practitioners must select the number and type of centrifuge tubes. The tube choice depends upon the scope of the procedure and the ABP(s) required. Smooth plastic centrifuge tubes do not initiate clotting and are selected when liquid PRF is desired. In contrast, glass tubes do induce clotting and are chosen when plug- or membrane-formed PRF is needed. In many cases (Figures 3,4,7-9), practitioners utilize a combination of plastic and glass tubes to prepare multiple ABP types.

Although not specifically written for outpatient/ambulatory care, Infusion Therapy Standards of Practice, particularly those aiming for prevention of complications, are meant to be adapted and applied in any setting where infusion therapy is administered [24,31]. Peripheral IV catheter placement has been associated with adverse events such as phlebitis, local infiltration/extravasation, loss of patency, dislodgement, and infection [23,24,31-33]. Phlebitis is a poorly defined term, and consensus regarding identification and management of this complication is lacking. Thus, the incidence of phlebitis varies widely across studies—from <1% to 100% [33]. Phlebitis occurrence has been attributed to mechanical, chemical, and infective factors [23]. Rapid infusion and movement of the catheter intravenously may induce phlebitis by generating shear stress at the vessel wall [23]. Utilizing a catheter excessively large relative to the selected vein may contribute to mechanical phlebitis [13,23], especially in the presence of shear stress related to blood collection. The catheter should be free-floating within the lumen of the vessel [13,23]. Thus, periodontists may prefer a relatively large-diameter vein in the antecubital fossa—typically the basilic vein, median cubital vein, or cephalic vein—when planning blood collection. Chemical phlebitis relates to properties of medications delivered through IV catheters, particularly osmolality, pH, and solubility [23]. Among sedatives commonly used by periodontists, diazepam is associated with a comparatively high phlebitis rate [34].

Interestingly, incidence of infiltration and extravasation appears substantially higher in emergency departments (ED) compared with other hospital divisions [33]. This excess incidence may be attributable to use of IV catheters to draw blood, large-bore catheters, and high-volume fluid delivery in EDs [33]. Nevertheless, ability to collect blood through the same catheter used for fluid and medication delivery is a well-supported practicality that increases the convenience of ABP preparation [17-20]. No instances of infiltration/extravasation have occurred while applying the described protocol in the APDS from 2020 through 2023.

To maintain patency, it is recommended to flush peripheral IV catheters with 2 to 10 ml of normal saline or another appropriate fluid after each IV medication administered, or in the hospital setting, every 4 to 12 hours [12-14]. To manage an occluded catheter, practitioners should gently aspirate with a 5-ml syringe, then flush with 2 to 10 ml of normal saline. If resistance is encountered when performing the saline flush, the catheter should be removed, and a new catheter should be placed at another site [24,35].

Peripheral IV catheter dislodgement is generally preventable by properly securing the IV line with tape, thus countering forces that would remove the catheter from the vein. Dislodgement is a nuisance because it may interrupt sedation, surgery, and blood collection but also a threat to the practitioner’s ability to deliver reversal agents and other emergency medications. Thus, securing the IV line after catheter placement should be routine practice. Infection—including infective phlebitis—is considered a rare complication of peripheral IV catheter placement when Aseptic Non-Touch Technique (ANTT) is observed [23,31]. The skin overlying the site should be thoroughly disinfected before catheter placement, and a transparent sterile dressing should protect the site of venous access intraoperatively [23,24].

Conclusion

---

For clinicians providing moderate IV sedation, barriers to routine clinical ABP use are minimal. Blood volumes required for ABP preparation amount to a fraction of published recommended total blood draw volume limits. The blood collection method described in this report, which is consistent with published standards of practice, necessitates few additional steps and supplies for practitioners already placing peripheral IV catheters. By incorporating routine ABP use into their practices, clinicians may consistently accelerate wound healing in a variety of procedure types, and for some indications, enhance clinical outcomes.

Footnotes
^¶Cytoplast, Osteogenics, Lubbock, TX
^#Chromic Gut Monofilament Suture, Ethicon, Raritan, NJ
**Osseotite Certain, ZimVie, Westminster, CO
^††Polypropylene Suture, Ethicon, Raritan, NJ

Disclosure statement
The views expressed in this manuscript are those of the authors and do not necessarily reflect the official policy of the United States Government, the Department of Defense, the Defense Health Agency, or Uniformed Services University.
Funding source: The Defense Health Agency funded this research entirely. The authors received no extramural funding for this work.
Author contribution statement: All authors have contributed substantially to conceptualization of the article, writing the original draft, critical review, and editing. All authors have approved the final version of the manuscript.
Conflict of interest statement
The authors report no financial, economic, or professional interests that may have influenced the design, execution, or presentation of this work.

References

---

1. Urist MR. (1965) Bone: formation by autoinduction. Science. 150(3698): 893-899. [PubMed.]
2. Bang G, Urist MR. (1967) Bone induction in excavation chambers in matrix of decalcified dentin. Arch Surg. 94(6): 781-789. [PubMed.]
3. Bowers GM, Chadroff B, Carnevale R, et al. (1989) Histologic evaluation of new attachment apparatus formation in humans. Part III. J Periodontol. 60(12): 683-693. [PubMed.]
4. Susin C, Wikesjö UM. (2013) Regenerative periodontal therapy: 30 years of lessons learned and unlearned. Periodontol 2000. 62(1): 232-242. [PubMed.]
5. Suárez-López Del Amo F, Monje A. (2022) Efficacy of biologics for alveolar ridge preservation/reconstruction and implant site development: An American Academy of Periodontology best evidence systematic review. J Periodontol. 93(12): 1827-1847. [PubMed.]
6. Avila-Ortiz G, Ambruster J, Barootchi S, et al. (2022) American Academy of Periodontology best evidence consensus statement on the use of biologics in clinical practice. J Periodontol. 93(12): 1763-1770. [PubMed.]
7. Carlson NE, Roach RB Jr. (2002) Platelet-rich plasma: clinical applications in dentistry. J Am Dent Assoc. 133(10): 1383-1386. [PubMed.]
8. Marx RE. (2001) Platelet-rich plasma (PRP): what is PRP and what is not PRP? Implant Dent. 10(4): 225-228. [Ref.]
9. Pocaterra A, Caruso S, Bernardi S, et al. (2016) Effectiveness of platelet-rich plasma as an adjunctive material to bone graft: a systematic review and meta-analysis of randomized controlled clinical trials. Int J Oral Maxillofac Surg. 45(8): 1027-1034. [PubMed.]
10. Dohan DM, Choukroun J, Diss A, et al. (2006) Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I: technological concepts and evolution. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 101(3): e37-44. [PubMed.]
11. Palaiologou A, Keeling F. (2022) Autologous blood products: Usage and preparation protocols. Clin Adv Periodontics. 12(4): 287-293. [PubMed.]
12. Fernandez RS, Griffiths RD, Murie P. (2003) Peripheral venous catheters: a review of current practices. J Infus Nurs. 26(6): 388-392. [PubMed.]
13. Frank RL, Wolfson AB, Ganetsky M. (2023) Peripheral venous access in adults. In: Post T, ed. UpToDate. [Ref.]
14. Hadaway LC. (1998) Major thrombotic and nonthrombotic complications. Loss of patency. J Intraven Nurs. 21(5 Suppl): S143-S160. [PubMed.]
15. Halm MA, Gleaves M. (2009) Obtaining blood samples from peripheral intravenous catheters: best practice? Am J Crit Care. 18(5): 474-478. [PubMed.]
16. Sotnikova C, Fasoi G, Efstathiou F, et al. (2020) The efficacy of normal saline (N/S 0.9%) versus heparin solution in maintaining patency of peripheral venous catheter and avoiding complications: a systematic review. Mater Sociomed. 32(1): 29-34. [PubMed.]
17. Dietrich H. (2014) One poke or two: can intravenous catheters provide an acceptable blood sample? A data set presentation, review of previous data sets, and discussion. J Emerg Nurs. 40(6): 575-578. [PubMed.]
18. Hadaway L. (2003) Can I stop a drug infusion to draw blood? Nursing. 33(5): 14. [PubMed.]
19. Lesser FD, Lanham DA, Davis D. (2020) Blood sampled from existing peripheral IV cannulae yields results equivalent to venepuncture: a systematic review. JRSM Open. 11(5): 2054270419894817. [PubMed.]
20. Himberger JR, Himberger LC. (2001) Accuracy of drawing blood through infusing intravenous lines. Heart Lung. 30(1): 66-73. [PubMed.]
21. Quinn JG, Tansey EA, Johnson CD, Roe SM, Montgomery LE. (2016) Blood: tests used to assess the physiological and immunological properties of blood. Adv Physiol Educ. 40(2): 165-175. [PubMed.]
22. Czoski-Murray C, Lloyd Jones M, McCabe C, et al. (2012) What is the value of routinely testing full blood count, electrolytes and urea, and pulmonary function tests before elective surgery in patients with no apparent clinical indication and in subgroups of patients with common comorbidities: a systematic review of the clinical and cost-effective literature. Health Technol Assess. 16(50): i-xvi, 1-159. [PubMed.]
23. Nickel B. (2019) Peripheral Intravenous Administration of high-risk infusions in critical care: A risk-benefit analysis. Crit Care Nurse. 39(6): 16-28. [PubMed.]
24. Gorski LA. (2017) The 2016 infusion therapy standards of practice. Home Healthc Now. 35(1): 10-18. [PubMed.]
25. Zingg W, Barton A, Bitmead J, Eggimann P, Pujol M, et al. (2023) Best practice in the use of peripheral venous catheters: A scoping review and expert consensus. Infect Prev Pract. 5(2): 100271. [PubMed.]
26. Learoyd P. (2012) The history of blood transfusion prior to the 20th century--part 1. Transfus Med. 22(5): 308-314. [PubMed.]
27. World Health Organization. (2012) Blood Donor Selection: Guidelines on Assessing Donor Suitability for Blood Donation. Geneva: World Health Organization. [Ref.]
28. Kiechle FL. (2017) So You’re Going to Collect a Blood Specimen: An Introduction to Phlebotomy. 15th ed. CAP Press. PUB225. [Ref.]
29. University of Pennsylvania Human Research Protections Program. (2023) Maximum allowable blood draw volumes. [Ref.]
30. Peplow C, Assfalg R, Beyerlein A, et al. (2019) Blood draws up to 3% of blood volume in clinical trials are safe in children. Acta Paediatr. 108(5): 940-944. [PubMed.]
31. Gorski LA, Hadaway L, Hagle ME, et al. (2021) Infusion Therapy Standards of Practice, 8th Edition. J Infus Nurs. 44(Suppl 1): S1-S224. [PubMed.]
32. Helm RE, Klausner JD, Klemperer JD, Flint LM, Huang E. (2015) Accepted but unacceptable: peripheral IV catheter failure. J Infus Nurs. 38(3): 189-203. [PubMed.]
33. Marsh N, Webster J, Ullman AJ, et al. (2020) Peripheral intravenous catheter non-infectious complications in adults: A systematic review and meta-analysis. J Adv Nurs. 76(12): 3346-3362. [PubMed.]
34. Litchfield NB. (1983) Venous complications of intravenous diazepam. J Oral Maxillofac Surg. 41(11): 701-705. [PubMed.]
35. Barrus DH, Danek G. (1987) Should you irrigate an occluded i.v. line? Nursing. 17(3): 63-64. [PubMed.]