Platelet rich fibrin: A novel treatment for trophic ulcer in Hansen's disease

Material and methods

We included ten leprosy patients with trophic, non-healing ulcers, extending upto the subcutaneous tissue, present for >8 weeks in treated cases of Hansen's disease. Exclusion criteria included infected ulcers, presence of bony sequestrum within the ulcer, any bleeding diathesis, treatment with anticoagulants (including aspirin), pregnant and lactating females and the presence of uncontrolled diabetes mellitus.

The ulcer margins were freshened using a sterile #15 blade and any callouses present were pared. The ulcer was cleaned daily for three consecutive days with normal saline before the commencement of the procedure. The size of the ulcer was measured by tracing its outline over transparent paper, and its area calculated by superimposing graph paper over the transparency (Figure I). Under aseptic precautions, 10 ml of venous blood was drawn into a plain vacutainer, and then immediately subjected to centrifugation at 2000 rpm for ten minutes. Three layers were thus obtained: straw-coloured platelet-poor plasma uppermost, platelet-rich fibrin in the middle and red cells at the bottom.

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Figure I.

1A & 1B: area calculation using transparency and graph paper. IC: PRF obtained. ID: PRF transferred to sterile gauze. IE: PRF applied on ulcer.

After thoroughly cleaning the ulcer with povidone-iodine and normal saline, PRF was separated from red cells by a sterile forceps and blade, over a piece of sterile gauze, and placing it over the entire ulcerated area with the help of sterile jeweller's forceps. Sterile gauze was then placed over the ulcerated lesion and the wound dressed with roller bandage (Figure 1). A course of oral antibiotics was given empirically preceding the PRF therapy, in case the ulcer was secondarily infected.

Bed rest was ordered, with avoidance of weight bearing over the ulcerated area. Weekly follow-up allowed ulcer assessment (Figure 2). The percentage reduction in ulcer area was documented and weekly dressing with PRF therapy continued till adequate response was seen.

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Figure 2.

2A: Ulcer at baseline. 2B: Ulcer after 1^stweek's dressing of PRF. 2C: Ulcer after 2ⁿweekly dressing of PRF. 2D: Ulcer after 3^rdweekly dressing of PRF. 2E: Ulcer after 4^thweekly dressing of PRF. 2F: Healed ulcer after 6^thweekly dressing of PRF.

Results

The mean age of the patients was 44.3 years, mean duration of ulcer existence 7.4 months, and mean ulcer size 6.25 cm² at baseline. Demographic details are shown in Tables 1 and 2. The maximum ulcer size was 12 cm² and the minimum 2 cm². The maximum number of weekly dressings required for significant healing was six (Table 3, Figure 3), and the minimum number was three, with the mean being four. We found that after the third weekly dressing, there was a significant reduction in the ulcer size (p  =  0.015). No adjuvant treatment was given for the treatment of these ulcers. There was complete healing in eight patients while the remaining two had seen significant improvement but were lost to follow up in the fifth and seventh week when ulcer size had reduced to 0.5cm² (from initial sizes of 4 & 11cm² respectively). No patient experienced any adverse event using PRF. Even after a year of follow up, no recurrence was reported in any patient

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Figure 3.

Image showing the progressive improvement per session. The yellow shading shows a statistically significant improvement as compared to baseline.

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Table 1.

Demographic data of patients.

S. No.	Age	Sex	Type of hansen	Duration of Ulcer	Site of Ulcer	Size of ulcer (cm2) at first visit
1.	53 years	Male	LL	3 months	Planter aspect of left big toe	4
2.	54 years	Male	LL	12 months	Planter aspect of right big toe	12
3.	38 years	Female	BL Hansen	7 months	Planter aspect of right big toe	2
4.	35 years	Female	LL	5 Months	Planter aspect of left big toe	4
5.	60 years	Male	LL	9 Months	Planter surface of left foot	4.5
6.	40 years	Male	BL Hansen	6 months	Planter aspect of left big toe	3.5
7.	45 years	Male	LL	12 months	Tip of Right big toe	6.5
8.	36 years	Female	LL	6 months	Heel of Left foot	11
9.	40 Years	Male	BL Hansen	7 months	Planter surface near little toe	7
10.	42 years	Male	LL	7 months	Lateral aspect of right foot	8

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Table 2.

Summary of the data.

No. of patients	10
Mean age	44.3 years
Gender	Male: 7, Female: 3
Mean duration of ulcer	7.4 months
Mean size	At baseline	At 2nd visit	At 3rd visit	At 4th visit	At 5th visit	At 7th visit	At 7th visit
	6.25 cm2	4.5	3	1.55	1	0.375
No. of patient healed	At 2nd visit	At 3rd visit	At 4th visit	At 5th visit	At 6th visit	At 7th visit
	0	0	2	2	3	1
P value at different sittings	2nd visit (p value)			3rd visit (p value)
	0.234			0.015

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Table 3.

Size Of ulcer at subsequent visits.

S.No.	Area (cm2) at 1st visit	Area (cm2) at 2nd visit	Area (cm2) at 3rd visit	Area (cm2) at 4th visit	Area (cm2) at 5th visit	Area at 6th visit	Area at 7th visit
1.	4	2	1.5	0
2.	12	10	6	4	2	1	0
3.	2	1	0.5	0
4.	4	2	1.5	0.5
5.	4.5	3	2	0.5	0
6.	3.5	2.5	2	0.5	0
7.	6.5	5	3.5	2.5	1	0
8.	11	9	6.5	4	1.5	0.5
9.	7	4.5	3	1.5	1	0
10.	8	6	3.5	2	0.5	0

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Table 4.

Classification Of platelet concentrate:. ⁹

Pure Platelet-Rich Plasma (P-PRP)	Liquid platelet suspensions, without leukocytes
Leukocyte- & Platelet-Rich Plasma (L-PRP)	Liquid platelet suspensions, with leukocytes
Pure Platelet-Rich Fibrin (P-PRF)	Solid fibrin biomaterials, without leukocytes
Leukocyte- and Platelet-Rich Fibrin (L-PRF)	Solid fibrin biomaterials, with leukocytes

Discussion

All our patients showed significant reduction of ulcer size as reported in similar studies.^1–3

Trophic ulceration is seen in 10% of leprosy patients. Various treatment modalities have been used for their treatment, namely vacuum assisted closure, hyperbaric oxygen therapy, moist wound dressing and reconstructive surgery. Conventional treatment comprises of surgical debridement with or without a plaster cast The cost of such reconstructive techniques as well as rehabilitation procedures prescribed naturally adds to the psychological burden of the disease.

Topical growth factors derived from platelets have recently been used to treat these kind of ulcers.

Platelet-rich fibrin (PRF) is a second-generation platelet concentrate, prepared from centrifuged blood. It was first developed in France by Choukroun et al. ⁴ It is a fibrin clot rich in platelets without addition of thrombin during preparation. It differs from platelet rich plasma (PRP) in being less fragile and greater in volume when made from an equal quantity of blood. The PRF membrane was intact even after one week of incubation in culture media whereas PRP membrane completely dissolved. The PRF membrane slowly releases large amounts of TGFβ 1, PDGF-AB, VEGF, thrombospondin-1 and fibronectin for at least seven days, whilst PRGF/PPGF membranes release healing factors only during the first few days at much lower quantities. In PRF, living leucocytes are entrapped within the membrane and keep synthesizing growth factors. PRF has been used in the treatment of a number of other ulcers (Table 4).^5–8

A significant limitation of our study is its being a small observational case series from one centre. Much larger number of patients are required for true validation; however, the rapid healing of chronic ulcers was deemed sufficiently impressive. We were not able to exclude all confounding factors, nor compare PRF treatment with normal saline dressings alone.