Systematic pathological component scores for skin-containing vascularized composite allografts

Figures & data

Figure 1. Development of the pathologic component scoring system and evaluation for prognostic value and reproducibility.

Table 1. Definitions of pathological components and scores.

Component	Definition and Scoring
pc	perivascular cells – number of cells surrounding dermal vessels (capillaries, venules and arterioles) in the superficial and deep dermis; scored on the most involved vessels
	pc0 <10 cells/vessel
	pc1 10–25 cells/vessel
	pc2 26–50 cells/vessel
	pc3 >50 cells/vessel
pa	perivascular dermal infiltrate area – expressed as percent area occupied by the most involved dermal vessels at 40x magnification (example on Fig. 4D)
	pa0 <25%
	pa1 25–50%
	pa2 50–75%
	pa3 >75%
ei	epidermal infiltrate – total number of mononuclear cells per 4 20x fields
	ei0 <10 cells
	ei1 10–20 cells
	ei2 >20 cells
	ei3 transepidermal infiltrate
	eix no epidermis
v	endarteritis – mononuclear cells underneath arterial endothelium; scored on the most involved artery, arterioles not scored
	v0 none
	v1 <25% of lumen/vessel
	v2 >25% of lumen/vessel
	v3 fibrinoid necrosis/transmural involvement
	vx no arteries
e	epidermal injury and necrosis – presence of keratinocyte apoptosis and necrosis
	e0 none
	e1 apoptosis
	e2 focal necrosis
	e3 sloughed
cav	chronic allograft vasculopathy – intimal thickening with luminal reduction; scored as percent
	luminal reduction
	cav0 none
	cav1 <25% luminal reduction
	cav2 25–50% luminal reduction
	cav3 >50% luminal reduction
	cavx no arteries
c	capillaritis – maximum number of cells per capillary cross section; scored on most involved capillaries
	c0 0–1/capillary
	c1 2–4/capillary
	c2 5–10/capillary
	c3 >10/capillary
	ct thrombi

Table 2. Pathologic component scores of first biopsies between 2 groups.

			Pathologic Component Scores
Animal number	Gross Appearance	Days post-transplant	pc	pa	ei	e	v	c	cav	Graft survival (number of days)
Group A
A1	Indistinguishable from surrounding skin	POD 56	0	0	0	0	0	0	0	486 days^*
A2	Indistinguishable from surrounding skin	POD 58	0	0	0	0	0	0	0	139 days^*
A3	Mild edema	POD 13	0	1	0	0	0	0	0	275 days^*
A4	Mild edema	POD 13	1	1	0	0	0	0	0	275 days^*
Group B
B1	Edema	POD 14	1	1	1	2	0	2	0	40 days
B2	Edema	POD 15	2	3	2	1	0	2	0	50 days
B3	Erythema, induration	POD 42	3	3	2	1	0	2	0	63 days
B4	Erythema	POD 2	2	2	2	1	2	2	0	8 days

* No evidence of rejection at end of experimental follow up

Figure 2. Number of epidermal mononuclear cells per 4 medium power (20x) fields in native and autologous skin flaps

Figure 3. Pathologic component scores across 20 VCA biopsies with different Banff grades of rejection.

Figure 4. Histologic features of porcine skin and skin-containing VCA and pathologic component scores. A) Normal pig skin, punch biopsy. B) Minimal perivascular infiltrate (pc0) with capillaritis (c1) (arrows) in the superficial dermis. C) Moderately dense perivascular inflammation (pc2) with more evident capillaritis (c2) (arrows) at 40x magnification. D) The perivascular infiltrate surrounds dilated superficial dermal capillaries (pc1) occupying more than half of the field at 40x magnification (pa2) (outlined in blue). Dilated capillaries show occasional intraluminal mononuclear cells (c1) (arrows).

Figure 5. Epidermal inflammation (ei) and injury (e) in skin-containing VCA. A) Epidermis at 40x magnification with no epidermal infiltrate (ei0). Dilated superficial dermal capillaries with minimal perivascular inflammation are also present. B) One focus of prominent epidermal involvement without apoptosis (ei3, e0). C and D) Focal keratinocyte apoptosis (e1) (arrows). E) Frank epidermal necrosis. Moderate perivascular inflammation in the superficial dermis is also present.

Figure 6. Endothelialitis (v) and chronic allograft vasculopathy (cav). A) Small artery with endothelialitis (v1) (arrow). B) Artery with endothelialitis (arrow) and transmural inflammation in an arteriole (arrowheads) (v3). C) Transmural arteritis (v3). Fibrin (arrows) can be seen in v lesions. D) Early chronic allograft vasculopathy (cav1) in a small artery. E) Severe chronic allograft vasculopathy with luminal reduction (cav3).

Figure 7. Pathologic component scores across accepted and rejected allografts over time (in days). Grafts that rejected and survived for less than 100 d (black circles) have higher pathologic component scores than grafts that were accepted (white circles).

Figure 8. A) Animal A3 VCA post-surgical day 13 showing mild edema. B) Animal A3 POD 13 skin biopsy showing minimal perivascular inflammation (pc1, pa1) and C) no epidermal involvement (ei0, e0). D) Animal B3 POD 14 showing mild edema. E) Animal B3 POD 14 skin punch biopsy showing mild perivascular inflammation (pc1, pa1) but with F) epidermal apoptosis (arrow) and focal necrosis (not shown) (ei1, e2). G) Animal B1 POD 42 showing erythema. H) Animal B1 POD 42 skin biopsy showing severe perivascular inflammation (pc3, pa3) and I) lymphocytic infiltration and focal apoptosis (not shown) (ei2, e1).

Figure 9. Other pathologic features in rejection. A) Intracapillary mononuclear cells and neutrophils (arrow) in superficial dermal capillaries and a B) capillary thrombus (arrow) in high grade rejection. Mononuclear cell infiltrates along C) a hair follicle and D) sweat glands.