Study Participants

The study protocol and consent forms were approved by the ethics committee at Hôpital Ste. Croix and the institutional review board at Centers for Disease Control and Prevention (CDC). Patients were eligible to participate if they were enrolled in the lymphedema treatment clinic at the hospital, had been examined by the clinic physician to rule out other causes of lower limb swelling, gave informed consent to collection of all biopsies, had no medical contraindications to biopsy, had no ADLA episodes during the previous 2 weeks, and lived within a 10-km radius of the hospital.

Patients were tested for filarial infection by using an immunochromatographic card test (ICT), which detects antigen of adult W. bancrofti in the blood (30). Lymphedema stage was assessed by using an adaptation of a three-stage system recommended by WHO (31). Stage 1 lymphedema is characterized by swelling that is reversible on elevation at night. Stage 2 lymphedema is not reversible upon elevation and has no papillomatous changes. Stage 3 lymphedema, sometimes called elephantiasis, is characterized by papillomatous lesions and pronounced dermatosclerosis.

Patients were instructed in lymphedema self-care (27), with emphasis on thorough daily washing of the limb, basic skin care to treat and prevent entry lesions, range-of-motion limb exercises, and elevation of the leg during the day when possible and at night while sleeping. Participants were provided with basic supplies (e.g., soap, towels, wash basin) as needed. To monitor lymphedema self-care, patients returned to the clinic or were visited at home every 4–6 weeks and were asked about compliance with the regimen since the previous visit. Patients were encouraged to seek antimicrobial drugs and symptomatic treatment at the hospital during ADLA attacks. Thus, most ADLA attacks were observed by clinic staff; a few attacks were recorded on the basis of patient history and the presence of residual clinical signs (e.g., peeling of the skin, swelling) at the next follow-up visit.

Biopsy Collection and Analysis

A total of 91 patients agreed to undergo skin-punch biopsy of their lymphedematous leg or legs, and 26 of these patients also agreed to a biopsy of their non-lymphedematous leg (control). Twenty-seven patients agreed to have follow-up biopsies of their lymphedematous limb ≈12 months later. Biopsies were taken from the anterolateral surface of the leg from a site that was representative in appearance of the rest of the leg. Irregular protrusions and skin lesions were avoided. After the skin was cleaned with betadine and anesthetized with 1% lidocaine, a 4-mm skin-punch biopsy was obtained, and suture or 3M Steri-Strips were used to close the skin at the biopsy site. A short course of oral amoxicillin (250 mg three times a day for 5 days) was given to help prevent bacterial infection.

Skin-punch biopsies were fixed in formalin in Léogâne, Haiti and sent to CDC in Atlanta, Georgia, where they were embedded in paraffin. Five-micrometer sections were cut and stained with hematoxylin and eosin. All biopsy sections were read by the same pathologist (JG), who was blinded with respect to patient identification, lymphedema stage, and whether the specimen was from an initial or follow-up biopsy. Each biopsy specimen was evaluated for the presence or absence of the histopathologic characteristics presented in Table 1.

Statistical Analysis

Statistical analysis was performed using EpiInfo 6.0. The chi-square and Fischer exact tests were used to compare differences in the proportions of specimens with histopathologic features.

Participants

Of the 91 patients enrolled in this study, 73 (80%) were female. Women and men did not differ significantly with regard to age, lymphedema stage, or histologic features (data not shown). Median age was 39 years (range 16–75 years). One patient had bilateral lymphedema; the others had unilateral disease. Two patients had filarial antigen detected in the blood by ICT. Both were treated with diethylcarbamazine, the drug of choice for W. bancrofti infection. The median length of time between enrollment in the lymphedema management program and the first biopsy was 21 days (range 0–866 days).

Microbial Findings

No clinical signs of infection or inflammation were evident at the biopsy site. Microscopic examination of tissue sections stained with hematoxylin and eosin revealed no evidence of bacterial infection and no W. bancrofti adult worms or microfilariae.

Initial Biopsies and Histopathologic Features

One hundred eighteen biopsies were collected, 92 from lymphedematous legs and 26 from nonlymphedematous control legs. Biopsies were collected a median of 14 cm (range 5–20 cm) above the sole of the foot. The number of biopsies taken from control legs and from legs with stage, 1, 2, or 3 lymphedema was 26, 12, 60, and 20, respectively. No postoperative infections developed. Among the biopsy specimens, the proportion with histopathologic features (prevalence) increased with lymphedema stage (Table 2).

Epidermis

The prevalence of hyperkeratosis, hypergranulosis, and acanthosis increased significantly with stage of lymphedema. Hyperkeratosis was the predominant pathologic feature within the epidermis, regardless of stage, and was found in 80% of biopsies from legs with stage 3 lymphedema. No biopsies from unaffected control legs showed hypergranulosis or acanthosis.

Superficial Dermis

Within the superficial dermis, the prevalence of fibrolamellar hyperplasia increased from 19% in nonlymphedematous legs to 50% in stage 1 lymphedema but remained similar for stages 1–3. The prevalence of condensed collagen steadily increased from 0% in unaffected control legs to 30% in stage 2 lymphedema (p = 0.002). Most of the perivascular infiltrate in the superficial dermis was composed of mononuclear cells, primarily lymphocytes and macrophages. Plasma cells were found only in stage 2 and 3 lymphedema and were significantly less common in biopsies from women (4 [4%] of 93) than from men (7 [28%] of 25) (p = 0.002). The prevalence of chronic mononuclear infiltrate increased steadily from 42% in nonlymphedematous biopsies to 95% in stage 3 biopsies (p = 0.0002). In all areas where cellular infiltrate was observed, lymphocyte and macrophage cell populations were assessed together with respect to infiltrate intensity because activated lymphocytes were difficult to differentiate microscopically from macrophages. The prevalence of pronounced lymphocyte and macrophage infiltrate increased with lymphedema severity: 4% of biopsies from nonlymphedematous legs had pronounced infiltrate compared to 35% of stage 3 biopsies (p = 0.008).

Deep Dermis

Perivascular fibrosis was recorded in 27% and 40% of skin biopsies from patients with stage 2 and stage 3 lymphedema, respectively; it was more prevalent within the deep dermis than the superficial dermis at nearly every stage of lymphedema. The prevalence of perivascular infiltrate, composed entirely of mononuclear cells, increased from 50% in stage 1 lymphedema to 90% in stage 2 (p = 0.003). Pronounced chronic infiltrate and plasma cells were also more common in the deep dermis than in the superficial dermis at every stage of lymphedema. The prevalence and intensity of periadnexal infiltrate, all of which was mononuclear, increased with stage of lymphedema. Plasma cells were rarely observed in periadnexal infiltrate.

Subcutaneous Tissue

All cellular infiltrate in fibrous septa surrounding subcutaneous adipose tissue was mononuclear, and its prevalence increased with lymphedema stage, particularly between nonlymphedematous controls and stage 1 lymphedema (4% to 25%). Plasma cells were rarely observed in the infiltrate surrounding subcutaneous tissue.

Follow-up Participants and Biopsies

Follow-up biopsies were collected from the lymphedematous limb of 27 patients a median of 365 days (range 317–656 days) after their first biopsy. Of these 27 patients, 20 (74%) were women; the median age was 38 years (range 16–61 years). They did not differ significantly by sex, age, or lymphedema stage from the 65 participants who only had one skin biopsy.

Compliance with self-care practices during the interval between biopsies was high. At 96%, 94%, 87%, and 98% of monthly follow-up visits, respectively, patients reported that, since the previous visit, they had washed the leg daily, practiced range-of-motion exercises, elevated the leg during the daytime, and raised the foot of the bed at night. No changes in lymphedema stage were observed.

The second biopsy was obtained a median of 14 cm (range 6–17 cm) above the sole of the foot, and a median of 1 cm (range <1–5 cm) from the first biopsy site. No postoperative infections developed. Of the 27 biopsies that were collected for follow-up, 2 were from legs with stage 1 lymphedema, 18 from stage 2 lymphedema, and 7 from stage 3 lymphedema.

Of the 27 patients, 21 (78%) had reported one or more ADLA attacks during the 12-month period before entering the program. In contrast, only eight (30%) reported one or more attacks during the interval between biopsies (1, 4, and 3 patients with stage 1, 2, and 3 lymphedema, respectively). The mean reported incidence of attacks during the year before entering the program was 1.7 (range, 0–8) per person-year, compared to 0.5 (range, 0–3) observed between biopsies (p = 0.0009).

Histopathologic Changes with Lymphedema Management

Figure1

Figure1. Representative sample of skin punch biopsy specimens from patients with lymphedema before (A, B, C) and after (D, E, F) 1 year of basic lymphedema management. Pretreatment abnormalities of the epidermis...

The prevalence and intensity of histopathologic abnormalities tended to be greater in initial skin biopsies (Figure 1A, B, and C) than in follow-up biopsies (Figure 1D, E, and F) (Table 3). Hyperkeratosis and hyperplasia of the epidermis were more prominent on initial biopsies (Figure 1A) than on follow-up specimens (Figure 1D). The thick collagen bundles observed in the dermis of initial biopsy samples (Figure 1A) were less obvious in biopsies after 1 year of lymphedema management (Figure 1D). In the superficial dermis, substantial decreases were observed between the first (Figure 1B) and second (Figure 1E) biopsies, both in the prevalence of chronic perivascular infiltrate (100% to 59%, p = 0.0002, Table 3) and in the proportion of specimens with pronounced infiltrate intensity (37% to 11%, p = 0.03).

Perivascular fibrosis in the deep dermis was less common in follow-up biopsy samples (22%, Figure 1E) than in initial biopsies (52%, Figure 1B). Additionally, significant decreases were observed in the prevalence of chronic periadnexal infiltrate (70% to 33%, p = 0.007) and the percentage of biopsies with pronounced infiltrates of lymphocytes and macrophages in periadnexal areas of the deep dermis (26% to 0%, p = 0.005, Table 3, and Figures 1C and F). The replacement of thick collagen bundles around adnexi in the first biopsy (Figure 1C) with adipose cells in the second biopsy (Figure 1F) was also noted in the subcutaneous tissue.

Figure 2

Figure 2. Individual histologic responses of the 27 patients with follow-up biopsy specimens who participated in a lymphedema management program for ≈1 year. Black boxes indicate histopathologic regression or improvement, gray boxes indicate...

Figure 2 summarizes the histologic changes in each of the 27 patients with follow-up biopsies. Histopathologic regression or improvement was defined as the disappearance of a histopathologic characteristic on follow-up biopsy, while histopathologic progression or worsening was defined as the appearance of a previously unnoted histopathologic characteristic on follow-up biopsy. All but one patient (96%) showed histopathologic regression in one or more characteristics. Histopathologic improvement was observed among all stages of lymphedema. However, 4 (57%) of 7 legs with stage 3 lymphedema showed regression of chronic perivascular infiltrate in the deep dermis, compared to none of 20 legs with stage 1 or 2 lymphedema (p = 0.003). Eleven (41%) patients showed regression in four or more characteristics; they did not differ significantly from the other 16 patients with respect to sex, age, stage, or duration of lymphedema; duration of participation in the lymphedema program before the first biopsy; interval between biopsies; or ADLA incidence between biopsies (Table 4). Sixteen (59%) patients showed histopathologic progression in one or more characteristics. None of the 27 patients had progression of chronic perivascular infiltrate.