Pubic symphysitis after transurethral resection of the prostate.

PURPOSE: Pubic symphysitis (PS) after urological operations is uncommon. This is a systematic single-institution review of patients with transurethral resection of the prostate (TUR-P) with the aim to determine the incidence of PS after TUR-P and to identify a risk profile. MATERIALS AND METHODS: In the past 15 years, 12,118 transurethral operations were performed in our department, 33.4% (n = 4045) were TUR-P, and 84.6% (n = 3421) had routine suprapubic trocar placement. A systematic retrospective analysis identified 12 patients, who developed PS (0.297%). RESULTS: Median age was 69.5 years (64-83). All patients had voiding difficulties. Urine culture had been positive in three cases. All 12 TUR-Ps were monopolar resections, and n = 11 patients had a suprapubic trocar. Median resection weight was 47.5 g (10-100). Two patients had a perforation of the capsule. Histopathological examination revealed chronic prostatitis in nine cases. After 1.0 ± 1.2 months, all patients developed pain in the pubic region. All patients underwent MRI, which suggested PS. Symptomatic and antibiotic medications were administered. Final outcome was resolution of symptoms in all patients after 3.8 ± 5.6 months. No patient retained voiding difficulties. CONCLUSION: PS remains a rare complication after TUR-P. We could not identify a single cause for developing PS. In our study, suprapubic trocar placement (11/12), chronic prostatic inflammation (9/12), previous UTI (3/12) and extended resection (2/12) were overrepresented. Inflammatory, thermic and/or surgical damage of the capsule may be causative. Patients require antibiotic and symptomatic medication. However, prognosis for remission is excellent.

[Management of complications after sling and mesh implantations]. / Komplikationsmanagement nach Band- und Netzimplantationen.

Tension-free alloplastic slings (TFAS) have revolutionized surgery for female stress urinary incontinence for more than 15 years. The procedure is easy to perform, minimally invasive with short operation time in an ambulatory setting, and has proven efficacy comparable to the gold standard procedure of retropubic colposuspension.Possible TFAS complications are potentially underestimated with respect to prevalence and manageability. We report our experience with major complications following TFAS and mesh implantation in patients referred to our interdisciplinary continence center. Patient history, risk factors, and preoperative diagnostics were analyzed for development of individualized treatment strategies. Overcorrections with formation of postvoid residual (PVR) can occur in retropubic TFAS as well as in transobturator TFAS. However, the most prevalent and challenging complication is de novo urgency. Major complications like urethrovaginal fistula, sling arrosions of the urethra, bladder, and vagina as well as infected gangrene and complete urethral loss requiring urinary diversion were seen at a frequency suggesting underrepresentation of these complications in the literature. The large amount of implanted artificial mesh material used for pelvic organ prolapse (POP) correction represents a particular challenge in cases of dyspareunia or persisting pelvic pain.Complication management has to be based on cystoscopic, urodynamic, and physical examination findings to be individualized to each patient and must take potential risks of recurrent incontinence or persisting complaints into account.To prevent TFAS or mesh complications, every patient should have tried all conservative treatment options and should be completely evaluated (including urodynamics) preoperatively. Artificial meshes should only be used in cases of prolapse recurrence or in otherwise inoperable patients. Postoperative urodynamics may help to document treatment success and to identify and quantify complications.

Manifestation of congenital urethral diverticula in a 57-year-old male.

Congenital urethral diverticula are a rare finding in adult males. Most cases are diagnosed in childhood or adolescence because of voiding symptoms such as urinary dribbling. Diagnostic workup should include radiography and urethroscopy. The standard therapeutic approach is open surgical excision or endoscopic marsupialization. An unusual case of male congenital urethral diverticula that remained asymptomatic until age 57 is presented.

[Prevalence of lymph node metastases in non-muscle-invasive bladder cancer. Delay of radical cystectomy and upstaging in the cystectomy specimen as risk factors]. / Prävalenz von Lymphknotenmetastasen beim nicht muskelinvasiven Blasenkarzinom. Verzögerung der Zystektomie und Upstaging im Zystektomiepräparat als Risikofaktoren.

OBJECTIVE: To study clinical and histopathologic parameters after cystectomy and lymphadenectomy in non-muscle-invasive transitional cell carcinoma (TCC) of the bladder and their association with the prevalence of lymph node metastases (N+). PATIENTS AND METHODS: Of 866 patients treated with radical cystectomy and lymphadenectomy, 219 had non-muscle-invasive TCC of the bladder. The prevalence of N+ was related to parameters such as gender, age, number of transurethral resections of the bladder (TURBs), intervals between first TURB and cystectomy, adjuvant therapy, maximum histopathologic tumor stage and grade at TURB, and tumor upstaging in the cystectomy specimen by univariate and multivariate analysis. RESULTS: A total of 33 patients (15%) had N+. By multivariate analyses, tumor upstaging and the number of TURBs were independent predictors of N+ at cystectomy. The number of TURBs increased the prevalence of N+ from 8% (one TURB) to 24% (two to four TURBs). Tumor upstaging in the cystectomy specimen increased the prevalence of N+ from 4% to 36%. CONCLUSION: Inappropriate delay and staging errors of"high risk" non-muscle-invasive TCC of the bladder contribute to an increased prevalence of N+ and should be avoided. In our series, the number of TURBs and tumor upstaging in the cystectomy specimen were independent predictors for N+ by multivariate analysis.

[Which are reasonable diagnostic procedures in the evaluation of urinary incontinence in the elderly?]. / Welche Untersuchungen sind beim alten Menschen mit Harninkontinenz sinnvoll?

The ageing of our society continuously increases the number of frail elderly patients in the incontinence cohort. Shortage of financial and personnel resources demands reasonable and purposeful use of the diagnostic armamentarium. All intended diagnostic procedures should follow an algorithm hierarchized for invasiveness and should be limited to the minimum extent necessary for initiation of a conservative first-line treatment. Reasonable diagnostics objectify patients' complaints, differentiate between subgroups, reveal underlying pathologies and comorbidities, classify incontinence severity, support the therapeutic strategy, identify possible treatment complications and serve as follow-up tools. Diagnostic results have to be documented in detail and the procedures must be as easy and minimally invasive as possible. Basic diagnostics in urinary incontinence comprise patient history, clinical examination, urinalysis, uroflowmetry and sonographic post-void residual measurement, voiding diary and evaluation of the mental status. With these procedures, the vast majority of elderly patients can be classified correctly and a conservative first-line treatment can be started. Only a minority of patients with incongruent diagnostic results or recurrent incontinence refractory to conservative therapy should undergo further special diagnostics (urethrocystoscopy, urodynamics, morphologic and functional radiologic imaging, perineal or introital ultrasound) if they lead to therapeutic consequences. If not, expensive special diagnostics should be omitted in elderly patients due to their inherent morbidity.

[Established treatment options for male stress urinary incontinence]. / Etablierte Methoden in der Behandlung der Belastungsinkontinenz des Mannes.

Nowadays, male stress urinary incontinence is rare and almost always of iatrogenic origin (radiotherapy, pelvic surgery). However, the prognosis of urinary incontinence following surgery is good and can be improved by pelvic floor muscle exercises in combination with biofeedback systems. For the remaining patient cohort with persistent urinary incontinence, several established surgical treatment options are available. Suburothelial injections of bulking agents can easily be performed in an ambulatory setting. However, regardless of the material used, long-term results are disappointing. Moreover, the residual urethral function deteriorates due to cicatrization of the suburothelial plexus with consequent loss of urethral elasticity. The fascial sling procedure in males has to be performed in preoperated areas and is as technically demanding for the surgeon as it is burdening for the patient. Alloplastic material is not used, thus minimizing risks for arrosion or infection. Since the sling tension can neither be standardized nor postoperatively readjusted, the risk of overcorrection is considerable and the success of the procedure is heavily dependent on the surgeon's experience. Despite wear and high revision rates, the technically mature artificial sphincter produces excellent continence results and has become the gold standard in the therapy of male stress urinary incontinence. The circumferential and continuous urethral compression by the cuff is highly effective, but at the price of an almost inevitable urethral atrophy. To overcome this problem, various surgical techniques have been developed (tandem cuff, cuff downsizing, transcorporal cuff placement). However, the expensive artificial sphincter is not a nostrum for every incontinent man, since it requires certain minimal cognitive and manual capabilities. Therefore, the search for less demanding treatment alternatives seems to be necessary, even if one has to accept lower continence rates.

[Urethroplasty and simultaneous perineal prostatectomy after traumatic urethral disruption and carcinoma of the prostate]. / Harnröhrenplastik nach Urethraabriss und simultane perineale Prostatektomie.

We present a case of post-traumatic posterior urethral stricture and localized prostate cancer, which could be treated successfully with simultaneous radical perineal prostatectomy and membranous urethral stricture excision. After 6 months follow-up, the patient is continent with no evidence of stricture recurrence. Post-traumatic posterior urethral strictures can be managed surgically through a perineal approach with high success rates. Prostate surgery after pelvic fracture with posterior urethral distraction defects does not necessarily lead to stress urinary incontinence.

Hypoxia-induced up-regulation of angiogenin in human malignant melanoma.

Angiogenesis is essential for tumor progression and metastasis, however, the angiogenesis regulators that are biologically relevant for human melanoma are still unknown. In this study, we analyzed the expression of the potent angiogenic factor angiogenin (ANG) in human melanoma in vitro and in vivo. Four different human melanoma cell lines and two normal melanocytes were kept either under normoxic or hypoxic conditions. After 24 h of hypoxic culture conditions, ANG was up-regulated in the melanoma cell lines but not in normal melanocytes. Induction levels correlated with the metastatic potential of the cell lines. These data were confirmed by Northern blot analysis. In contrast, induction of vascular endothelial growth factor by hypoxia was equally strong in the examined highly aggressive melanoma cell lines and in one nonaggressive cell line. Other angiogenic factors tested as well as the melanoma growth stimulatory activity (Gro-alpha) showed no up-regulation. Thus, in the present study, hypoxia-induced up-regulation in melanoma cells was only observed for ANG and vascular endothelial growth factor. Immunohistochemical studies showed that 8 of 10 melanomas and all 15 metastases were positive for ANG, particularly in the vicinity of small vessels, whereas all benign nevi were negative. Reverse transcription-PCR detected only weak ANG mRNA in nevi but strong signals in primary melanomas and metastases. In conclusion, we demonstrate for the first time enhanced expression of ANG in highly metastatic cell lines as well as in melanomas and metastases in vivo, suggesting that ANG expression is associated with the metastatic potential.

Chemokines in cutaneous wound healing.

Healing of wounds is one of the most complex biological events after birth as a result of the interplay of different tissue structures and a large number of resident and infiltrating cell types. The latter are mainly constituted by leukocyte subsets (neutrophils, macrophages, mast cells, and lymphocytes), which sequentially infiltrate the wound site and serve as immunological effector cells but also as sources of inflammatory and growth-promoting cytokines. Recent data demonstrate that recruitment of leukocyte subtypes is tightly regulated by chemokines. Moreover, the presence of chemokine receptors on resident cells (e.g., keratinocytes, endothelial cells) indicates that chemokines also contribute to the regulation of epithelialization, tissue remodeling, and angiogenesis. Thus, chemokines are in an exclusive position to integrate inflammatory events and reparative processes and are important modulators of human-skin wound healing. This review will focus preferentially on the role of chemokines during skin wound healing and intends to provide an update on the multiple functions of individual chemokines during the phases of wound repair.

[Drug therapy of female urinary incontinence]. / Medikamentöse Therapie der weiblichen Harninkontinenz.

Drug treatment for female urinary incontinence requires a thorough knowledge of the differential diagnosis and pathophysiology of incontinence as well as of the pharmacological agents employed. Pharmacotherapy has to be tailored to suit the incontinence subtype and should be carefully balanced according to efficacy and side effects of the drug. Women with urge incontinence require treatment that relaxes or desensitizes the bladder (antimuscarinics, estrogens, alpha-blockers, beta-mimetics, botulinum toxin A, resiniferatoxin, vinpocetine), whereas patients with stress incontinence need stimulation and strengthening of the pelvic floor and external sphincter (alpha-mimetics, estrogens, duloxetine). Females with overflow incontinence need reduction of outflow resistance (baclofen, alpha-blockers, intrasphincteric botulinum toxin A) and/or improvement of bladder contractility (parasympathomimetics). If nocturia or nocturnal incontinence are the major complaints, control of diuresis is obtained by administration of the ADH analogue desmopressin. Future developments will help to further optimize the pharmacological therapy for female urinary incontinence.

Differential expression of the calpactin I subunits annexin II and p11 in cultured keratinocytes and during wound repair.

Transforming growth factor beta1 (TGF-beta1) is an important modulator of skin morphogenesis and cutaneous wound repair. To gain insight into the mechanisms of TGF-beta1 action in the skin, we used the differential display RT-PCR technique to identify genes that are regulated by this factor in cultured human keratinocytes. We obtained several partial cDNA clones. One of them was identical to the 3'-end of p11, the small and regulatory subunit of the calpactin I complex [(annexin II)2(p11)2]. RNase protection and northern blot analysis revealed specific regulation of expression of both subunits of this heterotetrameric protein (p11 and annexin II) by TGF-beta1 as well as by other growth factors, although the time course and degree of induction or suppression were different for each gene. Furthermore, we analyzed p11 and annexin II expression in normal and wounded skin. Both p11 and annexin II mRNAs were found in the dermal and epidermal compartments of normal human skin. Immunohistochemical studies demonstrated the presence of p11 at equally high levels in all layers of normal epidermis and in the hyper-proliferative epithelium at the wound edge. By contrast, annexin II expression was high in the basal layer of normal epidermis but low in the suprabasal layers and in the hyper-proliferative epithelium at the wound edge, suggesting a differentiation-specific regulation of this calpactin I subunit. The differential expression and regulation of p11 and annexin II subunits in keratinocytes suggest the existence of different ratios of monomeric versus p11-complexed annexin II that might be associated with different cellular functions.

Decreased production of interferon in whole blood cultures derived from patients with psoriasis.

Patients suffering from psoriasis show many alterations with respect to their immune system as documented by in vitro test systems. In the present study we investigated the in vitro production of interferons (IFN) of leukocytes from psoriatic patients to stimulation with a variety of IFN inducers. Furthermore, the lymphoproliferative responses were tested. Whole blood cultures of 30 psoriatic patients showing moderate to severe disease activity and 21 cultures from healthy controls were stimulated with the mitogens PHA, ConA, and PWM, with PPD and Tetanus Antigen as IFN gamma inducers and with C. parvum, PolyI-PolyC, and Herpes simplex virus as inducers of IFN alpha. Interferon activity was tested in the supernatant of 48-h cultures by using an antiviral assay. Lymphoproliferation was assayed in 5-d cultures in parallel. Psoriatic patients showed a significantly decreased IFN production to all the stimuli tested. There were no significant differences in the lymphoproliferative responses; only the response to PWM was slightly decreased. The decreased IFN production by leukocytes from psoriatic patients seems to be very remarkable since increased susceptibility to infections is not generally known in these patients.

MIG is a dominant lymphocyte-attractant chemokine in lichen planus lesions.

Dense accumulation of mononuclear cells (lymphocytes >> macrophages) in the dermal-epidermal interface and a T cell-mediated cytotoxic reaction against basal keratinocytes are hallmarks of lichen planus lesions. In this study, we focused on the chemotactic signals responsible for the selective recruitment of these cells. Using in situ hybridization and immunohistochemistry, the expression and localization of the lymphocyte-and/or monocyte/macrophage-attractant CC chemokines macrophage chemoattractant protein-1 (MCP-1), regulated on activation, normal T cell expressed, and secreted (RANTES), macrophage inflammatory protein-1alpha and -1alpha (MIP-1alpha/beta), I-309 and the CXC chemokines monokine induced by interferon-gamma (MIG), interferon-gamma-inducible protein-10 (IP-10), interleukin-8 (IL-8), epithelial-derived neutrophil attractant-78, and growth-related oncogene-alpha were investigated. Strong mRNA expression of MIG, IP-10, and MCP-1 and moderate mRNA expression of RANTES and MIP-1alpha were detected exclusively within foci characterized by strong infiltration with CD3+ lymphocytes (CD4+ cells > CD8+ cells) and CD68+ macrophages. All other chemokines investigated were minimally expressed or absent. With more than 11% of total cells strongly expressing MIG transcripts, this selectively lymphotactic chemokine was by far the dominant chemokine and thus may significantly contribute to the inflammatory reaction in lichen planus lesions. According to the mRNA expression profiles, MIG, IP-10, and MCP-1 were expressed by both basal keratinocytes above and mononuclear cells within the inflammatory foci. Our findings indicate that a set of chemokines composed of IP-10, MCP-1, RANTES, MIP-1alpha, and especially MIG contributes to the cytokine network and preferential trafficking of mononuclear cells to the interface region of lichen planus lesions.

Differential expression of GRO-alpha and IL-8 mRNA in psoriasis: a model for neutrophil migration and accumulation in vivo.

Dense focal accumulation of neutrophils in the upper epidermis is a hallmark of psoriasis. Because the signals for neutrophil diapedesis and migration in vivo are not fully understood, psoriatic lesions with pronounced migration of neutrophils may serve as an important model for studying neutrophil chemotaxis. In this study, we present evidence for differential expression of the neutrophil chemotactic cytokines growth-related oncogene alpha, interleukin-8, and ENA-78 (epithelial cell derived and neutrophil-activating properties, 78 amino acids) in psoriatic lesions. In situ hybridization and immunohistochemistry of serial sections were employed to identify and microanatomically localize the cells producing these chemokines. High levels of focal interleukin-8 message were found to be expressed in the upper epidermis by keratinocytes and, most importantly, neutrophils themselves. Growth-related oncogene alpha transcripts were detected in clusters of keratinocytes of the upper epidermis at the same sites where interleukin-8 mRNA was abundant. In contrast to interleukin-8, growth-related oncogene alpha was also detected in the papillary dermis produced by vessel-associated cells. Sites of interleukin-8 and growth-related oncogene alpha mRNA expression were associated with infiltration of neutrophils. Interestingly, mRNA expression of the highly homologous chemokine ENA-78 was quiescent. In conclusion, our data indicate that growth-related oncogene alpha is an important chemoattractant for neutrophil diapedesis in vivo, whereas further migration of neutrophils and formation of micropustules appears to be influenced by the cooperative action of both growth-related oncogene alpha and interleukin-8.

The chemokine repertoire of human dermal microvascular endothelial cells and its regulation by inflammatory cytokines.

Activation of endothelium is a critical event during the initiation of inflammatory processes and is associated with the induction of cell adhesion molecules and cytokines. The latter include chemotactically active cytokines (chemokines) that promote leukocyte diapedesis from the circulation to sites of evolving inflammation. In this study we evaluated the chemokine repertoire of human endothelial cells derived from the skin (HDMECs) and regulation of these chemokines by cytokines. HDMECs and an immortalized human dermal microvascular endothelial cell line, HMEC-1, were investigated for the expression of C-X-C and C-C chemokines at mRNA and protein levels. Upon stimulation with interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), both HDMECs and HMEC-1 expressed high levels of IL-8, GRO, and monocyte chemoattractant protein-1 (MCP-1). RANTES was only weakly induced; however, concomitant treatment with TNF-alpha and interferon-gamma (IFN-gamma) led to upregulation of RANTES, indicating a synergy between these two cytokines. The C-X-C chemokine IFN-inducible protein-10 was upregulated by IFN-gamma but not by other cytokines studied. Macrophage inflammatory protein-1alpha and beta, 1-309, and ENA-78 could not be induced. The chemokine repertoires of HDMECs and HMEC-1 were compared to those of human umbilical vein endothelium and found to be rather similar with the important exception that IFN-gamma and IL-4 up-regulated MCP-1 only in macrovascular endothelium. Our data indicate that HDMECs contribute to the dermal cytokine network by selective production of MCP-1, IL-8, GRO, RANTES, and IP-10, which may critically influence the site-specific recruitment of leukocyte subsets.

Upper keratinocytes of psoriatic skin lesions express high levels of NAP-1/IL-8 mRNA in situ.

In order to better understand the factors regulating disease promotion and activity in psoriasis (PS), we searched for the in situ expression of mRNA for various cytokines in long-standing PS skin lesions. Specific hybridization with a NAP-1/IL-8 anti-sense RNA probe was keratinocyte associated and yielded strong and specific signals exclusively in the upper layers of the lesional epidermis, but not in uninvolved skin from psoriatic patients or normal skin from non-psoriatics. Interestingly, NAP-1/IL-8 transcripts were focally clustered in a spotty pattern predominantly between the tips of elongated papillae, but were absent in the lower epidermal region and the dermal compartment. We consistently failed to detect appreciable numbers of TNF-alpha and/or IL-6 mRNA-containing cells in psoriatic lesions. These results support the notion that IL-8, rather than IL-6, is an important disease-promoting cytokine in PS. In view of the known in vitro and in vivo effects of IL-8, it is conceivable that this substance greatly contributes to the major pathologic changes seen in psoriatic skin, i.e., keratinocyte hyperproliferation and leucocyte infiltration. In this case, local pharmacologic down-regulation of NAP-1/IL-8 activity could be a promising therapeutic strategy in PS.

Endemic Kaposi's sarcoma in human immunodeficiency virus type 1-seronegative persons: demonstration of retrovirus-like particles in cutaneous lesions.

In 1984, Greek physicians reported on the clustering of cases of Kaposi's sarcoma (KS) on the Peloponnesus peninsula. To gain more insight into its pathogenesis, we studied the seroepidemiologic and clinicopathologic characteristics of 12 Greek KS patients (eight male/four female) five of whom were residents of an endemic area on the Peloponnesus. These patients were in good general health with ages ranging from 48 to 80 years, had no clinical signs of immunodeficiency, and combined the features of both classic and epidemic KS in that they displayed not only involvement of acral areas but also widespread mucocutaneous lesions. Routine laboratory data were within normal limits; no patient had HTLV-1 and HIV-1/2 antibodies, but all patients had antibodies to several herpesviruses. The histopathology was characteristic of KS with the peculiar feature of a dense infiltrate composed predominantly of CD4+ T lymphocytes. Immunoenzymatic/morphologic studies of the KS cells were consistent with their origin from lymphatic endothelium. Outstanding ultrastructural findings were tubuloreticular structures and cylindrical confronting cisternae, structures that are indicative of an ongoing viral infection. Indeed, extensive electronmicroscopic studies resulted in the detection of retrovirus-like particles in close association to KS cells in five of 12 patients. This in situ observation opens the possibility that this retro-virus contributes to KS development.

MCP-1 mRNA expression in basal keratinocytes of psoriatic lesions.

In addition to hyperproliferation of keratinocytes, psoriasis is characterized by pronounced leukocytic infiltration. In contrast to the epidermal localization of neutrophils and T lymphocytes, macrophages are almost exclusively restricted to the dermal compartment. By immunohistologic analysis, these dermal macrophages were mainly encountered in the papillary dermis and arranged along the rete ridges in close proximity to proliferating keratinocytes. Monocyte chemoattractant protein (MCP-1) anti-sense RNA probes yielded abundant signals over the proliferating basal keratinocytes of the tips of the rete ridges, and, to a lesser extent, in cells in the papillae. Thus, the strongest MCP-1 message in psoriatic lesions is found above the dermal-epidermal junction and this may explain the characteristic sub-basal distribution of dermal macrophages. These results suggest that MCP-1 is important in regulating the interaction between proliferating keratinocytes and dermal macrophages in psoriasis pathogenesis.

Relative advantages and disadvantages of radical perineal prostatectomy versus radical retropubic prostatectomy.

In recent years prostate cancer has become the predominant malignancy in men. With the introduction of prostate specific antigen (PSA) the disease can be diagnosed at an early stage, at which surgical therapy can be curative. In the past century, the retropubic and the perineal routes were established as alternatives of surgical access to the gland for clinically localized prostate cancer. The selection of the operative route is mostly decided individually on the basis of surgical training and experience. The revived interest in perineal radical prostatectomy is explained by the fact that this technique has been associated with low morbidity. The differences of both surgical approaches of radical prostatectomy are elucidated and compared regarding tumor control and short and long term complication rates. Taking these results into consideration, specific advantages and disadvantages of radical perineal prostatectomy are emphasized.

A reliable method for simultaneous demonstration of two antigens using a novel combination of immunogold-silver staining and immunoenzymatic labeling.

We have developed a reliable and sensitive immunohistochemical staining technique which allows the simultaneous demonstration of two different antigens expressed in or on the same cell (referred to as mixed labeling), together with the evaluation of the general histopathological appearance of the tissue. The staining procedure combines a three-step (streptavidin-biotin) immunogold-silver staining (IGSS) with a three-step immunoenzymatic labeling. For this purpose, we investigated the compatibility of IGSS with various substrates of peroxidase or alkaline phosphatase (AP). Highly reliable and discernible mixed labeling was achieved only after initial labeling with IGSS followed by AP labeling using the substrates naphthol AS-MX phosphate/Fast Blue or naphthol AS-BI phosphate/New Fuchsin, respectively. To ensure utmost specificity, we applied FITC-conjugated mouse monoclonal antibodies and rabbit anti-FITC immunoglobulins visualized by AP-labeled immunoglobulins and the respective substrate in a final step. This novel approach provides an excellent means for demonstration of immunocompetent cells and unequivocal determination of the percentage of specific cell subsets in infiltrated tissue. The advantages of this method, as compared with double immunofluorescence or double immunoenzymatic labeling, were investigated and are discussed.