CLI Patients Research Articles

Morphometric analysis of gastrocnemius muscle biopsies from patients with peripheral arterial disease: objective grading of muscle degeneration

Peripheral arterial disease (PAD), which affects ~10 million Americans, is characterized by atherosclerosis of the noncoronary arteries. PAD produces a progressive accumulation of ischemic injury to the legs, manifested as a gradual degradation of gastrocnemius histology. In this study, we evaluated the hypothesis that quantitative morphological parameters of gastrocnemius myofibers change in a consistent manner during the progression of PAD, provide an objective grading of muscle degeneration in the ischemic limb, and correlate to a clinical stage of PAD. Biopsies were collected with a Bergström needle from PAD patients with claudication (n = 18) and critical limb ischemia (CLI; n = 19) and control patients (n = 19). Myofiber sarcolemmas and myosin heavy chains were labeled for fluorescence detection and quantitative analysis of morphometric variables, including area, roundness, perimeter, equivalent diameter, major and minor axes, solidity, and fiber density. The muscle specimens were separated into training and validation data sets for development of a discriminant model for categorizing muscle samples on the basis of disease severity. The parameters for this model included standard deviation of roundness, standard deviation of solidity of myofibers, and fiber density. For the validation data set, the discriminant model accurately identified control (80.0% accuracy), claudicating (77.7% accuracy), and CLI (88.8% accuracy) patients, with an overall classification accuracy of 82.1%. Myofiber morphometry provided a discriminant model that establishes a correlation between PAD progression and advancing muscle degeneration. This model effectively separated PAD and control patients and provided a grading of muscle degeneration within clinical stages of PAD.

Clinical presentation and outcome after failed infrainguinal endovascular and open revascularization in patients with chronic limb ischemia

Failure of prior endovascular (EV) interventions for chronic limb ischemia has been reported to negatively affect patency and limb salvage after subsequent revascularization procedures. The goal of our study was to compare the clinical presentation of patients who failed infrainguinal EV and open revascularizations (OR) and the effect of the initial intervention on final outcomes. From June 2001 to October 2010, 216 patients (237 limbs; 66 disabling claudication [DC], 171 critical limb ischemia [CLI]) presented with failed infrainguinal OR or EV revascularization for chronic limb ischemia. Clinical presentation, reinterventions, patency and limb salvage rates, and final outcomes were analyzed. The EV group (n = 143) had more diabetes (44% vs 57%; P = .048) and ulcers (26% vs 38%; P = .039), whereas the OR group (n = 94) had more multilevel revascularizations (59% vs 33%; P < .001), rest pain (23% vs 9%; P = .002), and infrapopliteal interventions (58% vs 38%; P = .038). Presentation at time of failure was non-limb-threatening ischemia in 70% of DC and 16% of CLI patients (P < .001), with no difference in those initially treated with EV or OR. In CLI, 23% presented with acute limb ischemia in the OR group vs 10% in the EV group (P = .024). Early failure (<3 months) occurred in 15% of DC and in 36% of CLI patients and was more in the OR than in the EV group (30% vs 7% for DC [P = .011] and 71% vs 38% for CLI [P = .024]). Overall, 195 (82%) had attempted reinterventions (79% in DC and 85% in CLI; P = .245). In DC patients, 48% of OR had OR + EV and 26% had EV; 32% of EV had OR + EV and 47% had EV reinterventions. In CLI patients, 40% of OR had OR + EV and 42% had EV; 17% of EV had OR + EV; and 70% had EV reinterventions. A patent revascularized limb was achieved in 66% of OR and in 92% of EV patients (P < .001). Patency and limb salvage were significantly better in the EV group, mainly due to the difference in CLI patients, whereas survival was identical. Clinical presentation after failed infrainguinal revascularization is determined by the initial indication. CLI patients are more likely to present early with acute limb ischemia, especially after OR. EV reinterventions play a significant role in the management of patients with failed revascularization, and EV failure is associated with better outcomes than those after OR failure, likely due to OR patients having more disadvantaged anatomy and advanced disease at the time of their initial presentation.

Abstract 504: Drug-Eluting Stent Placement for Treatment of Below-The-Knee Critical Limb Ischemia: A Report of Parameter for Wound Healing with Measurement of Transcutaneous Oxygen Tension

Objectives The aim of this study is to evaluate the efficacy of drug-eluting stents (DES) placed below-the-knee level in the parameters for wound healing. Background Critical limb ischemia (CLI) has a serious impact on quality of life with a major amputation rate of 30 percent within 12 months. Endovascular treatment has been used to perform revascularization of limbs over the past decade. However, the role of drug-eluting stent (DES) placement at below-the-knee level to improve wound healing is still inconclusive. Methods A single center retrospective study was conducted at Faculty of Medicine, Siriraj Hospital, Mahidol University. The outcomes of infrapopliteal DES placement among forty-six patients with critical limb ischemia (category 4-6 of Rutherford’s classification) from 2007 to 2011 were retrospectively reviewed. The primary endpoints were pre- and post-procedural Ankle-Brachial index (ABI) and transcutaneous oxygen tension (TcPO2). The secondary endpoints were limb salvage rate, time-to-major amputation and healing of ulcers. The differences between pre- and post-procedural parameters were analyzed with paired T-test. Results The mean follow-up time in the study was 490.21 days with limb salvage rate of 93.8 percent. Complete wound healing was observed in 81.3 percent of patients. Three of the patients underwent major amputations with the time-to-amputation ranging from 40 to 277 days post-procedure. The overall ABI was significantly improved from 0.64 ± 0.12 to 0.88 ± 0.28 (p-value &lt;0.001). Among subjects with isolated below-the-knee DES placement, ABI was also significantly improved from 0.62 ± 0.12 to 0.82 ± 0.26 (p-value 0.004). Furthermore, TcPO2 was also increased from 25.82 ± 15.77 to 40.18 ± 13.21 mmHg (p-value 0.023). Conclusions DES placement at below-the-knee level is both a safe and effective limb saving therapy in CLI patients. It has been shown to significantly improve parameters for wound healing: ABI and transcutaneous oxygen tension (TcPO2).

Abstract 227: Adipose Stromal Vascular Fraction (SVF) Vasculogenesis Demonstrates a Dose Response Through the Assembly of Endothelial Networks

Objective Critical limb ischemia (CLI) is a challenging clinical entity complicated by chronic microvascular disease. Stromal vascular fraction (SVF), a heterogeneous population of cells derived from adipose tissue, has garnered interest as a means of an autologous cell-based approach to vasculogenesis in these patients. The SVF concentration needed to achieve this goal is still unknown. Our goal was to determine if SVF vasculogenesis exhibits a dose-response using in vitro and murine in vivo models. Methods SVF was isolated from adult male Sprague Dawley transgenic-GFP+ Rat epididymal fat pads and serially diluted using SVF growth media and then added to a respectively labeled matrigel-coated plates. Wells were then labeled with Griffonia simplicifolia-1 (GS-1) rhodamine lectin. Phase and fluorescent microscopy images of each well were then taken. The same SVF concentrations were suspended in collagen gels and subcutaneously implanted into the flanks of 6 Rag-1 deficient mice and imaged with confocal microscopy 2 weeks later to determine if a dose-response could be seen in-vivo. Tubule length was calculated using Metamorph software. Results Vasculogenic networks showed monolayering at concentrations of 5.0 x 10 5 and 1.0 x 10 6 cells/mL, where as the intermediate dose exhibited greater complexity to its network. Total tubule length, measured in μm, peaked with the 2.50 x 10 5 cells/mL concentration. Tubule length increased significantly up to this peak, and then gradually decreased at higher concentrations. GS-1 staining was positive when viewing the tubules under fluorescence microscopy. Preliminary results from the in-vivo model showed increasing vasculogenesis up to a maximal tubule length for the 1 x 10 6 cells/mL concentration. Conclusion SVF vasculogenesis exhibits a dose-response. Tubule formation is of endothelial phenotype and tubule length is directly proportional to cell concentration up to a peak concentration of 2.5 x 10 5 cells/mL. Preliminary in-vivo data also suggests that a dose-reponse exists. We are encouraged by these results that suggest that cell based therapy may be tailored for appropriate dosing in CLI patients.

Anatomic Findings and Outcomes Associated With Arteriography and Thrombolysis for Acute Finger Ischemia

bone marrow then concentrated and injected intramuscularly into the ischemic limb. Results: BMA patients had a higher incidence of prior smoking (90% vs 43%; P < .05) with a trend towards fewer diabetics (47% vs 71%; P 1⁄4 .07). In the BMA cohort, 84% had rest pain and 79% had tissue loss. 68% had a prior bypass or endovascular intervention. Mean follow-up was 334 days (6170). A second BMA treatment was performed in 21% due to clinical deterioration. ABI improvement was 0.23 (60.25). Rest pain improved in 87.5% and completely resolved in 56%. Wound healing occurred in 67%. 3 patients went on to amputation (Freedom from major amputation or death at 1.5 years 89% vs 59% (Fig; P < .05). Conclusions: Bone marrow aspirate injection therapy is a potential option in CLI patients who are not candidates for bypass or endovascular intervention. Limb salvage is unexpectedly high in this population with few other options.

Differential Proliferative and Migratory Activity of Human Myoblasts Isolated from CLI and Control Patients

Differential Proliferative and Migratory Activity of Human Myoblasts Isolated from CLI and Control Patients

Commentary: Heading for the Backdoor: An Extreme Approach to Foot Salvage in CLI Patients

Commentary: Heading for the Backdoor: An Extreme Approach to Foot Salvage in CLI Patients

The Endurant Stent-Graft U.S. Clinical Trial: 2-Year AAA Diameter Outcomes Are Predicted by 6-Month Volume Outcomes

formed to evaluate rates and predictors of 30-day readmission from a multicenter trial data set. Methods: We analyzed the PREVENT III data set of 1404 CLI patients undergoing LEB at 83 North American centers. The primary end point was readmission 30 days of discharge. Secondary end points included graft patency and limb salvage evaluated in the context of readmission. Results: We analyzed 1356 patients, of these, 23 (1.7%) died inhospital and were excluded from re-admission analyses. Overall, 327 of 1333 patients (24.5%) were readmitted 30 days of discharge. Reasons for readmission included 127 (39%) wound infections in the index leg, 75 (23%) nonvascular reasons, 68 (20.9%) additional procedures in the index leg, and 19 graft failures (5.8%). Univariate predictors are shown in the Table. Adjusted independent predictors of 30-day readmission included wound infection (odds ratio [OR], 4.1; 95% confidence interval [CI], 3.0-5.4; P .0001), renal failure (OR, 4.1; 95% CI, 1.9-8.8; P .0004), early lost patency (OR, 1.9; 95% CI, 1.2-2.9; P .003), dialysis (OR, 1.8; 95% CI, 1.2-2.6; P .003), and female gender (OR, 1.3; 95% CI, 1.0-1.8; P .03). Patients readmitted 30 days had lower rates of limb salvage at 1 year (78% 2.4% vs 91% 0.9%, P .0001). Thirty-day readmission was predictive of limb loss (HR, 2.25; 95% CI, 1.6-3.1; P .0001) at 1 year, after adjustment for other factors. Conclusions: Readmission after LEB for CLI is common (24%) and associated with defined clinical predictors. Readmission is associated with long-term limb loss. These data provide benchmark values for this complex patient population and may prove useful as disease-specific bundling strategies are derived.

A Phase II Trial of Autologous Transplantation of Bone Marrow Stem Cells for Critical Limb Ischemia: Results of the Naples and Pietra Ligure Evaluation of Stem Cells Study

Critical limb ischemia (CLI) is a vascular disease affecting lower limbs, which is going to become a demanding challenge because of the aging of the population. Despite advances in endovascular therapies, CLI is associated with high morbidity and mortality. Patients without direct revascularization options have the worst outcomes. To date, 25%-40% of CLI patients are not candidates for surgical or endovascular approaches, ultimately facing the possibility of a major amputation. This study aimed to assess the safety and efficacy of autologous bone marrow (BM) transplantation performed in "no-option" patients, in terms of restoring blood perfusion by collateral flow and limb salvage. A multicenter, prospective, not-controlled phase II study for no-option CLI patients was performed. Patients were subjected to intra-arterial infusion of autologous bone marrow and followed for 12 months after the treatment. Variation of blood perfusion parameters, evaluated by laser Doppler flowmetry or transcutaneous oximetry, was set as the primary endpoint at 12 months after treatment and amputation-free survival as the secondary endpoint. Sixty patients were enrolled and treated with BM transplantation, showing improvement in objective and subjective measures of perfusion. Furthermore, survival analysis demonstrated improved amputation-free survival rates (75.2%) at 12 months after the treatment. This study provides further evidence that autologous bone marrow transplantation is well tolerated by CLI patients without adverse effects, demonstrating trends toward improvement in perfusion and reduced amputation rate, confirming the feasibility and safety of the procedure.

Abstract 119: Potential Role for Bone Marrow Niche and Endothelial Progenitor Cell Dysfunction in Critical Limb Ischemia

Background Critical limb ischemia (CLI) is characterized by obstruction of lower extremity arteries and a largely unexplained impaired ischemic neovascularization response. Bone marrow (BM) derived endothelial progenitor cells (EPC) contribute to postnatal neovascularization. We hypothesize that reduced levels and function of circulating progenitor cells and a dysfunctional BM environment contribute to impaired neovascularization in CLI. Methods Levels of primitive (CD34+ and CD133+) progenitors and CD34+KDR+ haemangioblastic EPC were analyzed using flow cytometry in peripheral blood (PB) and BM from 101 CLI patients in the JUVENTAS trial ( NCT00371371 ) and healthy controls (n=37 and n=12 for PB and BM, respectively). Endothelial damage markers (sE-selectin, sICAM-1, sVCAM-1, thrombomodulin) and PB levels of progenitor cell mobilizing (VEGF, SDF-1α, SCF, G-CSF) and inflammatory (IL-6, IL-8, IP-10) factors were assessed by ELISA and multiplex. Levels and activity of the EPC mobilizing protease MMP-9 were assessed in BM plasma by ELISA and zymography. Circulating angiogenic cells (CAC) were cultured from PB, and CAC paracrine function was assessed. Results Endothelial damage markers were higher in CLI ( p&lt; 0.01). PB levels of VEGF, SDF-1α, SCF, G-CSF ( p&lt; 0.05) and of IL-6, IL-8 and IP-10 were higher in CLI ( p&lt; 0.05). Circulating EPC and CD133+ cells and BM CD34+ cells were significantly lower in CLI (all p &lt;0.05), BM levels and activity of MMP-9 were lower in CLI (both p&lt; 0.01). Multivariate regression analysis showed an inverse association between IL-6 levels and BM CD34+ cell levels ( p= 0.007). CAC outgrowth did not differ significantly between CLI patients and healthy controls ( p= 0.137), however CAC from CLI patients had profoundly reduced migration stimulating potential ( p&lt; 0.0001). Conclusion CLI patients have reduced levels of circulating EPC despite profound endothelial injury and an EPC mobilizing response. Moreover, CLI patients have lower BM CD34+ cell levels, which were inversely associated with the inflammatory marker IL-6, and lower BM MMP-9 levels and activity. Our data suggest that reduced levels and function of circulating progenitor cells and BM dysfunction contribute to the defective neovascularization response in CLI.

Early endothelial progenitor cells in bone marrow are a biomarker of cell therapy success in patients with critical limb ischemia

Endothelial progenitor cells (EPC) have been proposed for autologous angiogenic therapy. The objectives of this study were to quantify EPC in the peripheral blood and bone marrow mononuclear cells (BM-MNC) of patients with critical limb ischemia that had received BM-MNC as a cell therapy product, and to study the putative relationship between the presence of EPC and the process of neovascularization in toe or transmetatarsal amputation specimens. Early and late endothelial progenitor cells (CFU-EC and ECFC) were cultivated and quantified according to published methods in peripheral blood and BM-MNC from patients with critical limb ischemia (CLI; n = 11) enrolled in the OPTIPEC trial ( http://clinicaltrials.gov/ct2/show/NCT00377897 ) to receive BM-MNC as a cell therapy product. Eight out of the 11 patients had undergone amputations. Three of the patients displayed a neoangiogenic process that was associated with a higher number of CFU-EC in BM-MNC, while CD3+ , CFU-GM and CD34+ in BM-MNC, and EPC in peripheral blood, did not correlate with the appearance of newly formed vessels. As expected, circulating CFU-EC and ECFC counts were significantly lower in CLI patients compared with age-matched controls. In patients with critical limb ischemia, EPC in peripheral blood were decreased compared with healthy individuals. However, in BM-MNC we found that relative numbers of CFU-EC could be used as an indicator to discriminate patients with neoangiogenic processes. These results need to be confirmed in a randomized study.

重症下肢虚血患者におけるModified Transmetatarsal Amputation前後の血行動態の検討

重症下肢虚血患者におけるModified Transmetatarsal Amputation前後の血行動態の検討

Endovascular Management as First Therapy for Chronic Total Occlusion of the Lower Extremity Arteries:Comparison of Balloon Angioplasty, Stenting, and Directional Atherectomy

To evaluate the role of endovascular therapy in the management of infrainguinal arterial chronic total occlusions (CTOs). Data on all patients with CTOs treated at a single center from 2004 to 2010 were extracted from a prospectively maintained database for retrospective analysis. Patient demographics, angiographic studies, noninvasive vascular test results, and clinical outcomes were evaluated. In this time frame, 481 patients (283 men; mean age 71.7±11.5 years, range 52-85) with claudication (n = 177) or critical limb ischemia (CLI, n = 304) were treated for 688 CTOs. Lesions were segregated according to location [SFA (n = 193), popliteal (n = 67), tibial (n = 217), and multilevel (n = 211)] and analyzed based on treatment mode (angioplasty, angioplasty with stenting, or atherectomy) and clinical indication. Primary patency, assisted primary patency, and secondary patency, as well as limb salvage rates for CLI patients, were calculated. At 2 years in claudicants with CTOs confined to the SFA, primary patency ranged from 44% to 58% and secondary patency to 92% depending on treatment type; there were no significant differences among the treatments. However, in CLI patients with SFA CTOs, atherectomy produced better outcomes at 2 years (p = 0.002 for primary and p = 0.012 for secondary patency) than angioplasty alone. The limb salvage rates ranged from 73% to 91% (no differences among treatment types). In diabetics, CTOs treated with angioplasty and stent had improved secondary patency rates over angioplasty alone. The endovascular management of CTO results in reasonable primary patency; moreover, secondary patency at 2 years is excellent. Endovascular therapy should be the first-line option for many patients with peripheral artery disease, including those with CLI, claudicants with poor bypass conduit, or patients at high medical risk for surgery. The presence of CTOs does not appear to change these recommendations. Although multiple reinterventions may be required, endovascular therapies can be considered a primary therapy for many patients with CTO.

Hypocellularity and insufficient expression of angiogenic factors in implanted autologous bone marrow in patients with chronic critical limb ischemia

The aim of this study was to identify the clinical parameters of absolutely poor-prognosis patients with chronic critical limb ischemia (AP-CLI). Sixteen no-option CLI patients with arteriosclerosis obliterans: ASO (nine) and non-ASO patients (seven) treated with bone marrow-mononuclear cell implantation (BMI) were analyzed. There were three AP-CLI patients (all ASO). The mRNA expression of several angiogenic factors in the implanted cells was analyzed in comparison with normal donor bone marrow. To observe the response of bone marrow components to hypoxia, normal bone marrow cells were cultured for 24 h in 2.5% O(2), and mRNA expression of angiogenic factors were measured. AP-CLI patients exhibited extraordinary low bone marrow cellularity as well as the percentage of CD34-positive cells. Among angiogenic factors, only VEGF expression was maintained in response to HIF-1, while other factors such as HGF, Ang-1, PLGF, and SDF-1 decreased in the implanted bone marrow cells of the patients with CLI compared to normal bone marrow cells. HIF-1 and all of the five angiogenic factors increased in vitro in response to hypoxia. Thus it is highly likely that angiogenic factors except VEGF do not respond to chronic ischemia in bone marrow in vivo. An organ-protection system against tissue ischemia may be applied for acute hypoxia, but it may be insufficient for chronic ischemia.

Critical limb ischemia.

Opinion statementCritical limb ischemia (CLI), defined as chronic ischemic rest pain, ulcers, or gangrene attributable to objectively proven arterial occlusive disease, is the most advanced form of peripheral arterial disease. Traditionally, open surgical bypass was the only effective treatment strategy for limb revascularization in this patient population. However, during the past decade, the introduction and evolution of endovascular procedures have significantly increased treatment options. In a certain subset of patients for whom either surgical or endovascular revascularization may not be appropriate, primary amputation remains a third treatment option. Definitive high-level evidence on which to base treatment decisions, with an emphasis on clinical and cost effectiveness, is still lacking. Treatment decisions in CLI are individualized, based on life expectancy, functional status, anatomy of the arterial occlusive disease, and surgical risk. For patients with aortoiliac disease, endovascular therapy has become first-line therapy for all but the most severe patterns of occlusion, and aortofemoral bypass surgery is a highly effective and durable treatment for the latter group. For infrainguinal disease, the available data suggest that surgical bypass with vein is the preferred therapy for CLI patients likely to survive 2 years or more, and for those with long segment occlusions or severe infrapopliteal disease who have an acceptable surgical risk. Endovascular therapy may be preferred in patients with reduced life expectancy, those who lack usable vein for bypass or who are at elevated risk for operation, and those with less severe arterial occlusions. Patients with unreconstructable disease, extensive necrosis involving weight-bearing areas, nonambulatory status, or other severe comorbidities may be considered for primary amputation or palliative measures.

The Role for Cryoplasty in the Treatment of Infrainguinal Artery Disease:Case Studies

Cryoplasty has been shown to be safe and effective for the treatment of atherosclerotic lesions in the peripheral vasculature and offers the promise of improving on the results of percutaneous transluminal angioplasty (PTA) by limiting dissection, vessel recoil, and restenosis. The PolarCath Peripheral Dilatation System utilizes nitrous oxide rather than the standard mixture of saline and contrast medium to inflate and cool the balloon to the desired temperature of approximately -10 degrees C. Cryoplasty can be used in combination with other therapies, can be repeated, and offers the advantage of not leaving any foreign objects in the body. In a multicenter registry of claudicants with de novo or restenotic femoropopliteal lesions and in a multicenter trial of CLI patients with infrapopliteal lesions (86.9% mean diameter stenosis, 33.9% occlusions), favorable results with primary cryoplasty included minimal need for bailout stenting, avoidance of repeat revascularization, and high amputation-free survival. We review cases in which cryoplasty was used for diffuse superficial femoral and popliteal artery disease, for infrapopliteal stenosis and occlusion, for in-stent restenosis, and as part of a hybrid strategy for treating multilevel occlusive disease. As these clinical cases demonstrate, cryoplasty therapy can be employed effectively as a primary strategy, or in conjunction with debulking, for the treatment of lesions of varying severity in different segments of the infrainguinal vasculature. Further studies will be required to better define the role of this therapy relative to the other modalities that are currently available for the treatment of femoropopliteal and infrapopliteal disease.

3P-0876 The organs microcirculation structural changes in atherogenesis

Endothelial progenitor cells (EPC) have been proposed for autologous angiogenic therapy. The objectives of this study were to quantify EPC in the peripheral blood and bone marrow mononuclear cells (BM-MNC) of patients with critical limb ischemia that had received BM-MNC as a cell therapy product, and to study the putative relationship between the presence of EPC and the process of neovascularization in toe or transmetatarsal amputation specimens.Early and late endothelial progenitor cells (CFU-EC and ECFC) were cultivated and quantified according to published methods in peripheral blood and BM-MNC from patients with critical limb ischemia (CLI; n = 11) enrolled in the OPTIPEC trial (http://clinicaltrials.gov/ct2/show/NCT00377897) to receive BM-MNC as a cell therapy product.Eight out of the 11 patients had undergone amputations. Three of the patients displayed a neoangiogenic process that was associated with a higher number of CFU-EC in BM-MNC, while CD3+, CFU-GM and CD34+ in BM-MNC, and EPC in peripheral blood, did not correlate with the appearance of newly formed vessels. As expected, circulating CFU-EC and ECFC counts were significantly lower in CLI patients compared with age-matched controls.In patients with critical limb ischemia, EPC in peripheral blood were decreased compared with healthy individuals. However, in BM-MNC we found that relative numbers of CFU-EC could be used as an indicator to discriminate patients with neoangiogenic processes. These results need to be confirmed in a randomized study.

Biological markers as classifiers for depression: A multivariate study

1. 1. Delta TSH, REM latency, 4 pm and 11 pm post-dexamethasone Cortisol values were determined after a wash-out period in a group of 74 non-selected depressed patients who were diagnosed (according to RDC with the SADS) as follows: 46 definite and 10 probable MD, 4 minor and 14 intermittent depression. 2. 2. These biological variables, as well as gender, age and basal TSH were introduced in a principal component analysis. The four first PC scores explaining up to 77% of the data set were further calculated for each patients and used in a cluster analysis. A three clusters solution was retained. 3. 3. DST escape and increased TSH response to TRH each identified subgroups of depressed patients. Conversely, blunted TSH response or REM latency were inefficient to classify patients. 4. 4. Thus, HPA hyperactivity characterized CL-I patients (n = 29). These were more severely depressed, displayed more endogenous features and were reported as being more anxious. 5. 5. Increased TSH response to TRH identified CL-III, exclusively composed of female patients (n = 10) that displayed more apparent sadness and tended to be older. 6. 6. In CL-II, the usual sex-ratio for depressive illness was reversed and patients (n = 35) exhibited the least HPA axis disturbances and the same rate of blunted TSH response than in CL-I. They were also less severely depressed, displayed less endogenous characteristics and were rated as more mood reactive. 7. 7. These results suggest heterogeneity in biological disturbances in depression and further stress the importance for controlling age, gender and severity of illnes in studies investigating biological markers in depression.