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Preparation Systems
Abstract
The first FDA-cleared platelet-rich plasma (PRP) collection devices were developed for use in orthopedic surgery in the 1990s, but recently there has been an explosion in off-label use of newer devices for aesthetic indications. The protocol for PRP preparation involves blood collection, platelet isolation via centrifugation, platelet activation, and administration into target tissues. Each of these steps introduces a source of variation that affects the composition and efficacy of the final PRP product, and this explains the significant heterogeneity seen among the preparation systems currently on the market. A number of classification systems have been proposed to help systematize the reporting of PRP outcomes, but they can also be used to compare devices. Aesthetic clinicians must understand the components of a PRP preparation system in order to critically evaluate the scientific literature on this topic and to offer the most effective treatment regimen to patients. The nuances pertaining to each of the preparatory factors discussed in this chapter are sources of great debate and require further study.
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Background:
Platelet rich plasma (PRP), processed from autologous peripheral blood, is used to treat androgenetic alopecia (AGA).
Objective:
To determine the efficacy of PRP for hair growth promotion in AGA patients in a randomized, blinded, placebo controlled, pilot clinical trial (NCT02074943).
Methods:
The efficacy of an 8 week, 5 session, PRP treatment course was determined by measuring hair density and hair caliber changes in 10 AGA affected patients. For each PRP sample, the concentrations of selected growth factors were determined using a multiplex assay system. The clinical results were then correlated to the growth factor concentrations in PRP.
Results:
At 16 weeks, 8 weeks after the last PRP injection, treated areas exhibited increased mean hair density (+12.76%) over baseline compared to placebo (+0.99%). Mean hair caliber decreased in both treated and placebo regions (-16.22% and -19.46% respectively). Serial analysis of PRP significant variability in concentrations between patients. Overall, there was a positive correlation between GDNF concentration and hair density (p= 0.004). Trends, though not statistically significant, were also observed for FGF2 and VEGF.
Limitations:
Small sample size and lack of comparative cohorts receiving protocol variations limit confidence in the study data.
Conclusions:
This small pilot clinical trial suggests PRP treatment may be beneficial for AGA. However, the variable hair growth responses between patients indicate there is a significant opportunity to improve PRP therapy protocols for hair growth promotion. The variability in growth factor concentration in PRP suggests standardization of growth factors post-processing might improve hair growth responses.
Autologous biologics, defined as platelet-rich plasma (PRP) and bone marrow aspirate concentrate (BMC), are cell-based therapy treatment options in regenerative medicine practices, and have been increasingly used in orthopedics, sports medicine, and spinal disorders. These biological products are produced at point-of-care; thereby, avoiding expensive and cumbersome culturing and expansion techniques.
Numerous commercial PRP and BMC systems are available but reports and knowledge of bio-cellular formulations produced by these systems are limited. This limited information hinders evaluating clinical and research outcomes and thus making conclusions about their biological effectiveness. Some of their important cellular and protein properties have not been characterized, which is critical for understanding the mechanisms of actions involved in tissue regenerative processes. The presence and role of red blood cells (RBCs) in any biologic has not been addressed extensively. Furthermore, some of the pathophysiological effects and phenomena related to RBCs have not been studied. A lack of a complete understanding of all of the biological components and their functional consequences hampers the development of clinical standards for any biological preparation.
This paper aims to review the clinical implications and pathophysiological effects of RBCs in PRP and BMC; emphasizes hemolysis, eryptosis, and the release of macrophage inhibitory factor; and explains several effects on the microenvironment, such as inflammation, oxidative stress, vasoconstriction, and impaired cell metabolism.
Background:
Platelet-rich plasma (PRP) is a blood-derived preparation whose use has grown exponentially in orthopaedic practice. However, there remains an unclear understanding of the biological properties and effects of PRP on musculoskeletal healing. Heterogeneous processing methods, unstandardized nomenclature, and ambiguous classifications make comparison among studies challenging. A comprehensive assessment of orthopaedic clinical PRP trials is key to unraveling the biological complexity of PRP, while improving standardized communication. Toward this goal, we performed a systematic review of the PRP preparation protocols and PRP composition utilized in clinical trials for the treatment of musculoskeletal diseases.
Methods:
A systematic review of the literature was performed from 2006 to 2016. Inclusion criteria were human clinical trials, English-language literature, and manuscripts that reported on the use of PRP in musculoskeletal/orthopaedic conditions. Basic-science articles, editorials, surveys, special topics, letters to the editor, personal correspondence, and nonorthopaedic applications (including cosmetic use or dental application studies) were excluded.
Results:
A total of 105 studies (in 104 articles) met the inclusion criteria for analysis. Of these studies, only 11 (10%) provided comprehensive reporting that included a clear description of the preparation protocol that could be used by subsequent investigators to repeat the method. Only 17 studies (16%) provided quantitative metrics on the composition of the final PRP product.
Conclusions:
Reporting of PRP preparation protocols in clinical studies is highly inconsistent, and the majority of studies did not provide sufficient information to allow the protocol to be reproduced. Furthermore, the current reporting of PRP preparation and composition does not enable comparison of the PRP products being delivered to patients. A detailed, precise, and stepwise description of the PRP preparation protocol is required to allow comparison among studies and provide reproducibility.
Chronic wounds, such as diabetic foot ulcers, represent a serious clinical problem for patients and clinicians. Management of these wounds has a strong economic impact worldwide. Complications resulting from injuries are a frequent cause of morbidity and mortality. Chronic wounds lead to infections, painful dressings and prolonged hospitalisation. This results in poor patient Quality of Life and in high healthcare costs. Platelet concentrates (PC) are defined as autologous or allogeneic platelet derivatives with a platelet concentration higher than baseline. PC are widely used in different areas of Regenerative Medicine in order to enhance wound healing processes; they include platelet-rich plasma (PRP), platelet gel (PG), platelet-rich fibrin (PRF), serum eye drops (E-S), and PRP eye drops (E-PRP). This review highlights the use of platelet-rich plasma (PRP) and platelet gel (PG) preparation for clinical use.
Background/aim Significant biological differences in platelet-rich plasma (PRP) preparations have been highlighted and could explain the large variability in the clinical benefit of PRP reported in the literature. The scientific community now recommends the use of classification for PRP injection; however, these classifications are focused on platelet and leucocyte concentrations. This presents the disadvantages of (1) not taking into account the final volume of the preparation; (2) omitting the presence of red blood cells in PRP and (3) not assessing the efficiency of production.
Methods On the basis of standards classically used in the Cell Therapy field, we propose the DEPA (Dose of injected platelets, Efficiency of production, Purity of the PRP, Activation of the PRP) classification to extend the characterisation of the injected PRP preparation. We retrospectively applied this classification on 20 PRP preparations for which biological characteristics were available in the literature.
Results Dose of injected platelets varies from 0.21 to 5.43 billion, corresponding to a 25-fold increase. Only a Magellan device was able to obtain an A score for this parameter. Assessments of the efficiency of production reveal that no device is able to recover more than 90% of platelets from the blood. Purity of the preparation reveals that a majority of the preparations are contaminated by red blood cells as only three devices reach an A score for this parameter, corresponding to a percentage of platelets compared with red blood cells and leucocytes over 90%.
Conclusions These findings should provide significant help to clinicians in selecting a system that meets their specific needs for a given indication.
Platelet-rich plasma (PRP) is blood plasma that has been enriched with platelets. It holds promise for clinical use in areas such as wound healing and regenerative medicine, including bone regeneration. This study characterized the composition of PRP produced by seven commercially available separation systems (JP200, GLO PRP, Magellan Autologous Platelet Separator System, KYOCERA Medical PRP Kit, SELPHYL, MyCells, and Dr. Shin's System THROMBO KIT) to evaluate the platelet, white blood cell, red blood cell, and growth factor concentrations, as well as platelet-derived growth factor-AB (PDGF-AB), transforming growth factor beta-1 (TGF-β1), and vascular endothelial growth factor (VEGF) concentrations. PRP prepared using the Magellan Autologous Platelet Separator System and the KYOCERA Medical PRP Kit contained the highest platelet concentrations. The mean PDGF-AB concentration of activated PRP was the highest from JP200, followed by the KYOCERA Medical PRP Kit, Magellan Autologous Platelet Separator System, MyCells, and GLO PRP. TGF-β1 and VEGF concentrations varied greatly among individual samples, and there was almost no significant difference among the different systems, unlike for PDGF. The SELPHYL system produced PRP with low concentrations of both platelets and growth factors. Commercial PRP separation systems vary widely, and familiarity with their individual advantages is important to extend their clinical application to a wide variety of conditions.
Platelet gel is used to facilitate wound healing in virtue of the growth factors released from activated platelets at the site of lesion, but little is known about the specific mechanisms underlying cellular repair.
To evaluate, in vitro, cellular effects of different concentrations of platelet gel -released supernatant on endothelial cells.
Platelet concentrate was produced at the Service of Immunohaematology and Transfusion of San Salvatore Hospital of L'Aquila, using multiple bags. Platelet gel was obtained by adding thrombin and calcium gluconate to the concentrates and then centrifuging to recover the supernatant. Human umbilical vein endothelial cells were isolated from umbilical cord veins and grown in appropriate conditions. To study their viability, cells were treated with different concentrations of supernatant and XTT assays were performed on the 3 days following treatment. Endothelial cell motility and invasiveness were assayed using modified Boyden chambers with filters coated with 0.1% gelatin (for the motility test) or with a thick layer of the reconstituted basement membrane Matrigel (for the invasion test). The supernatant, added at various concentrations to the lower compartment of the chamber, was used as an attractant. Umbilical cells were added to the upper compartment of the chamber. After 4 hours (for the motility test) or 6 hours (for the invasion test), filters were stained and the migrated cells in five high-power fields were counted.
When used at specific concentrations, platelet gel-released supernatant is able to induce proliferation and to stimulate motility and invasiveness of endothelial human cells. Higher concentrations induce a reversion of the stimulatory processes.
There is a large body of evidence indicating that platelets and their derivatives have the potential for a substantial therapeutic role in tissue regeneration. The results of this in vitro study highlight the need for an in-depth analysis of technical protocols for the most appropriate and effective use of platelet gel for in vivo applications.
Platelet-rich plasma (PRP) has been increasingly used in sports medicine applications. Platelets are thought to release growth factors important in wound healing, including transforming growth factor (TGF-β1), platelet-derived growth factor (PDGF-AB), and vascular endothelial growth factor (VEGF). However, little is known about the effect of platelet activator choice on growth factor release kinetics.
The choice of platelet activator would affect the timing and level of growth factor release from PRP.
Controlled laboratory study.
Platelet-rich plasma aliquots were activated with either thrombin or collagen. A control group of whole blood aliquots was clotted with thrombin. Supernatant containing the released growth factors was collected daily for 1 week. Levels of TGF-β1, PDGF-AB, and VEGF were measured using enzyme-linked immunosorbent assay (ELISA).
The use of thrombin as an activator resulted in immediate release of TGF-β1 and PDGF-AB, while the collagen-activated PRP clots released similar amounts each day for 5 days. The use of collagen as an activator resulted in an 80% greater cumulative release of TGF-β1 from the PRP aliquots over 7 days (P < .001). Concentrating platelets to 3 times the systemic blood level resulted in a 3-fold higher release of TGF-β1, 2.5-fold greater release of PDGF, and 5-fold greater release of VEGF (all P < .0001) when compared with whole blood control clots, but no significant differences in the timing of release were noted.
These experiments demonstrated that the choice of platelet activator can significantly influence the release kinetics of cytokines from PRP, with thrombin resulting in an immediate release and collagen having a more sustained release pattern.
The level and rate of growth factor release depends on the selected platelet activator, a factor that should be considered when selecting a PRP system for a given application.
Background:
Platelet-rich plasma (PRP) is an increasingly popular treatment modality for various dermatologic conditions, but there are limitations in both the published literature and clinician knowledge.
Objective:
To create a high-yield, in-depth analysis of PRP in procedural dermatology by reviewing available data on its role in hair restoration, soft-tissue remodeling, resurfacing, and rejuvenation; identifying practice gaps and controversies; and making suggestions for future research that will establish dermatologists as pioneers of regenerative medicine.
Materials and methods:
A 2-part systematic review and expert analysis of publications before October 2018.
Results and conclusion:
Most studies on PRP report favorable outcomes with the strongest level of evidence existing for androgenetic alopecia followed by postprocedure wound healing, scar revision, striae, rejuvenation, and dermal filling. There is a dearth of large randomized controlled trials, considerable heterogeneity in the variables studied, and lack of specificity in the preparatory protocols, which may influence clinical outcomes. Future investigations should use consistent nomenclature, find ideal solution parameters for each cutaneous indication, determine significant outcome metrics, and follow double-blinded, randomized, controlled methodologies. Addressing these deficiencies will take sound scientific inquiry but ultimately has the potential to benefit the authors' specialty greatly.
Female pattern hair loss (FPHL) is a common nonscarring alopecia characterized by progressive loss of terminal hairs. FPHL is a major concern for women and has a high impact on quality of life. Therapeutic regimen is often challenging and requires multiple combinations of topical, systemic, and interventional therapies to control hair loss and produce satisfactory hair regrowth. This article reviews common treatments of FPHL and their efficacy.
Autologous and single donor allogenic platelet preparations are increasingly being used in many areas of regenerative medicine. However, there are few properly controlled randomized clinical trials, and the preparation, content and characteristics of platelet preparations are generally poorly defined and controlled. The Platelet Physiology Subcommittee of the Scientific and Standardization Committee (SSC) of the ISTH formed a working party of experts with the aim of producing consensus recommendations for guidance on the use of platelets in regenerative medicine. Due to a lack of investigations that provide definitive evidence for the efficacy, definition and use of different platelet preparations in regenerative medicine, there were insufficient data to develop evidence‐based guidelines. Therefore, the RAND method was used, which obtains a formal consensus among experts particularly when scientific evidence is absent, scarce and/or heterogeneous. Using this approach, each expert scored as “appropriate”, “uncertain” or “inappropriate” a series of 45 statements about the practice of regenerative medicine with platelets, which included different sections on general aspects, platelet preparations, clinical trial design; and potential utility in different clinical scenarios. After presentation and public discussion at SSC meetings, the assessments were further refined to produce final consensus recommendations which is the subject of the present report.
This article is protected by copyright. All rights reserved.
Background
Platelet‐rich plasma (PRP) injections have gained popularity in dermatology practice as a treatment for hair loss. As an autologous blood product, PRP is categorized as a minimally manipulated tissue by the U.S. Food and Drug Administration (FDA) and thus evades extensive regulation. As a result, there is a lack of standardization of its preparation and final composition.
Objective
This study aims to provide a systematic review of the various PRP preparation protocols and PRP compositions utilized in clinical trials for the treatment of hair loss.
Methods & Materials
A review of the literature was performed using PubMed and Ovid/Medline in November 2017 using the search terms “Platelet‐Rich Plasma” and (“Alopecia” or “Hair Loss”), including all publication dates. Human clinical trials in the English language were included.
Results
Nineteen studies (in 15 articles) met the inclusion criteria for analysis. Only 21% of these studies reported all PRP preparation factors analyzed, and only 32% of the protocols reported the platelet count for both the initial whole blood and final PRP product.
Conclusion
The current reporting of PRP preparation methodology and final composition is inconsistent and insufficient to enable comparison between studies and determination of efficacy for particular treatment applications.
Background
Platelet rich plasma (PRP) has attracted attention in a number of surgical fields due to a wide variety of potential clinical benefits. Yet PRP has not gained wide popularity in aesthetic surgery as a result of uncertainty surrounding objective clinical evidence.
Objectives
We aim to describe the current applications, define preparation and activation, explore effectiveness, and propose a classification system to facilitate comparisons across studies.
Methods
A comprehensive review of the literature regarding the use of platelet rich plasma in aesthetic surgery was performed. Data gathered included: PRP application, study type, subject number, centrifugation, anticoagulation, activation, PRP composition, and outcomes.
Results
Thirty-eight reports were identified. Applications included injection into aging skin (29%), scalp alopecia (26%), lipofilling (21%), fractional laser (13%), and facial surgery (11%). The majority of studies (53%) were case series without controls. Leucocytes were sparsely defined (32%). The concentration of injected and/or baseline platelets was rarely clarified (18%). The mechanism of activation was described in 27 studies (71%), while anticoagulation was uncommonly elucidated (47%). While most studies (95%) claim effectiveness, objective measures were only utilized in 17 studies (47%).
Conclusions
Current studies produce context-dependent results with a lack of consistent reporting of PRP preparation, composition, and activation in aesthetic applications, making meaningful meta-analysis unrealistic. Thus the method of PRP preparation warrants increased attention. We recommend a set of descriptors, FIT PAAW (described below), to produce scientifically grounded conclusions, facilitating a clearer understanding of the situations in which PRP is effective.
Level of Evidence: 4
Background:
Platelet-rich plasma (PRP) therapy is a novel procedure used to treat androgenetic alopecia (AGA).
Objective:
Propose a mechanism of action of PRP therapy for AGA.
Methods and materials:
A thorough literature search including PRP research for AGA therapy as well as PRP research in other areas of medicine was conducted.
Results:
A mechanistic model for the action of PRP on the hair follicle was created.
Conclusion:
Platelet-rich plasma therapy stimulates hair growth through the promotion of vascularization and angiogenesis, as well as encourages hair follicles to enter and extend the duration of the anagen phase of the growth cycle. The process is accomplished through growth factor-mediated increased activation of wingless (Wnt)/β-catenin, extracellular signaling regulated kinase (ERK), and protein kinase B (Akt) signaling pathways, which leads to the necessary cellular proliferation and differentiation.
Whole blood is composed of both cellular and plasma components, providing a rich source of therapeutic products. Of late, platelet derived biomaterial (platelet rich plasma) consisting of plasma proteins and platelets are increasingly being used for various indications. Protocols for preparation and nomenclature of this biomaterial vary widely amongst authors and are often not well defined. Additionally, they are not uniformly documented in the literature, making results difficult to compare or replicate. In this paper we review the evolution and type of these products available for clinical use. Further we will discuss the scientific rational and technical aspects in preparation of these platelet biomaterials in order to administer them in
various fields of medicine.
With increased utilization of platelet-rich plasma (PRP), it is important for clinicians to understand the United States, the Food and Drug Administration (FDA) regulatory role and stance on PRP. Blood products such as PRP fall under the prevue of FDA's Center for Biologics Evaluation and Research (CBER). CBER is responsible for regulating human cells, tissues, and cellular and tissue-based products. The regulatory process for these products is described in the FDA's 21 CFR 1271 of the Code of Regulations. Under these regulations, certain products including blood products such as PRP are exempt and therefore do not follow the FDA's traditional regulatory pathway that includes animal studies and clinical trials. The 510(k) application is the pathway used to bring PRP preparation systems to the market. The 510(k) application allows devices that are "substantially equivalent" to a currently marketed device to come to the market. There are numerous PRP preparation systems on the market today with FDA clearance; however, nearly all of these systems have 510(k) clearance for producing platelet-rich preparations intended to be used to mix with bone graft materials to enhance bone graft handling properties in orthopedic practices. The use of PRP outside this setting, for example, an office injection, would be considered "off label." Clinicians are free to use a product off-label as long as certain responsibilities are met. Per CBER, when the intent is the practice of medicine, clinicians "have the responsibility to be well informed about the product, to base its use on firm scientific rationale and on sound medical evidence, and to maintain records of the product's use and effects." Finally, despite PRP being exempted, the language in 21 CFR 1271 has caused some recent concern over activated PRP; however to date, the FDA has not attempted to regulate activated PRP. Clinicians using activated PRP should be mindful of these concerns and continued to stay informed.
Chronic complex musculoskeletal injuries that are slow to heal pose challenges to physicians and researchers alike. Orthobiologics is a relatively newer science that involves application of naturally found materials from biological sources (for example, cell-based therapies), and offers exciting new possibilities to promote and accelerate bone and soft tissue healing. Platelet-rich plasma (PRP) is an orthobiologic that has recently gained popularity as an adjuvant treatment for musculoskeletal injuries. It is a volume of fractionated plasma from the patient's own blood that contains platelet concentrate. The platelets contain alpha granules that are rich in several growth factors, such as platelet-derived growth factor, transforming growth factor-β, insulin-like growth factor, vascular endothelial growth factor and epidermal growth factor, which play key roles in tissue repair mechanisms. PRP has found application in diverse surgical fields to enhance bone and soft-tissue healing by placing supra-physiological concentrations of autologous platelets at the site of tissue damage. The relative ease of preparation, applicability in the clinical setting, favorable safety profile and possible beneficial outcome make PRP a promising therapeutic approach for future regenerative treatments. However, there is a large knowledge gap in our understanding of PRPs mechanism of action, which has raised skepticism regarding its potential efficacy and use. Thus, the aim of this review is to describe the various factors proposed to contribute to the biological activity of PRP, and the published pre-clinical and clinical evidence to support it. Additionally, we describe the current techniques and technology for PRP preparation, and review the present shortcomings of this therapy that will need to be overcome if it is to gain broad acceptance.
The topical use of platelet concentrates is recent and its efficiency remains controversial. Several techniques for platelet concentrates are available; however, their applications have been confusing because each method leads to a different product with different biology and potential uses. Here, we present classification of the different platelet concentrates into four categories, depending on their leucocyte and fibrin content: pure platelet-rich plasma (P-PRP), such as cell separator PRP, Vivostat PRF or Anitua's PRGF; leucocyte- and platelet-rich plasma (L-PRP), such as Curasan, Regen, Plateltex, SmartPReP, PCCS, Magellan or GPS PRP; pure plaletet-rich fibrin (P-PRF), such as Fibrinet; and leucocyte- and platelet-rich fibrin (L-PRF), such as Choukroun's PRF. This classification should help to elucidate successes and failures that have occurred so far, as well as providing an objective approach for the further development of these techniques.
Numerous studies have supported the use of topical blood components to improve wound healing and tissue regeneration. Platelet gel (PG), a hemocomponent obtained from mix of activated platelets (PLTs) and cryoprecipitate, is currently being used clinically in an attempt to improve tissue healing. The present study sought to define the most effective PG concentration to promote angiogenesis in vitro.
The effects of PG-released supernatant at different concentrations on human endothelial cells were studied using different in vitro assays (proliferation, migration, invasion, cord formation, and wound healing).
The concentration of PG-released supernatant had a significant influence on the angiogenic potential of endothelial cells. The optimal concentration for the stimulation of angiogenesis was 1.5 x 10(6) PLTs per microL in most of the in vitro experiments used in this study. Lower or higher concentrations of PG displayed a lower angiogenic potential.
An optimal concentration of PG to promote angiogenesis in human endothelial cells was identified. Excessively high PG concentrations may inhibit the angiogenic process, thereby being counterproductive for wound healing in a clinical setting.
This study analyzed the effect of the platelet count in platelet-rich plasma (PRP) on bone regeneration in vivo. Twenty male New Zealand white rabbits were used. PRP was produced using the Platelet Concentrate Collection System (PCCS) (3i, Miami, FL, USA). After inducing ketamine-xylazine anaesthesia, a self-tapping titanium screw (Branemark MK III TiUnite, 3.75 x 7 mm) was inserted in each distal femur; the femurs were randomized so that one side was treated with PRP while the other (control) was not. Intravital fluorochrome staining was performed on days 1, 7 (1.5 ml of 2% doxycycline/kg bodyweight), 14 (6% xylenol orange, 1.5 ml/kg), and 21 (1% calcein green, 5 ml/kg). Animals were euthanized on day 28 (n = 20). Specimens were prepared for histomorphological evaluation according to Donath and Breuner [J. Oral Pathol. 11 (1982) 318]. Comparing the bone regeneration (fluorochrome staining) in the 4-week implants (n = 19), the only significant difference (sign test, P = 0.004) was seen with intermediate platelet concentrations (n = 9,503,000-1,729,000 platelets/microl PRP). There were no differences in the bone/implant contact rates between the test and the control side among the three groups. The platelet concentration required for a positive PRP effect on bone regeneration seems to span a very limited range. Advantageous biological effects seem to occur when PRP with a platelet concentration of approximately 1,000,000/microl is used. At lower concentrations, the effect is suboptimal, while higher concentrations might have a paradoxically inhibitory effect. On the other hand, the effect of this type of platelet concentrate was not beneficial to accelerate the osseointegration of enosseous dental implants.
Treatment of male pattern alopecia with platelet-rich plasma: a double-blind controlled study with analysis of platelet number and growth factor levels
- B L Rodrigues
- Sal Montalvão
- Rbb Cancela
- Far Silva
- A Urban
- S C Huber
- Jlrc Júnior
- Jfsd Lana
- J M Annichinno-Bizzacchi
- BL Rodrigues