Immediate spermatogenesis assessment after testicular tissue retrieval for azoospermic males using touch print smear.

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Learn more: PMC Disclaimer | PMC Copyright Notice J Assist Reprod Genet . 2026 Feb 24;43(5):1441–1448. doi: 10.1007/s10815-026-03819-z Immediate spermatogenesis assessment after testicular tissue retrieval for azoospermic males using touch print smear Chen-Hao Hsu 1 , Cheng-Han Tsai 1, 2 , Eric Yi-Hsiu Huang 1, 2 , I-Shen Huang 1, 2 , Wei-Jen Chen 1, 2 , Yu-Ching Peng 3 , William J Huang 1, 2, 4, ✉ Author information Article notes Copyright and License information 1 Department of Urology, Taipei Veterans General Hospital, Taipei, Taiwan 2 Department of Urology, School of Medicine, College of Medicine and Shu-Tien Urological Research Center, National Yang Ming Chiao Tung University, Taipei, Taiwan 3 Department of Pathology and Laboratory Medicine, Taipei Veterans General Hospital, Taipei, Taiwan 4 Division of Urology, Department of Surgery, National Yang Ming Chiao Tung University Hospital, Yilan, Taiwan ✉ Corresponding author. Received 2025 Aug 30; Accepted 2026 Jan 22; Issue date 2026 May. © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ . PMC Copyright notice PMCID: PMC13221543  PMID: 41733871 Abstract Purpose Touch print smear (TPS) of testicular specimens provides immediate diagnostic results comparable to histopathology and in vitro fertilization laboratory findings. This study aimed to validate TPS as a rapid complement to histopathology for detecting post-meiotic germ cells in azoospermic patients. Materials and methods We retrospectively analyzed 495 azoospermic patients (274 with obstructive azoospermia [OA]) who underwent testis needle biopsy (2015–2022). Specimens were immediately smeared onto sterile slides, then transferred into Bouin’s solution for pathological diagnosis. Slides were stained with thionine and examined under light microscopy. Spermatogenesis development was compared between TPS findings and histopathology. Results TPS and histopathology were concordant in 98.2% (269/274) of OA and 75.6% (167/221) of non-obstructive azoospermia (NOA) cases. Among discordant NOA patients, 74.1% (40/54) showed more advanced spermatogenesis with TPS. Of 20 patients in whom post-meiotic germ cells were identified by TPS but not by histopathology, 95% (19/20) achieved successful sperm retrieval during subsequent microdissection testicular sperm extraction (mTESE). Conversely, in NOA patients where TPS suggested less advanced spermatogenesis and failed to identify post-meiotic germ cells, the sperm retrieval rate was 57.1% (4/7). Conclusion TPS allows rapid identification of post-meiotic germ cells with excellent concordance in OA and provides clinically relevant information in NOA. Patients in whom TPS demonstrates post-meiotic germ cells have a high likelihood of sperm retrieval, whereas their absence on TPS indicates lower retrieval success, in which case histopathology remains a useful backup. Keywords: Azoospermia, Touch print smear, Spermatogenesis, Testicular biopsy, Sperm retrieval Introduction Azoospermia, the condition in which no sperm is present in the ejaculate, is a significant contributor to male infertility, affecting around 1% of the general male population and 10–15% of infertile men [ 1 ]. It is classified into two types: obstructive azoospermia (OA), where sperm production occurs but cannot be ejaculated due to a physical blockage, and non-obstructive azoospermia (NOA), which results from impaired sperm production in the testes [ 2 ]. Determining the presence of viable sperm in azoospermic males is essential for guiding fertility treatments, particularly for those seeking assisted reproductive techniques such as in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) [ 3 ]. Testis biopsy is often used to assess spermatogenesis, but traditional histopathological analysis has limitations in providing immediate results and may not always correlate with successful sperm retrieval. Touch print smear (TPS) with 0.5% thionine stain solution of testicular tissue offers a rapid cytological assessment of spermatogenesis. TPS can expedite diagnostic decisions by providing an immediate evaluation of germ cells and their developmental stages. This technique is particularly advantageous when urgent decisions are needed during procedures like sperm retrieval for assisted reproductive techniques (ART), including testis biopsy and microdissection testicular sperm extraction (mTESE). Prior studies have indicated that TPS results correlate well with histopathology and IVF laboratory findings [ 4 ]. However, discrepancies between TPS and histopathological findings, particularly in NOA cases, necessitate further investigation to establish TPS as a reliable tool for identifying viable sperm in these patients. In this study, we aim to validate the diagnostic accuracy of TPS in assessing spermatogenesis compared to histopathology. Additionally, we explore the potential of TPS to provide critical, immediate interpretations of post-meiotic germ cells, which could directly influence decisions regarding sperm retrieval and subsequent fertility treatments. TPS may serve as a valuable complement to histopathology, helping to prevent both oversight and overdiagnosis of post-meiotic germ cells. Materials and methods Study population A total of 495 azoospermic patients who underwent percutaneous testis needle biopsy to determine the cause of their azoospermia at our institution between January 2015 and December 2022 were retrospectively enrolled through an electronic medical records review. This study received approval from the Human Research Ethics Committee at our institution (IRB: 2020–01–020 BC#1). Following diagnostic percutaneous testis needle biopsy, patients with OA proceeded to conventional testicular sperm extraction (TESE) with fresh sperm for IVF/ICSI, while those with NOA underwent subsequent mTESE for potential sperm retrieval. Touch print smear (TPS) During the percutaneous testis needle biopsy, a testicular sample was blotted dry on gauze before being gently touched and smeared onto a pre-sterilized slide. All slides prepared for TPS undergo pre-disinfection via autoclaving prior to use. The global specimen was then transferred into Bouin’s solution for fixation for later pathological diagnosis. The slide was immersed in a staining solution containing 0.5% thionine for 2 s, carefully rinsed with distilled water, and dried using filter paper. A drop of mounting medium was applied before placing the coverslip onto the smear. The prepared slide was then placed under a light microscope for interpretation (Fig. 1 ). The entire slide preparation process typically took less than 3 min. At our institution, testicular specimens are interpreted intraoperatively by the operating urologist using TPS, as embryologists are not routinely available in the operating room. This enables immediate cytological evaluation of spermatogenesis at the time of biopsy. Fig. 1. Open in a new tab a Percutaneous testis needle biopsy procedure, b staining with 0.5% thionine, and ( c ) light microscope setup used for TPS interpretation Touch print smear (TPS) interpretation After slide preparation, each specimen was examined under a light microscope at 40 ×, 100 ×, and 400 × magnification by two reproductive urologists, including one with over 15 years of TPS-reading experience. TPS slides were independently reviewed by both urologists. In cases of discrepant interpretation, the slides were re-evaluated jointly under microscopy, and a consensus diagnosis was reached. This consensus-based approach reflects routine clinical practice and was used to minimize operator-dependent variability. The TPS results were categorized into five diagnostic groups: (1) Normo-spermatogenesis (NS), characterized by the presence of all stages of germ cells, commonly seen in patients with OA (Fig. 2 ); (2) Hypospermatogenesis (HS), identified by a significant reduction in the quantity of germ cells, although all stages of germ cell development are still present; (3) Early Maturation Arrest (EMA), where spermatogenesis is arrested at an early stage, with only primary spermatocytes observed; (4) Late Maturation Arrest (LMA), marked by a failure to fully mature, with spermatids present but no spermatozoa found; and (5) Sertoli Cell-Only (SCO), where the seminiferous tubules contain only Sertoli cells without any germ cells [ 5 ]. Fig. 2. Open in a new tab TPS illustration of six different cell types observed in a patient with normo-spermatogenesis These five diagnostic categories can be further simplified into two broader groups based on the presence or absence of post-meiotic germ cells. Categories that include post-meiotic germ cells (i.e., round spermatids, elongated spermatids, immature sperm, and mature sperm) are LMA, HS, and NS (Fig. 3 ). Categories lacking post-meiotic germ cells are SCO and EMA (Fig. 4 ). To further standardize cytologic interpretation, the Sperm–Sertoli Index (SSI) was applied [ 6 , 7 ]. The SSI is defined as the number of mature sperm counted while enumerating 100 Sertoli cells in 10 representative non-overlapping × 400 fields. This semi-quantitative index provides an objective measure of germ-cell abundance relative to supporting cells. NS was characterized by the complete series of spermatogenic cells, with spermatogenic elements exceeding Sertoli cells and mature sperm forming the predominant population (SSI > 1). HS was defined by the presence of all germ-cell stages but a diffuse quantitative depletion, reflected by a reduced SSI (< 1). Fig. 3. Open in a new tab TPS images showing the presence of post-meiotic germ cells in patients with late maturation arrest (LMA), hypospermatogenesis (HS), and normo-spermatogenesis (NS) Fig. 4. Open in a new tab TPS images highlighting the absence of post-meiotic germ cells in cases of Sertoli cell-only (SCO) and early maturation arrest (EMA) Histopathologic diagnosis of testicular specimens Alongside TPS, the global testicular tissue samples were also collected for histopathologic analysis, which was performed in the department of pathology. These specimens were fixed in Bouin’s solution, processed into paraffin blocks, and sectioned for staining with hematoxylin and eosin (H-E). Pathologists then evaluated the samples, using the same classification system as TPS to assess the quality of spermatogenesis. TPS and histopathology were performed on the same biopsy specimen obtained during percutaneous testis needle biopsy, ensuring direct comparability of findings. The histopathologic diagnosis served as the gold standard for determining spermatogenic status, and the reports were retrospectively reviewed and directly compared to the TPS findings to evaluate the correlation between the two diagnostic methods. Endpoints and statistical analysis For patients with NOA who did not have sperm identified through percutaneous testis needle biopsy, a subsequent mTESE was performed for potential sperm retrieval. The successful retrieval of sperm during mTESE was considered the endpoint of the study and served as the basis for comparing the diagnostic efficacy between TPS and histopathology in percutaneous testis needle biopsies. Because of the retrospective design, not all patients with discordant TPS and histopathology findings proceeded to subsequent mTESE. The decision to undergo mTESE was made through shared clinical decision-making between the treating urologist and the patient. In routine practice, this decision is primarily driven by patient preference and whether the female partner is ready to proceed with IVF/ICSI. Consequently, only a subset of discordant cases underwent mTESE. Additionally, factors such as age, smoking status, testicular size, and hormone profiles were analyzed in patients showing discordant findings between TPS and histopathology. A Mann–Whitney U test was used for statistical comparison, and all analyses were performed using SPSS Statistics v29. Results Among the 495 azoospermic patients, 274 were diagnosed with OA, while 221 were classified as having NOA. As shown in Fig. 5 , 98.2% of the OA group displayed concordance between histopathology and TPS findings, whereas in the NOA group, 75.6% exhibited concordant results. Of the 54 NOA patients with discordant results between TPS and histopathology, 40 (74.1%) showed evidence of more advanced spermatogenesis based on TPS findings. Among these, 21 patients subsequently underwent mTESE. In contrast, 14 patients (25.9%) showed more advanced spermatogenesis according to histopathology, and 7 of them proceeded with mTESE. Fig. 5. Open in a new tab Flowchart of enrolled patients with TPS and histopathology findings Table 1 summarizes the demographic data, including age, smoking status, testis size, and hormone profiles, for patients with discordant TPS and histopathology findings. Notably, only follicle-stimulating hormone (FSH) levels showed a significant difference between the groups. Patients whose histopathology results indicated more advanced spermatogenesis than TPS had a significantly higher median FSH level (14.5 mIU/mL, IQR 12.2–17.5) compared to those for whom TPS indicated more advanced findings (median 6.7 mIU/mL, IQR 4.5–10.4; p = 0.042). Table 1. Demographic characteristics of patients with discordant TPS and histopathology results Patho < TPS ( n = 21) Patho > TPS ( n = 7) p value Age (years) mean ± SD 38.5 ± 7.6 37.4 ± 3.3 0.876 Smoking status (%) 7/21 (33.3%) 0/7 (0%) 0.208 Testis size (cc) mean ± SD 16.0 ± 4.0 17.4 ± 4.6 0.876 FSH (mlU/mL) median (IQR) 6.7 (4.5–10.4) 14.5 (12.2–17.5) 0.042 LH (mlU/mL) median (IQR) 4.1 (3.4–6.6) 5.8 (5.2–11.5) 0.090 Testosterone (ng/mL) median (IQR) 3.9 (3.5–4.9) 2.9 (2.4–4.7) 0.321 Prolactin (ng/mL) median (IQR) 7.2 (6.6–10.4) 7.5 (6.3–11.3) 0.756 Estradiol (pg/mL) median (IQR) 20.7 (16.9–31.5) 16.5 (12.2–23.4) 0.228 Open in a new tab TPS touch print smear, Patho histopathology, SD standard deviation, IQR interquartile range In the cohort of 21 patients whose TPS indicated more advanced spermatogenesis than histopathology, mTESE was subsequently performed. Among these patients, 20 showed the presence of post-meiotic germ cells detectable exclusively by TPS, and successful sperm retrieval was achieved in 19 (95%) of these cases. In 1 patient, despite TPS detecting post-meiotic germ cells, no sperm were retrieved during mTESE (Table 2 ). Table 2. Sperm retrieval outcomes by mTESE in patients with a more advanced spermatogenesis diagnosis through TPS compared to histopathology, categorized by the presence of post-meiotic germ cells detectable only by TPS and those without post-meiotic germ cells identified by either histopathology or TPS Patho < TPS ( n = 21) Presence of post-meiotic germ cells only by TPS No post-meiotic germ cells by either Patho or TPS Sperm (+) by mTESE n = 19 n = 1 Sperm (−) by mTESE n = 1 n = 0 Open in a new tab TPS touch print smear, Patho histopathology In the group of 7 patients whose histopathology indicated more advanced spermatogenesis than TPS, 5 were found to have post-meiotic germ cells detectable only by histopathology. However, sperm retrieval via mTESE was successful in only 3 of these cases. In one case, no post-meiotic germ cells were detected by either histopathology or TPS, but sperm was successfully retrieved by mTESE. Notably, when post-meiotic germ cells were not identified by TPS, the sperm retrieval rate dropped to 57.1% (4/7) (Table 3 ). Table 3. Sperm retrieval outcomes by mTESE in patients with a more advanced spermatogenesis diagnosis through histopathology compared to TPS, categorized by the presence of post-meiotic germ cells detectable only by histopathology and those without post-meiotic germ cells identified by either method Patho > TPS ( n = 7) Presence of post-meiotic germ cells only by Patho No post-meiotic germ cells by either Patho or TPS Sperm (+) by mTESE n = 3 n = 1 Sperm (−) by mTESE n = 2 n = 1 Open in a new tab TPS touch print smear, Patho histopathology Discussions TPS cytology of testicular specimens has been demonstrated in previous studies to yield diagnostic outcomes comparable to both histopathology and IVF laboratory findings [ 4 ]. In this study, we further validate TPS as not only a rapid imprint method but also a critical complement to histopathology in evaluating spermatogenesis. The ability to assess the presence or absence of post-meiotic germ cells during testicular biopsy emerges as a vital indicator for predicting successful sperm retrieval in subsequent procedures such as mTESE. Several staining methods have been developed to assist in the identification and morphological analysis of spermatozoa during TESE, including Papanicolaou staining, Diff-Quik staining, Shorr staining, H-E staining, Wright staining, and the wet prep technique [ 8 , 9 ]. While these methods provide valuable insights into sperm morphology and spermatogenesis, they are often regarded as labor-intensive and time-consuming, particularly in the intraoperative settings [ 10 ]. Among these techniques, TPS offers a notable advantage by simplifying the staining process through the use of thionine, which effectively highlights the DNA components of germ cells. This streamlined approach not only facilitates the rapid interpretation of spermatogenesis but also reduces the technical complexity associated with conventional staining methods, making TPS a practical and efficient option for real-time intraoperative evaluation. Among the 269 patients with OA, TPS exhibited a high concordance rate of 98.2% with histopathology findings, underscoring its reliability as a diagnostic tool for testicular biopsies in OA patients. The rapid identification of sperm through TPS provides immediate insights into spermatogenesis, accelerating the diagnostic process and alleviating patient anxiety by reducing the waiting period for definitive results. Moreover, TPS enables clinicians to make timely decisions regarding the need for further invasive procedures, such as TESE or mTESE, potentially avoiding unnecessary surgical intervention when sperm can be identified early for reproductive treatments. Regarding the 1.8% of discordant cases between TPS and histopathology, our findings suggest that TPS offered the more accurate diagnosis. In these cases, TPS was interpreted as late maturation arrest based on the presence of round spermatids as the only post-meiotic germ cells. In contrast, the corresponding paraffin sections were reported as normal spermatogenesis, likely reflecting the inherent limitations of high-power light microscopy (40 × objective) in reliably distinguishing post-meiotic germ cells within seminiferous tubules. Importantly, the TPS interpretations in these discordant cases precisely matched the germ-cell search findings of embryologists in the reproductive laboratory. At our institution, percutaneous needle biopsy is the standard diagnostic approach for evaluating azoospermia upon the initial clinical encounter. This office-based technique involves only a small puncture (0.2–0.3 cm) under local spermatic cord anesthesia, eliminating the need for general anesthesia or hospitalization. Previous studies have demonstrated its safety and diagnostic adequacy for assessing spermatogenesis [ 11 , 12 ]. The high concordance rate observed in OA cases supports its reliability, whereas in NOA cases, TPS provided complementary diagnostic information that may aid clinical decision-making. It should be noted that the present study was not designed to compare sperm retrieval techniques but rather to highlight the clinical utility of TPS in interpreting testicular tissue and identifying the stage of spermatogenesis. These findings suggest that TPS can serve as a valuable complementary method to histopathology, even from limited tissue obtained by needle biopsy. The rapid interpretation capability of TPS offers practical advantages in our clinical environment. In our institution, embryologists are not routinely stationed in the operating room to evaluate testicular tissue, primarily due to cost constraints and the limited availability of trained personnel in our country. Therefore, the ability of the reproductive urologist to interpret spermatogenesis directly using TPS enables immediate intraoperative assessment without embryologist assistance. This enables reproductive urologists to manage operative time more efficiently and make informed decisions regarding the optimal point to conclude the procedure. In addition to improving intraoperative efficiency, this feature is especially beneficial in office-based settings or when collaborating with external reproductive centers, where confirming the presence of sperm before specimen transfer helps reduce unnecessary costs and patient anxiety. Furthermore, providing rapid diagnostic feedback allows clinicians to counsel patients in real time, alleviating uncertainty and improving the overall treatment experience. In NOA patients, the utility of TPS becomes even more apparent. Our study found that NOA patients in whom TPS identified post-meiotic germ cells, even in the absence of histopathological confirmation, had a significantly higher sperm retrieval rate at 95% during mTESE. This highlights the importance of confirming the presence of post-meiotic germ cells, as they are essential for the success of advanced reproductive treatments such as ICSI or round spermatid injection (ROSI). By establishing TPS as a companion to histopathology, we can assess spermatogenesis instantaneously during the operation, enabling real-time decision-making that could improve treatment outcomes. In contrast, among the 7 patients whose histopathology indicated more advanced spermatogenesis than TPS, 5 exhibited post-meiotic germ cells that were detectable only by histopathology. However, sperm retrieval was successful in only 3 cases, suggesting that when post-meiotic germ cells are not identified by TPS, histopathology may potentially over-diagnose spermatogenesis and lead to unrealistic expectations for successful sperm retrieval. Although TPS and histopathology in this study were derived from the same biopsy specimen, these two methods interrogate spermatogenesis from fundamentally different analytic perspectives, and discordant findings do not necessarily imply true diagnostic error. Histopathology, while a cornerstone of testicular biopsy diagnostics, does have several limitations that TPS helps to address. First, histopathology may over-diagnose the presence of mature sperm. This may be due to the difficulty to differentiate spermatid from the mature sperm in the seminiferous tubule architecture in histopathological slide. In our study, patients diagnosed with the presence of sperm solely by histopathology, without confirmation by TPS, had a lower sperm retrieval rate. This suggests that TPS may provide a more accurate, real-time assessment of spermatogenesis in certain cases. Second, the interpretation of histopathology is based on the predominant cellular pattern observed in the biopsy sample, which may lead to the under-diagnosis of the most advanced germ cells, particularly post-meiotic ones. TPS, on the other hand, reports the presence of the most advanced germ cells directly, providing a clearer assessment of spermatogenesis progression. This distinction is critical in determining the patient’s fertility potential, especially in NOA cases. Additionally, TPS offers a practical advantage by allowing clinicians to actively search for regions of interest under a low-power microscope, followed by a high-power examination to confirm the presence of post-meiotic germ cells. The thionine staining used in TPS highlights the DNA components of germ cells more effectively than the standard H-E stain used in paraffin block sections, further enhancing the accuracy of TPS in identifying germ cells. Nevertheless, an important consideration is the potential variability in TPS results due to differences in operator experience and sample handling techniques. Achieving optimal staining and accurate interpretation requires proper training and standardized protocols. Future efforts should focus on developing comprehensive guidelines and training programs to ensure consistent and reliable TPS cytology results across different medical centers. Emerging technologies such as digital pathology and artificial intelligence (AI)-assisted image analysis hold promise for enhancing the consistency and scalability of TPS interpretation. Although not evaluated in the present study, our research group has previously demonstrated the feasibility of AI-based TPS cytology, showing that machine-learning models can detect and classify cells from TPS images [ 5 ]. With further training to identify and quantify post-meiotic germ cells, AI may reduce operator-dependent variability and facilitate wider adoption of TPS across institutions. The integration of AI-based analysis with TPS therefore represents an important future direction for intraoperative assessment in azoospermic patients. This study has some limitations. Its retrospective, single-center design may introduce selection bias, particularly in determining which patients with discordant TPS and histopathology findings proceeded to mTESE. TPS and histopathology were interpreted in routine clinical practice without formal blinding. In addition, only a subset of NOA patients with discordant results underwent mTESE, limiting more detailed secondary analyses. These findings should therefore be validated in future prospective and multicenter studies. Despite the advantages of TPS and histopathology, neither technique can replace the critical expertise of embryologists in interpreting spermatogenesis. Embryologists play a key role in confirming the viability of sperm for reproductive procedures, and their involvement remains essential in fertility treatments. TPS and histopathology should be viewed as complementary diagnostic tools that support, but do not substitute, the embryologist’s judgment. The integration of TPS into the diagnostic process can enhance the overall accuracy and efficiency of spermatogenesis assessment in azoospermic patients. Furthermore, combining histopathology and TPS may represent the optimal diagnostic approach for assessing spermatogenesis, as this combination demonstrates the highest concordance with embryologists’ findings in IVF laboratories. Conclusion In conclusion, TPS cytology provides a valuable and immediate diagnostic approach in testicular biopsy for azoospermic patients, particularly in identifying post-meiotic germ cells that are crucial for sperm retrieval and subsequent fertility treatments. While histopathology continues to play an important role, TPS enhances diagnostic precision, potentially reduces the need for additional invasive procedures, and improves patient outcomes by facilitating real-time decision making. Author contribution Conception and design of the study: WJH. Acquisition of data: all authors. Data curation and analysis: CHH, CHT, WJH. Funding acquisition: WJH. Investigation: WJH. Writing — original draft: all authors. Writing — review and editing: all authors. Funding Open access funding provided by Taipei Veterans General Hospital. This work was supported by Taipei Veterans General Hospital (VGH109-C-132, VGH112-C-159 and VGH114-C-162) and National Science and Technology Council of Taiwan (109–2314-B-075–073-MY2) to William J. Huang. Data Availability The data are available from the authors upon reasonable request. Declarations Ethical approval This study received approval from the Taipei Veterans General Hospital Human Research Ethics Committee (IRB: 2020–01-020 BC#1). Conflict of interest The authors declare no competing interests. Footnotes Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References 1. Cocuzza M, Alvarenga C, Pagani R. The epidemiology and etiology of azoospermia. Clinics (Sao Paulo). 2013;68(Suppl 1):15–26. 10.6061/clinics/2013(sup01)03. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 2. 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