Controlling genetic heterogeneity in gene-edited hematopoietic stem cells by single-cell expansion.

Gene editing using engineered nucleases frequently produces unintended genetic lesions in hematopoietic stem cells (HSCs). Gene-edited HSC cultures thus contain heterogeneous populations, the majority of which either do not carry the desired edit or harbor unwanted mutations. In consequence, transplanting edited HSCs carries the risks of suboptimal efficiency and of unwanted mutations in the graft. Here, we present an approach for expanding gene-edited HSCs at clonal density, allowing for genetic profiling of individual clones before transplantation. We achieved this by developing a defined, polymer-based expansion system and identifying long-term expanding clones within the CD201+CD150+CD48-c-Kit+Sca-1+Lin- population of precultured HSCs. Using the Prkdcscid immunodeficiency model, we demonstrate that we can expand and profile edited HSC clones to check for desired and unintended modifications, including large deletions. Transplantation of Prkdc-corrected HSCs rescued the immunodeficient phenotype. Our ex vivo manipulation platform establishes a paradigm to control genetic heterogeneity in HSC gene editing and therapy.

Variability of autonomic nerve activity in dry eye with decreased tear stability.

The autonomic nervous system plays a crucial role in the maintenance of homeostasis. Neurogenic inflammation due to decreased stability of tear film may induce changes in autonomic nerve activity, which could be associated with symptom expression.This study aimed to measure biological parameters that represent autonomic nerve activity in dry eye (DE)s caused by tear film dysfunction and investigate their relationship with symptom intensity. This prospective, cross-sectional, comparative study evaluated 34 eyes of 34 participants (mean age: 52.5 ± 13.4 years; range: 20-81 years) without keratoconjunctival damage. Nineteen eyes in the DE group showed DE symptoms and tear break-up time (TBUT) of ≤5 seconds (short TBUT DE); the 15 eyes in the non-DE group showed no DE symptoms. Autonomic nerve activity was measured for 10 minutes-starting and ending 5 minutes before and after instilling ophthalmic solution-and evaluated using the low-frequency component (LF) to the high-frequency component (HF) ratio of heart rate variability (autonomic balance). The pre-ophthalmic solution administration LF/HF ratio was not significantly different (P = 0.59) between the two groups, however, the standard deviation of the LF/HF ratio (LF/HF-SD) tended to be higher in the DE group than that in the non-DE group (P = 0.086). The DE symptom intensity was significantly related to LF/HF-SD (P = 0.005), which significantly decreased after ophthalmic solution administration in the DE group (P = 0.04). The large fluctuations in autonomic balance may be key for the understanding of the mechanism underlying DE symptoms.

A Novel Lid Hygiene Detergent Successfully Eliminated Demodex Mites, a Common Pathogen of Refractory Obstructive Meibomian Gland Dysfunction.

INTRODUCTION: Demodex mites are microscopic arthropods that have been shown to be responsible for chronic blepharitis. Although many reports have demonstrated positive effects of lid hygiene on demodicosis, some have produced conflicting results. We retrospectively evaluated the effect of lid margin cleansing with a novel lid hygiene detergent, Eye Shampoo, in patients with ocular demodicosis. METHODS: Outpatient clinic notes recorded over a 24-month period between June 30, 2016, and June 29, 2018, at Keio University Hospital in Japan were reviewed to identify patients diagnosed with ocular demodicosis who started cleansing their lid margins with Eye Shampoo at least once daily as a lid hygiene therapy. RESULTS: Five patients diagnosed with ocular demodicosis who started cleansing their lid margins using Eye Shampoo for lid hygiene were identified. In four cases, Demodex mites were successfully eliminated, and ocular conditions were ultimately improved with no recurrence. Active Demodex mites were still noted after 3 months of lid hygiene therapy in one case; further observation of this case was not available. There were no adverse effects of Eye Shampoo in any of these cases. CONCLUSION: Lid hygiene therapy using Eye Shampoo successfully eliminated Demodex mites, a common pathogen of refractory obstructive meibomian gland dysfunction.

Role of transient receptor potential melastatin 8 activity in menthol-induced cold sensitivity and its qualitative perception in dry eye.

PURPOSE: To evaluate the role of transient receptor potential melastatin 8 (TRPM8) activity in menthol-induced cold sensitivity and its qualitative perception in patients with dry eye (DE). METHODS: This prospective, cross-sectional, comparative study included 52 eyes of 52 subjects (mean age: 66.8 ± 9.2 years; range: 44-86) with a tear break-up time (TBUT) of ≤5 s. The participants were classified into three groups: 17 patients with DE symptoms and keratoconjunctival (KC) staining scores of ≥3 points (positive KC-DE group), 18 patients with DE symptoms and KC staining scores of <3 points (negative KC-DE group), and 17 individuals with KC staining scores of <3 points and no symptoms (non-DE control group). The menthol-induced cool sensation (M-cool) and TBUT were measured after administration of 2 µl of 1.0 mM menthol eye drops. Furthermore, participants answered a questionnaire regarding their stimulus perception (pleasant, unpleasant, or neither). RESULTS: M-cool values were similar in the three groups. TBUT significantly increased in the negative KC-DE and control groups (P < 0.05) and remained unchanged in the positive KC-DE group (P > 0.05) after menthol administration. DE patients reported the sensation as pleasant or unpleasant, whereas most control participants were indifferent (P < 0.05). CONCLUSIONS: While M-cold sensitivity was similar in DE and control groups, its qualitative perception differed between these groups. Thus, TRPM8 activation at the peripheral level alone may not be sufficient to account for the manifestation of discomfort symptoms associated with DE.

Capsaicin-induced pain sensitivity in short tear break-up time dry eye.

PURPOSE: To evaluate transient receptor potential vanilloid 1 (TRPV1)-mediated pain sensitivity in patients with short tear break-up time (TBUT) dry eye (DE) by using the capsaicin stimulus test. METHODS: This prospective cross-sectional comparative study included 22 eyes of 22 patients with short TBUT DE and 11 eyes of 11 non-DE control subjects. Patients were divided into two groups based on response to standard DE treatments: 10 non-responders (intractable DE) and 12 responders (responsive DE). Mechanical touch (M-touch) and mechanical pain (M-pain) were measured using a Cochet-Bonnet esthesiometer. Capsaicin-induced pain (C-pain) and C-pain duration (C-pain DT) were measured using a capsaicin stimulus test. Psychological distress was also assessed. RESULTS: M-touch sensitivity was similar among all three groups. M-pain sensitivity was higher in the responsive DE group than in the intractable DE and control groups (P < .001). C-pain sensitivity was lower (P < .001) in the intractable DE group than in the responsive DE and control groups, and C-pain DT was shorter (P = .006) in the intractable DE group than in the responsive DE group. Psychological distress was higher in the intractable DE group than in the control group (P < .001). CONCLUSIONS: Patients with intractable short TBUT DE were less sensitive to the effects of capsaicin than patients with responsive short TBUT DE and controls. Altered neural activation may contribute to the development of DE symptoms in the short TBUT DE subjects. The capsaicin stimulus test may be used to better understand pain sensitivity in short TBUT DE patients.

Long-term ex vivo expansion of mouse hematopoietic stem cells.

Utilizing multipotent and self-renewing capabilities, hematopoietic stem cells (HSCs) can maintain hematopoiesis throughout life. However, the mechanism behind such remarkable abilities remains undiscovered, at least in part because of the paucity of HSCs and the modest ex vivo expansion of HSCs in media that contain poorly defined albumin supplements such as bovine serum albumin. Here, we describe a simple platform for the expansion of functional mouse HSCs ex vivo for >1 month under fully defined albumin-free conditions. The culture system affords 236- to 899-fold expansion over the course of a month and is also amenable to clonal analysis of HSC heterogeneity. The large numbers of expanded HSCs enable HSC transplantation into nonconditioned recipients, which is otherwise not routinely feasible because of the large numbers of HSCs required. This protocol therefore provides a powerful approach with which to interrogate HSC self-renewal and lineage commitment and, more broadly, to study and characterize the hematopoietic and immune systems.

Tear Film Dynamics of Soft Contact Lens-Induced Dry Eye.

PURPOSE: This study aimed to prospectively assess tear dynamics in soft contact lens-induced dry eye while wearing and after removing the soft contact lenses. MATERIALS AND METHODS: We analyzed 17 right eyes of soft contact lens users at Wada Eye Clinic (11 men, 6 women; mean age: 36.8 ± 8.3 years; range: 24-57 years). Participants were divided into two groups depending on presence of dry eye symptoms. Video interferometry was used to observe tear dynamics while wearing and after removing soft contact lenses. Interference images focusing on tear spread and interference fringe were compared between groups. RESULTS: This study included 10 symptomatic and 7 asymptomatic eyes. Considering the symptomatic eyes, while wearing soft contact lenses, 8 eyes showed poor tear spread and multicolor interference fringe, i.e., swift flow of multicolor interference waves after a grayish monochromatic band-color after a blink, but 8 and 10 eyes showed good tear spread and a grayish monochromatic interference fringe when soft contact lenses were removed, respectively. As for asymptomatic eyes, 6 eyes exhibited good tear spread and a grayish monochromatic interference fringe while wearing soft contact lenses and after removing soft contact lenses. CONCLUSIONS: soft contact lens-induced dry eye was associated with tear dynamics only while wearing soft contact lenses. Observation of tear film dynamics in eyes with soft contact lenses could facilitate the understanding of dry eye-related symptoms.

Author Correction: Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.

Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation1, which is a curative therapy for numerous diseases including immunodeficiencies and leukaemias2. Although substantial effort has been applied to identifying HSC maintenance factors through the characterization of the in vivo bone-marrow HSC microenvironment or niche3-5, stable ex vivo HSC expansion has previously been unattainable6,7. Here we describe the development of a defined, albumin-free culture system that supports the long-term ex vivo expansion of functional mouse HSCs. We used a systematic optimization approach, and found that high levels of thrombopoietin synergize with low levels of stem-cell factor and fibronectin to sustain HSC self-renewal. Serum albumin has long been recognized as a major source of biological contaminants in HSC cultures8; we identify polyvinyl alcohol as a functionally superior replacement for serum albumin that is compatible with good manufacturing practice. These conditions afford between 236- and 899-fold expansions of functional HSCs over 1 month, although analysis of clonally derived cultures suggests that there is considerable heterogeneity in the self-renewal capacity of HSCs ex vivo. Using this system, HSC cultures that are derived from only 50 cells robustly engraft in recipient mice without the normal requirement for toxic pre-conditioning (for example, radiation), which may be relevant for HSC transplantation in humans. These findings therefore have important implications for both basic HSC research and clinical haematology.

Safety and efficacy of wiping lid margins with lid hygiene shampoo using the "eye brush", a novel lid hygiene item, in healthy subjects: a pilot study.

BACKGROUND: To evaluate the safety and efficacy of using a lid hygiene brush prototype to wipe the lid margins with lid hygiene shampoo in subjects with normal meibomian glands. METHODS: Twelve eyes of 6 subjects were all evaluated just before and after wiping lid margins using 1) tap water alone, 2) Eye Shampoo, 3) Eye Brush, or 4) both products, each during a different week. The results after using both products twice daily for 1 month were also evaluated. Wiping efficacy was determined by post-wiping scores for the remaining fluorescein-stained 0.3% Tarivid ointment fully applied to eyelids and lid margins under microscopic view illuminated by blue light just after performing each of the four lid hygiene methods described above. RESULTS: No significant deterioration in ocular conditions occurred. Eyestrain, eye discharge, and dryness decreased with tap water (P = 0.020), Eye Shampoo (P = 0.036), and Eye Brush (P = 0.014), respectively. Sensations of eye discharge increased after 1 month of using both products (P = 0.042). The wiping efficacy of Eye Brush, Eye Shampoo or both was significantly greater than that of tap water alone (two-sided test, P = 0.003, 0.003, 0.002), and using both significantly increased efficacy above Eye Shampoo use alone (one-sided test, P = 0.009). CONCLUSIONS: Wiping lid margins using Eye Brush enhanced the cleansing power of Eye Shampoo. A daily healthcare routine using both products could be a safe and effective option for daily lid hygiene. TRIAL REGISTRATION: UMIN000016905 . Registration date: March 24, 2015; the study was prospectively registered.

Effect of Eyelid Hygiene Detergent on Obstructive Meibomian Gland Dysfunction.

PURPOSE: To investigate the efficacy of eyelid margin cleansing with lid hygiene detergent in patients with obstructive meibomian gland dysfunction (MGD). METHODS: As a pilot study, we investigated the safety and efficacy of lid hygiene using tap water and/or lid hygiene shampoo in fourteen eyes of 7 normal subjects. All subjects were instructed to cleanse the eyelid margin with tap water and/or lid hygiene shampoo. As a main prospective clinical study, thirty-four eyes of 34 patients with obstructive MGD were enrolled. All subjects were instructed to cleanse the eyelid margin with lid hygiene shampoo at least once daily for 1 month. Ocular surface conditions were observed before and 1 month after study initiation. RESULTS: A significant exacerbating change (p < 0.05) was not detected after either method in the pilot study. In the main study, significant improvements were observed in tear break-up time (TBUT), lid margin lissamine green staining scores, vascular dilatation, and meibum status (p < 0.05). No significant improvements in corneal or conjunctival fluorescein staining scores, the mucocutaneous junction, lid margin deformation, or plugging were observed (p ≥ 0.05). Subjective improvements were observed in 27 subjects. Meibography revealed that 28 subjects had normal meibomian glands, and 6 subjects had meibomian gland atrophy. Significant improvements were observed in TBUT, vascular dilatation, and meibum status only in the group with normal meibomian glands (p < 0.05), but subjective symptoms and lid margin lissamine green staining scores improved in both groups (p < 0.05). No subjects experienced any problems throughout the study. CONCLUSION: Daily routine use of lid hygiene detergent can alleviate the symptoms of MGD regardless of meibomian gland atrophy.

Mesenchymal cell differentiation and diseases: involvement of translin/TRAX complexes and associated proteins.

Translin and translin-associated factor X (translin/TRAX) proteins have been implicated in a variety of cellular activities central to nucleic acid metabolism. Accumulating evidence indicates that translin/TRAX complexes participate in processes ensuring the replication of DNA, as well as cell division. Significant progress has been made in understanding the roles of translin/TRAX complexes in RNA metabolism, such as through RNA-induced silencing complex activation or the microRNA depletion that occurs in Dicer deficiency. At the cellular level, translin-deficient (Tsn-/- ) mice display delayed endochondral ossification or progressive bone marrow failure with ectopic osteogenesis and adipogenesis, suggesting involvement in mesenchymal cell differentiation. In this review, we summarize the molecular and cellular functions of translin homo-octamer and translin/TRAX hetero-octamer. Finally, we discuss the multifaceted roles of translin, TRAX, and associated proteins in the healthy and disease states.

Severe symptoms of short tear break-up time dry eye are associated with accommodative microfluctuations.

AIM: Validating the hypothesis that accommodative microfluctuations (AMFs) may be associated with severe symptoms in short tear break-up time (BUT) dry eye (DE). METHODS: This study included 12 subjects with short BUT DE (age: 49.6±18.3 years). Diagnoses were performed based on the presence of DE symptoms, BUT ≤5 s, Schirmer score >5 mm, and negative keratoconjunctival epithelial damage. Tear evaluation, AMF, and functional visual acuity (VA) examinations were conducted before and after DE treatment. The AMF parameters evaluated were: total high-frequency component (HFC), HFC with low accommodation for the task of staring into the distance (HFC1), HFC with high accommodation for deskwork (HFC2). A subjective questionnaire of DE symptoms was also performed. RESULTS: Mean BUT increased from 1.9±2.0 to 6.4±2.5 s after treatment (P<0.05). The mean logarithm of the minimum angle of resolution functional VA significantly improved (from 0.19±0.19 to 0.12±0.17; P<0.05). Mean power spectrum values for total HFC and HFC1 decreased (from 61.3±5.7 to 53.8±6.6 dB and from 62.9±10.5 to 52.4±6.2 dB, respectively; P<0.05), while the mean HFC2 power spectrum values did not differ before and after treatment (P>0.05). Subjective DE symptoms were reduced in nine patients. CONCLUSION: Along with the improvement of BUT after treatment, DE symptoms diminished and HFC1 and functional VA improved, suggesting that tear film instability is associated with deterioration of functional VA, AMF, and DE symptoms.

An All-Recombinant Protein-Based Culture System Specifically Identifies Hematopoietic Stem Cell Maintenance Factors.

Hematopoietic stem cells (HSCs) are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX) and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations.

Eyelid cleansing with ointment for obstructive meibomian gland dysfunction.

PURPOSE: To investigate the efficacy of eyelid margin cleansing with ofloxacin ophthalmic ointment in obstructive meibomian gland dysfunction (MGD) patients. METHODS: Thirty-one eyes of 31 patients diagnosed with obstructive MGD were enrolled. All subjects were instructed to rub the eyelid edge with ofloxacin eye ointment once daily prior to bathing. Lid margin abnormalities, meibum properties, and tear stability were observed before and 3 months after ointment treatment. A questionnaire relating to the severity of MGD symptoms was administered after the treatment. RESULTS: Vascular engorgement, meibum, dandruff-like debris, and fluorescein staining scores significantly decreased after the treatment, whereas tear break-up time significantly increased (p < 0.05). Symptoms related to MGD improved after the treatment in 86.2 % of cases. CONCLUSIONS: Cleansing treatment ofloxacin ophthalmic ointment at the eyelid margin may be effective for patients with obstructive MGD.

Relationship of Corneal Pain Sensitivity With Dry Eye Symptoms in Dry Eye With Short Tear Break-Up Time.

PURPOSE: The purpose of this prospective comparative study was to investigate corneal sensitivity in subjects with unstable tear film, with and without dry eye (DE) symptoms. METHODS: Forty-one eyes of 41 volunteers (mean age: 45.1 ± 9.4 years; age range, 23-57 years), with normal tear function and ocular surface except for tear stability, were studied. The eyes were divided into two groups depending on the presence or absence of DE symptoms: 21 eyes with DE symptoms (symptomatic group); and 20 eyes without DE symptoms (asymptomatic group). Three types of corneal sensitivity values were measured using a Cochet-Bonnet esthesiometer: the sensitivity for perception of touch (S-touch), the sensitivity for blinking (S-blink), and the sensitivity for pain (S-pain). RESULTS: Mean S-blink and S-pain were significantly higher in the symptomatic group than in the asymptomatic group (P < 0.05), whereas there was no significant difference in mean S-touch between these groups (P > 0.05). CONCLUSIONS: Corneal sensitivity for blinking and pain evoked by increased stimuli was higher in the symptomatic group (subjects with short break-up time DE) compared with subjects who have no DE symptoms despite decreased tear stability. The presence of both tear instability and hyperesthesia, rather than tear instability alone, may contribute to DE pathogenesis.

Short break-up time type dry eye has potential ocular surface abnormalities.

PURPOSE: To describe a case series in which corneal fluorescein staining (CFS) development occurred in short break-up time (s-BUT) dry eyes after a short period during prolonged opening of the eye. METHODS: The study was designed as a clinical case series. Ocular surface evaluations were performed on 13 individuals with s-BUT dry eye. Tear function examinations included Schirmer's test and BUT evaluation. RESULTS: In all 13 cases, the BUT was short, but the tear quantity was not so bad. In all cases, CFS developed following a single eye opening, and the staining was observed at sites that showed as dark spots. In several cases, the CFS disappeared later. CONCLUSION: In this study, we demonstrated that CFS could develop following a single eye opening. Based on our findings, CFS is a dynamic phenomenon rather than a stable indicator of ocular surface abnormalities. Moreover, s-BUT dry eye has the potential to show ocular surface abnormalities.

The effect of tinted soft contact lens wear on functional visual acuity and higher-order aberrations.

PURPOSE: To investigate the differences of functional visual acuity (FVA) and high order aberrations (HOAs) in relation to tinted and clear hydrogel soft contact lens (SCL) wear. METHODS: A prospective comparative study was performed in 16 eyes of 16 healthy volunteers. Dynamic visual acuity (using a FVA measurement system) and higher-order aberrations (using a wavefront sensor) were compared in subjects wearing two types of soft contact lenses: 1-day Acuvue(®) (Vistakon, Jacksonville, FL) clear and the 1-day Acuvue(®) Define(TM) (Vistakon, Jacksonville, FL) tinted lens. The blink rates were recorded during FVA testing. The correlation between the difference of HOAs and differences in FVA values was analyzed. RESULTS: The mean LogMAR FVA scores with clear and tinted SCLs were 0.07 ± 0.13 and 0.14 ± 0.17 (P<0.05). The mean blink frequencies with clear and tinted SCL wear were 18.4 ± 8.3 and 25.3 ± 4.7 blinks/min (P<0.05). Both 3rd-order aberrations and total HOAs showed statistically significant differences between the two types of soft contact lenses for 6mm pupil measurements (P<0.05). A significant positive linear correlation was observed between ΔHOAs and ΔLogMAR FVA for 6mm pupil measurements (R=0.53, P=0.04). CONCLUSIONS: Tinted contact lens wear appears to induce a reduction in optical quality. Functional visual acuity measurement is a useful procedure to study the changes of visual performance and quality in tinted contact lens wear.

Short-term effects of instillation of a rebamipide suspension on visual function.

PURPOSE: To investigate the short-term adverse effects of using rebamipide for the treatment of dry eye by assessing visual function and optical quality. METHODS: This interventional noncomparative study included 14 right eyes of 14 healthy volunteers. Serial measurements of visual acuity (VA) and higher-order aberrations were obtained prior to instillation of the rebamipide suspension (baseline) and immediately after and at 5, 10, 20, and 30 min after instillation. Functional VA measurement was performed over a 60-s period with the subject blinking naturally. Ocular aberrations were measured for 10 s while the subject was told not to blink, but no topical anesthesia was applied. Each patient also filled out a questionnaire exploring the rebamipide-associated adverse effects. RESULTS: There was no significant difference between functional VA measured at baseline and at each time point after the instillation of rebamipide. In contrast, the root mean square of third-order and total higher-order aberrations increased significantly immediately after drug instillation (P<0.05). The severity of higher-order aberrations at baseline was similar to that observed at 5, 10, 20, and 30 min after instillation (P>0.05). CONCLUSIONS: The transient reduction in optical quality immediately after administration of rebamipide is corrected by the patient's natural blink reflex. The adverse effects observed in this study do not outweigh the benefits of rebamipide treatment.

Comparison of retention rates and complications of 2 different types of silicon lacrimal punctal plugs in the treatment of dry eye disease.

PURPOSE: To investigate the retention rate and complications of 2 different types of silicon lacrimal punctal plugs with the focus on the relationship to punctum size. DESIGN: A prospective, nonrandomized, interventional, comparative study. METHOD: Forty-four patients with dry eye syndrome received lacrimal punctal plug insertion with the Punctal Plug F (FCI Ophthalmics) and 30 patients received plug insertion with the Super Flex plug (Eagle Vision; Softplug-Oasis Medical Inc). In total, 110 plug insertion regions were performed using the Punctal Plug F (group A) and 110 insertion regions were carried out using Super Flex plug (group B). The gauging system of Eagle Vision was used before the lacrimal punctal plug insertion in all subjects. Plug conditions (retention rates, spontaneous plug loss, and removal) and complications related to lacrimal punctal sizes were compared between the 2 groups. RESULTS: Retention rates were 70.4% in group A and 30.1% in group B at 6 months. Spontaneous plug loss occurrence was significant with the larger lacrimal punctum size in group A (P < .05), whereas no significant differences were observed between lacrimal punctum sizes and spontaneous loss in group B. Spontaneous plug loss occurrence was significant in elderly patients in group B (P < .05). Granuloma and injector troubles were observed in 1 and 2 cases in group A, respectively. CONCLUSIONS: The Punctal Plug F seems to be more applicable for insertion into the smaller punctal sizes. Punctum size and age seem to be important factors in determining the appropriate types of lacrimal punctal plugs.