Dear Editor,

We read with interest the paper published by Boschi, et. al.[¹] in which immunohistochemistry was performed on orbital tissues from patients with thyroid associated ophthalmopathy (TAO) and compared to non-diseased orbital tissue.

Our laboratory recently reported positive TSH receptor staining within normal human muscle fibres, using one of the same antibodies (3G4) as Boschi et. al., (supplied by Costagliola) and a commercial antibody (3B12) [²].

Our findings differ from Boschi et. al.’s as no staining of the muscle fibres was visible in their experience.

Assessing the techniques used gave some possibilities as to why our findings differ. Our paraffin-embedded tissues were subjected to a proteolytic antigen retrieval step, as commonly used in avidin-biotin staining.[³] The reason for this is that formalin used in fixation is notorious for altering protein immunoreactivity, and hence masking protein expression.[⁴][⁵]

Moreover, the amplification immunohistochemistry kit used in our experiments is possibly more sensitive than conventional immunohistochemistry used in the experiments of Boschi, et. al.[⁶]

We do not dispute the finding that TSHR expression is elevated in orbital connective tissue of diseased patients. Combined with our findings, Boschi et. al.’s paper also suggests that expression of TSH-R on normal muscle fibres is lower than in the connective tissue of diseased patients. Boschi et. al. has successfully produced more evidence that connective tissues in the orbit are active in TAO affected patients, however the potential role of the extraocular muscle in the pathogenesis of TAO should also be considered.

Yours sincerely,

Steven J. Kloprogge and Albert G. Frauman

References

A Mataftsi et al1 published an interesting article regarding punctal plugs in children. One of their aim was to establish the efficacy however they have not mentioned any test (Schirmer, Tear film break-up time, Rose Bengal staining, osmolarity) to confirm the diagnosis of dry eye and to calibrate the tear deficiency. It was only the clinical impression (flouresein staining is not specific for dry eyes and therefore cannot be used as diagnostic). In the follow up also, there was no yardstick to measure and compare the post procedure improvement with the pre procedure status. Only subjective feelings (patients or parents) are difficult to gauge and therefore a scientific test at this stage would have established the exact positive role of the plugs which could have been counterchecked and verified by other workers. These tests could have been done in the same sitting/ anesthesia and would not have required any additional visit or anaesthesia. A few points of this article differed markedly from previous articles. In "Blepharokeratoconjunctivitis in children: diagnosis and treatment"2 by M Viswalingam et al in which patients in Moorfields Eye Hospital, London, UK, were analyzed, there is no mention of dry eye in either the text or Table 1- Classification of the severity of blepharokeratoconjunctivitis (BKC) in children and Table - 3 Clinical symptoms and signs. Punctate erosions were present in only 9 % (all Asian) of their patients. Their patients improved without any dry eye treatment. Similarly in "Visual Outcome and Corneal Changes in Children with Chronic Blepharokeratoconjunctivitis"3 by Jones SM et al, in patients analyzed in Great Ormond Street Hospital for Children, London, UK, from1999 to 2005 (same hospital and almost same time period as is of the present article), there was no mention of dry eye and punctate epithelial erosions (PEE) were found in 31% of eyes. "In the authors' experience, effective treatment for BKC should include a combination of both systemic and topical antimicrobial therapy, along with topical steroids" was the authors' recommendation in the above mentioned article and no punctal plugs were mentioned. Now in the present article, authors have found a lot of dry eyes (out of which 14 required punctual plugs) in BKC (and PEE in 100%) among the almost same record which was used for the above mentioned article and now they claim "plugs were successful in treating a variety of causes of dry eye in our cohort, with more than half of the children presenting with lipid deficiency secondary to meibomian gland dysfunction".

Despite these observations, authors deserve appreciation for introducing the new concept of punctal plug use in children.

References: 1. Mataftsi A, Subbu RG, Jones S, Nischal KK. The use of punctal plugs in children. Br J Ophthalmol 2012;96:90-92. 2. Viswalingam M, Rauz S, Morlet N, Dart JK. Blepharokeratoconjunctivitis in children: diagnosis and treatment. Br J Ophthalmol. 2005 Apr;89(4):400- 3. 3. Jones SM, Weinstein JM, Cumberland P, Klein N, Nischal KK. Visual Outcome and Corneal Changes in Children with Chronic Blepharokeratoconjunctivitis. Ophthalmology 2007;114:2271-2280

Conflict of Interest:

None declared

Dear Editor

We read with interest the remarks of Crowston et al. [1] on our article entitled "Value of two mortality assessment techniques for organ cultured corneal endothelium: trypan blue versus TUNEL technique".[2] We showed that the TUNEL technique revealed a far higher percentage of endothelial cells (ECs) irreversibly engaged in a cell death process than that obtained by trypan blue staining.

The two techniques were performed sequentially: after observation of trypan blue staining, corneas were immediately fixed in formaldehyde for TUNEL. Crowston et al. suggest that the trypan blue itself and/or the time spent outside the organ-culture medium before fixing in formaldehyde may have caused an artefactual increase in the percentage of TUNEL-positive ECs. Two arguments counter this remark:
1. The trypan blue staining procedure is identical to that used in all European cornea banks that use organ culture during endothelial examination(s) of grafts. Neither the low concentration of trypan blue (0.4%) nor the short exposure time (about 1 minute) nor the short incubation in the presence of 0.9% NaCl has ever been incriminated in ECs over-mortality in routine practice.[3] Moreover, the innocuity of injections of trypan blue into the anterior chamber, a common feature during cataract surgery, has been well demonstrated.[4]

2. The time spent outside the organ-culture medium before fixing in formaldehyde, a period required for vital staining and microscopic examination of the endothelium, lasts only a few minutes. The cornea remains under the microscope for about one minute only, the time needed for image acquisition. Such rapidity is possible by using a prototype automatic analyser of the endothelium, which we developed and have recently published.[5] This is very probably insufficient time for DNA fragmentation to occur in the proportion we observed. Moreover, the fixing of the endothelial layer in 10% formaldehyde is immediate, and prevents any continuation of fragmentation phenomena. On balance, it is highly unlikely that the succession of markings is responsible for the discrepancy between the positivity percentages of the two techniques. In addition, we chose not to perform the two techniques simultaneously on paired corneas or on the halves of one cornea because we wanted to superimpose the two stains on the same cornea and thus obtain a double cell staining.

The second remark by Crowston et al. is particularly interesting. We too were surprised by the high percentage of TUNEL-positive ECs (mean 12.7%, SD 16.4). This may imply that all the cells died within 8 days, which was evidently not the case. We believe this apparent contradiction can be explained by the following theory. The TUNEL staining is positive during a relatively long window (24-48 hours [6]). The TUNEL index, measured at a given moment, provides a global view of all the cells with fragmented DNA. However, the DNA fragmentation may be at different stages, and the cells very likely spread according to a Gaussian distribution. Therefore the cells, which are TUNEL-positive at a given moment, will not all die instantaneously and simultaneously. Only the cells furthest to the right on the curve will die in the very short term, and it is probably these that are liable to be revealed by trypan blue. If it were possible to perform TUNEL on two consecutive days, the percentage of positive cells revealed would probably be very similar, but a large majority of the positive cells recorded on the second day would have already been counted on day one... It is, however, undeniable that the cells TUNEL-positive at a given moment will all die eventually. In other words, we believe that, at the end of storage, corneas contain a number of ECs engaged in an irreversible cell-death process far more extensive than the highly unreliable trypan blue staining technique suggests.

References

(1) Crowston JG, Healey PR, Maloof A, et al . Quantifying corneal endothelial cell death. Br J Ophthalmol 2002;86:1068.

(2) Gain P, Thuret G, Chiquet C, et al . Value of two mortality assessment techniques for organ cultured corneal endothelium: trypan blue versus TUNEL technique. Br J Ophthalmol 2002;86:306-10.

(3) Sperling S. Evaluation of the endothelium of human donor corneas by induced dilation of intercellular spaces and trypan blue. Graefes Arch Clin Exp Ophthalmol 1986;224:428-34.

(4) Norn MS. Per operative trypan blue vital staining of corneal endothelium. Eight years' follow up. Acta Ophthalmol 1980;58:550-5.

(5) Gain P, Thuret G, Kodjikian L, et al. Automated tri-image analysis of stored corneal endothelium. Br J Ophthalmol 2002;86:801-8.

(6) Mesner PW, Epting CL, Hegarty JL, et al. A timetable of events during programmed cell death induced by trophic factor withdrawal from neuronal PC12 cells. J Neurosci 1995;15:7357-66.

Dear Editor,

The article by Al-Hazmi et al.[1] states that combined trabeculotomy- trabeculectomy with mitomyocin C (CTTM) gave better results than trabeculotomy alone for primary congenital glaucoma (PCG) at the King Khaled Eye Specialist Hospital (KKESH) in Riyadh, Saudi Arabia, between 1982 and 2002. For moderate PCG the success rate is stated as 40% and 80% for trabeculotomy and CTTM, respectively. For severe PCG the stated success rate is 10% and 70% for trabeculotomy and CTTM, respectively. However, without more specifics regarding when the trabeculotomies were performed at KKESH, the authors cannot advocate CTTM over trabeculotomy for moderate and severe PCG.

As the article states, over the years at KKESH the success rate for trabeculotomy for PCG dramatically improved (29% from 1982-1990; 47% from 1991-94; 82% from 1995-2002) The authors attribute these improved results over the years to improved primary healthcare facilities within the kingdom, earlier referrals, better equipment availability, and surgeons becoming more adapt at surgical intervention. In contrast, CTTM for PCG was first performed at KKESH in 1994 with less of a “learning curve”; the success rate from 1994 -2002 was 72%. The complication rate, however, was higher for CTTM than for trabeculotomy.

Because initial trabeculotomy success for PCG at KKESH has dramatically increased with time, it is important to know how many of the reported trabeculotomy failures for moderate and severe PCG were from the earlier periods of the hospital. This information was not in the manuscript. It may be that trabeculotomy as currently performed at KKESH for moderate and severe PCG has a success rate similar to that of CTTM for the same patient population with less surgical complications.

References

1. Al-Hazmi A, Awad A, Zwaan Z, et al. Correlation between surgical success rate and severity of congenital glaucoma. Br J Ophthalmol 2005;89:449–453.