Ten Tenets of Diagnostic Vitrectomy

Diagnosis of posterior segment inflammatory or infectious disease is mostly based on clinical presentation, ocular imaging and specific laboratory tests. However, in the absence of a characteristic clinical picture of a specific disease the diagnosis of uveitis can be challenging. It has been reported that upto 33% of uveitis remain undiagnosed. Moreover, some of the malignant or infective diseases of the eye may mimic an inflammatory pathology leading to delay in diagnosis and subsequently poor outcomes. Intraocular biopsy may be helpful in clinching the diagnosis in these situations. With the improvement in laboratory testing and vitrectomy instrumentations, diagnostic vitrectomy is increasingly being performed in clinical practice.

1. Indications 1,2

Diagnostic dilemma
Patient fails to respond to standard systemic anti-inflammatory therapy
Atypical clinical presentations
Inconclusive non-invasive laboratory work-up
Significant vitreous inflammation concerning for malignancy, infectious endophthalmitis or intraocular foreign body

2. Preparation

Before going for vitrectomy one needs to be sure about a) what are the laboratory tests that need to be run on the collected specimen b) availability of pathologist who is familiar with ocular pathology c) a discussion with the pathologist regarding the specimen collected and suspected diagnosis helps in better yield.

3. Surgical Steps 3,4

The procedure can be done under local or general anesthesia. 20, 23 or 25 gauge PPV can be done using a wide angle viewing system. The lens status (pseudophakia, phakia, aphakia) must be considered before surgery. Sometimes, the inflammatory debris may collect on the posterior lens capsule obscuring the view of the fundus. Standard 3 pars plana ports are made and an undiluted vitreous sample is collected. The cutter is kept in the mid vitreous cavity under visualization and the assistant is asked to collect the sample manually by aspiration using a 2cc syringe. Some surgeons advocate a lower cutting rate and higher duty cycle because larger and more intact pieces of vitreous may have a higher yield. This is specially suited for collecting samples in cases with suspected intraocular lymphomas. The undiluted vitreous sample is usually taken under air to reduce the risk of hypotony. After collecting the sample the infusion line is opened and a standard total vitrectomy is done after inducing a posterior vitreous detachment. The vitreous sample is sent immediately for analysis. The vitrectomy cassette may also be sent for analysis.

4. Role of PVD completeness of PPV

Removal of the entire vitreous has the advantage of removing all of the pathological material, inflammatory cells, malignant cells or microorganisms from the eye which helps in reducing the inflammation faster. More vitreous sample also increases the diagnostic yield. Hence, wherever possible a PVD should be induced and total vitrectomy should be done including removal of vitreous from the base.
Total vitrectomy also enhances post operative view of the fundus which helps in better management and better penetration of drugs.
In cases of poor media clarity due to corneal edema or lenticular opacity a limited vitrectomy should be done to avoid causing iatrogenic breaks.

5.Retinal or Choroidal Biopsy 5,6

Retinal or choroidal biopsy is considered when the pathology involves the sensory retina or the RPE
It is useful in the diagnosis of tuberculosis, sarcoidosis and lymphoma
The advantage of biopsy is more material available for immunohistochemistry
The biopsy can be obtained by transscleral or transvitreal approach depending on the location of the lesion
In presence of a detached retina the biopsy is taken at the junction of the attached and detached retina. In presence of an attached retina, before taking a biopsy a localized retinal detachment is created by subretinal injection of BSS using a 39G or 41G cannula.
Superior and nasal quadrants are preferred
The retina/choroidal tissue is harvested using vertical scissors and forceps using bimanual technique and laser is done
The case is closed under gas or silicone oil

6. Vitreous Sample Collection 7

Undiluted samples have a higher yield. However, in cases of hypotony diluted samples can be taken.

Undiluted sample: 2 cc syringe is connected directly to the vitreous cutter handpiece. Three-way stopcock attached to the vitreous cutter eases the sample collection. The vitreous sample, approximately 1.5ml is aspirated before turning on the infusion.

Diluted sample: the vitreous is aspirated after turning on the infusion and the sample is collected in a different syringe.

7. Vitreous Sample Handling 7

To prevent degeneration of lymphoma cells, the vitreous specimen is placed in RPMI (Roswell Park Memorial Institute) culture medium
Lymphoma cells degenerate early so the sample should be sent immediately to the pathologist
In cases of infectious etiology, the sample should be inoculated in appropriate culture media immediately to increase the recovery of organisms

8. Vitreous Sample Analysis

Undiluted sample is used for cytological analysis, immunohistochemical staining and for PCR testing. The supernatant of the undiluted sample is used for cytokine anlaysis and antibody levels. Diluted sample can be used for gram stain, culture, flow cytometry and PCR testing.

Microbiological analysis 2

This is useful in cases of infectious uveitis for identifying the causative organism and antibiotic sensitivity using various stains, smears and culture.
The diluted vitreous sample is passed through a millipore filter and the filter is used for culture
In cases of suspicion of slow growing organisms like Propionibacterium acne and fungi the microbiologist should be informed to keep the culture plates for a longer duration about 2 weeks to 1 month.
Endophthalmitis vitrectomy study showed a yield of 66% for culture and 41% for gram stain

PCR analysis of the vitreous 1

PCR is used to amplify the number of copies of a specific region of DNA in order to produce enough DNA to be adequately tested.
PCR testing of aqueous and vitreous plays an important role in the diagnosis of viral retinitis due to high sensitivity, rapidity of the assay and low false positive rates.
The infectious agents that are detected using PCR are viruses (CMV, HSV, VZV, EBV, adenovirus), protozoans (Toxoplasma gondii), fungi (Candida albicans, aspergillus), bacteria (Mycobacterium species, Staphylococcus species, Streptococcus species)
Goldmann Witmer Coefficient (GWC) is calculated to supplement the results of PCR testing. It is calculated as target IgG in the ocular fluid/total ocular fluid IgG divided by target IgG in the serum/total serum IgG. GWC greater than 1 indicates local antibody production but most of the authors designate GWC greater than 3 as indicative of local antibody production.

Cytopathological analysis, flow cytometry and gene rearrangement studies 7

Cytopathology helps to differentiate infectious and autoimmune uveitis from malignancy (masqueraders)
In primary intraocular lymphoma, microscopy demonstrates atypical lymphoid cells that are large with scarce cytoplasm, prominent nucleoli, frequently large, segmented nuclei, and a high nucleus to cytoplasm ratio.
Papinicolaou stain and Giemsa stain are useful to identify the malignant cells.
Flow cytometry demonstrates cell surface markers and monoclonal B cells. Diluted vitreous is centrifuged and resuspended in cell culture medium. Cells are stained with antibodies to detect markers that identify leucocytes, T lymphocytes, B lymphocytes, monocytes/macrophages and lymphocyte activation.
A kappa: lambda ratio of >3 or <0.6 is a highly sensitive marker of lymphoma
Cytokine analysis shows an elevation of IL 10 in patients with primary intraocular lymphoma whereas inflammatory conditions show elevated IL 6. IL 10 is immunosuppressive cytokine and IL6 is proinflammatory cytokine.
A IL10/IL6 ratio of greater than 1 is suggestive of diagnosis of B cell lymphoma
In B-cell lymphomas, molecular analysis can detect IgH gene rearrangements, while in T-cell lymphomas, T-cell receptor gene rearrangements can be detected.

9. Diagnostic Yield

Overall diagnostic yield ranges from 12.4 to 64.3% 2
The sensitivity of culture in infectious uveitis after diagnostic vitrectomy is 16.7 to 96%2
The sensitivity of PCR is 80.9% and specificity is 97.4% in viral uveitis8
Cytology has a sensitivity ranging from 31 to 66.7% of detecting an intraocular malignancy7
The diagnostic yield can be increased by taking undiluted vitreous sample, taking biopsy closer to the retinal/choroidal lesion, careful sample handling to transfer the sample to the laboratory immediately.

10. Challenges and Complications

Media haze due to corneal edema, pupillary membranes and cataract may cause difficulty in visualizing the infusion cannula and may limit vitrectomy
Hypotony in uveitis eyes may lead to difficulty in making ports and obtaining undiluted vitreous sample
Difficulty in inducing a PVD and increased risk of iatrogenic breaks due to necrotic retina in few of the pathologies like acute retinal necrosis
Postoperative hypotony may cause worsening of inflammation and also lead to choroidal detachments, it is hence important to suture the sclerotomy wounds even when performing small gauge vitrectomy.

References:

1. Papadaki T. Vitreous biopsy for the diagnosis of uveitis. intraocular Immunol uveitis Found Doc. 2002;7.

2. Jeroudi A, Yeh S. Diagnostic vitrectomy for infectious uveitis. Int Ophthalmol Clin. 2014;54(2):173.

3. Davis JL, Miller DM, Ruiz P. Diagnostic testing of vitrectomy specimens. Am J Ophthalmol. 2005;140(5):822-829.

4. Margolis R. Diagnostic vitrectomy for the diagnosis and management of posterior uveitis of unknown etiology. Curr Opin Ophthalmol. 2008;19(3):218-224.

5. Cole CJ, Kwan AS, Laidlaw DAH, Aylward GW. A new technique of combined retinal and choroidal biopsy. Br J Ophthalmol. 2008;92(10):1357-1360.

6. Johnston RL, Tufail A, Lightman S, et al. Retinal and choroidal biopsies are helpful in unclear uveitis of suspected infectious or malignant origin. Ophthalmology. 2004;111(3):522-528.

7. Hwang CS, Yeh S, Bergstrom CS. Diagnostic vitrectomy for primary intraocular lymphoma: when, why, how? Int Ophthalmol Clin. 2014;54(2):155.

8. Harper TW, Miller D, Schiffman JC, Davis JL. Polymerase chain reaction analysis of aqueous and vitreous specimens in the diagnosis of posterior segment infectious uveitis. Am J Ophthalmol. 2009;147(1):140-147.