**6. Selected examples**

had 24% sensitivity and flow cytometry had a sensitivity of 36%. [46] In addition, PCR may be used to detect bcl-2 gene translocations in PIOL that were shown to occur in younger patients,

Flow cytometry is a diagnostic technique that allows for simultaneous analysis of several different cell surface markers. It involves centrifuging diluted vitreous and re-suspension in cell culture medium. The cells are then counted and stained with antibodies to detect cellular

It has been shown to be useful in the diagnosis of PIOL. [16] It relies on the fact that most PIOLs are composed of monoclonal populations of B-lymphocytes that stain positively for B cell

Davis et al [49] correlated different flow cytometric markers with lymphoma, infection, and idiopathic uveitis. They found that the most sensitive marker for lymphoma was a κ:λ ratio ≥3 or ≤0.6, while CD22 and CD20 were specific but not sensitive for lymphoma. For infection they found that the CD8, CD14, and CD11c markers that indicate monocytes and cytotoxic

B-cell malignancies can secrete high levels of interleukin-10 (IL-10), an immunosuppressive cytokine. Inflammatory conditions are associated with high levels of interleukin-6 (IL-6), a proinflammatory cytokine. [50, 51] IL-10 in PIOL tends to be high, with IL-10:IL-6 ratios greater than 1.0 being suggestive of the disease. This ratio may serve as a useful adjunctive test in the diagnosis of suspected PIOL, while also showing whether there is a significant response to

Cassoux et al [52] found that mean IL-10 values were 2205.5 pg/mL in the vitreous and 543.4 pg/mL in the aqueous humor in patients with PIOL, while in uveitis patients mean values were 26.6 pg/mL in the vitreous and 21.9 pg/mL in the aqueous. This difference was highly signif‐

Since the measurement of cytokine levels is fairly easy, measurement of IL-10 and IL-6 levels

This indirect method of diagnosing infection is often negative early in the course of the disease as well as in immunocompromised patients. [31] Intraocular-specific antibody secretion has

A helpful concept in antibody measurement is the Goldmann-Witmer coefficient (GWC). It can be calculated to compare intraocular antibody production with serum antibody levels. A

T lymphocytes were specific, but not sensitive. A CD4:CD8 ratio of ≥4 was highly

markers (CD19, CD20, CD22) and have restricted expression of κ or λ chains [48]

suggesting a more aggressive treatment approach. [47]

surface markers that identify leukocytes. [20]

sensitive and specific for inflammatory uveitis.

is recommended for patients with suspected PIOL. [20]

been shown to confirm the etiology in 23-32% of cases. [53, 54]

**5.6. Cytokine measurement**

**5.7. Antibody measurement**

**5.5. Flow cytometry**

170 Advances in Eye Surgery

CD8<sup>+</sup>

treatment. [31]

icant.

#### **6.1. Infectious etiologies**

#### *6.1.1. Bacterial and fungal endophthalmitis*

In cases of suspected bacterial or fungal endophthalmitis, Gram stain and culture (aerobic, anerobic, and fungal) of the vitreous sample are performed in order to identify the causative organisms and their susceptibilities.

As mentioned above, PCR analysis of aqueous and vitreous fluid have also been applied in case series of patients with acute and delayed postoperative endophthalmitis, sometimes with better detection of the causative agent than cultures.

#### *6.1.2. Mycobacterium tuberculosis*

Diagnosis of ocular tuberculosis (TB) is possible with the use of various tests for the detection of systemic TB, including chest radiography, Purified Protein Derivative (PPD) tuberculin skin test, Interferon Gamma Release assays (IGRA), and analyses of extraocular sites. [58, 59] Intraocular fluid analysis may help.

Traditional fluid analysis with Ziehl-Neelsen staining and culture on Lowenstein-Jensen medium is not ideal, as the former has low yields and the latter takes up to 6-8 weeks, limiting its clinical utility. [58, 59] The yield of PCR analysis of aqueous and vitreous fluid has been shown to range from 37.7% to 72% in a series of Indian patients. [60-62] It was shown that 77-80% of PCR-positive patients in these series were PPD positive, and 90-100% of PCRpositive patients who were treated with antitubercular treatment had resolution of inflamma‐ tion. Similar rates were shown by a Mexican group, [63] where PCR testing in 22 patients with a known diagnosis of TB uveitis showed a yield of 77.2%. All patients improved with antitu‐ bercular treatment.

#### *6.1.3. Toxoplasma gondii*

While diagnosis of ocular toxoplasmosis is typically made by a characteristic clinical presen‐ tation and supported by positive serology, there are cases that pose a diagnostic dilemma in which an invasive ocular diagnostic procedure may be needed. For example, in immunocom‐ promised and elderly patients the disease may mimic viral necrotizing retinitis. [64, 65]

Culture of *T. gondii* from the vitreous may be lengthy and ranges from 2 to 23 days for positive cultures. [66] The rapid detection of toxoplasmosis DNA using PCR techniques on aqueous fluid has a yield of 13% to 55% according to literature, with positive results occurring more often with larger chorioretinal lesions, immunosuppressed patients, and active anterior segment inflammation. [21] Antibody levels in the aqueous may supplement PCR results by calculating the GWC, as described above. In one series, calculation of the aqueous GWC for toxoplasmosis antibody at the onset of clinical manifestation had a yield of 57%, rising to 70% after 3 weeks. [67]

The utility of GWC is decreased in immunocompromised patients. In one series of 34 immu‐ nocompetent patients with negative PCR tests for toxoplasmosis, 25 had a positive GWC, whereas none of the immunocompromised patients exhibited a positive test. [56] In a similar fashion, another series showed 93% positivity with use of this test in immunocompetent patients, in comparison with a yield of only 57% in immunocompromised patients. [68]

While these results deal with aqueous analysis, less data appears in the literature regarding diagnostic vitrectomy for this purpose. Available data shows a trend towards improved yields for PCR from vitreous specimens. [21]

#### *6.1.4. Viral retinitis*

The diagnosis of infectious viral retinitis caused by herpes simplex virus (HSV), cytomegalo‐ virus (CMV), or varicella zoster virus (VZV) is not always straightforward. As with the other infectious entities just mentioned, growth on culture may take a long time. PCR analysis of aqueous and vitreous fluid plays an important role thanks to its high sensitivity, low falsepositive rates, and the rapidity of the assay. [69, 70]

The sensitivities of PCR for VZV, HSV, and CMV were reported to exceed 90%, with specificities in excess of 95%. [39] Knox et al. performed PCR on specimens from 38 eyes of 37 patients with an inflammation of unknown etiology suggestive of an infectious posterior segment disease. In 24 of these cases CMV, HSV, or VZV were detected. [71] Sugita et al. collected 68 aqueous humor samples and 43 vitreous fluid samples from 100 patients with uveitis. The samples were assayed for human herpes viruses using multi‐ plex PCR and real-time PCR. Out of 16 patients with ARN, either HSV1, HSV2, or VZV genomes were detected. In another 10 patients with anterior uveitis with iris atrophy, the VZV genome was detected. Epstein-Barr virus was detected in 17% of samples, and (CMV) was detected in three patients with anterior uveitis of immunocompetent patients and in one immunocompromised CMV retinitis patient. [72]

As was shown above for toxoplasmosis, calculation of the GWC may also be of use for HSV, VZV, and CMV, although variable results have been reported in the literature. In one series of immunocompromised patients with posterior uveitis and panuveitis, analysis of an aqueous sample demonstrated a detection rate of 94% for PCR aimed for the detection of CMV and VZV versus only 18% with GWC. [73] Another series demonstrated an identification of 92% of HSV-associated and 87.5% of VZV-associated infectious uveitis using GWC, in comparison with 54% of HSV and 75% VZV cases that were identified using PCR. [74]

#### **6.2. Non-infectious inflammatory conditions**

#### *6.2.1. Sarcoidosis*

Culture of *T. gondii* from the vitreous may be lengthy and ranges from 2 to 23 days for positive cultures. [66] The rapid detection of toxoplasmosis DNA using PCR techniques on aqueous fluid has a yield of 13% to 55% according to literature, with positive results occurring more often with larger chorioretinal lesions, immunosuppressed patients, and active anterior segment inflammation. [21] Antibody levels in the aqueous may supplement PCR results by calculating the GWC, as described above. In one series, calculation of the aqueous GWC for toxoplasmosis antibody at the onset of clinical manifestation had a yield of 57%, rising to 70%

The utility of GWC is decreased in immunocompromised patients. In one series of 34 immu‐ nocompetent patients with negative PCR tests for toxoplasmosis, 25 had a positive GWC, whereas none of the immunocompromised patients exhibited a positive test. [56] In a similar fashion, another series showed 93% positivity with use of this test in immunocompetent patients, in comparison with a yield of only 57% in immunocompromised patients. [68]

While these results deal with aqueous analysis, less data appears in the literature regarding diagnostic vitrectomy for this purpose. Available data shows a trend towards improved yields

The diagnosis of infectious viral retinitis caused by herpes simplex virus (HSV), cytomegalo‐ virus (CMV), or varicella zoster virus (VZV) is not always straightforward. As with the other infectious entities just mentioned, growth on culture may take a long time. PCR analysis of aqueous and vitreous fluid plays an important role thanks to its high sensitivity, low false-

The sensitivities of PCR for VZV, HSV, and CMV were reported to exceed 90%, with specificities in excess of 95%. [39] Knox et al. performed PCR on specimens from 38 eyes of 37 patients with an inflammation of unknown etiology suggestive of an infectious posterior segment disease. In 24 of these cases CMV, HSV, or VZV were detected. [71] Sugita et al. collected 68 aqueous humor samples and 43 vitreous fluid samples from 100 patients with uveitis. The samples were assayed for human herpes viruses using multi‐ plex PCR and real-time PCR. Out of 16 patients with ARN, either HSV1, HSV2, or VZV genomes were detected. In another 10 patients with anterior uveitis with iris atrophy, the VZV genome was detected. Epstein-Barr virus was detected in 17% of samples, and (CMV) was detected in three patients with anterior uveitis of immunocompetent patients and in

As was shown above for toxoplasmosis, calculation of the GWC may also be of use for HSV, VZV, and CMV, although variable results have been reported in the literature. In one series of immunocompromised patients with posterior uveitis and panuveitis, analysis of an aqueous sample demonstrated a detection rate of 94% for PCR aimed for the detection of CMV and VZV versus only 18% with GWC. [73] Another series demonstrated an identification of 92% of HSV-associated and 87.5% of VZV-associated infectious uveitis using GWC, in comparison

with 54% of HSV and 75% VZV cases that were identified using PCR. [74]

after 3 weeks. [67]

172 Advances in Eye Surgery

*6.1.4. Viral retinitis*

for PCR from vitreous specimens. [21]

positive rates, and the rapidity of the assay. [69, 70]

one immunocompromised CMV retinitis patient. [72]

The frequency of sarcoidosis involving the posterior segment varies in different series. One group reported that as many as 89% of patients with ocular sarcoidosis demonstrated posterior segment involvement, with vitritis as the most common manifestation, present in 69% of these patients. [75] As the manifestations of sarcoidosis are varied, a diagnosis of this inflammatory entity is not always straightforward, and may require an invasive procedure such as diagnostic pars plana vitrectomy.

An increased CD4+ helper T-cell type 1 lymphocyte subset in bronchoalveolar lavage (BAL) fluid and a high CD4/CD8 ratio are helpful for the diagnosis of sarcoidosis. [76] Kojima et al. demonstrated that this ratio may also be applied for vitreous specimens, when a CD4/CD8 ratio of vitreous-infiltrating lymphocytes greater than 3.5 provided a diagnosis of ocular sarcoidosis with a sensitivity of 100% and a specificity of 96.3% (in comparison with a sensi‐ tivity of 53% and specificity of 94% in analysis of BAL fluid). [77]

#### **6.3. Neoplastic processes**

#### *6.3.1. Primary intraocular lymphoma*

PIOL is considered one of the masquerade syndromes, or diseases that mimic inflammatory conditions in presentation, leading to a diagnostic dilemma. [78] When the diagnosis of a neoplastic process such as PIOL is suspected, reaching a diagnosis is of utmost importance in terms of prognosis and the choice of treatment.

A definitive tissue diagnosis is required to make the diagnosis of PIOL. If lymphoma is identified from a lumbar puncture, an invasive diagnostic ocular procedure may not be required. If, on the other hand, lumbar puncture results are inconclusive and neuroimaging is not consistent with CNS lymphoma in a patient with a high index of suspicion for PIOL, invasive diagnostic procedures are appropriate. [3]

Histologic identification of malignant lymphoid cells is the gold standard for diagnosing PIOL. [3] As stated above, it is pertinent to communicate with the pathologist before the procedure, as any delay in delivery of the sample may result in death of acquired cells.

The characteristic features of PIOL using microscopic analyses include large atypical lymphoid cells with scarce cytoplasm, prominent nucleoli, frequently large segmented nuclei, and a high nuclear to cytoplasm ratio. [3] Cytology has a sensitivity ranging from 31% to 66.7% for detecting intraocular malignancy, and one report showed a sensitivity of 83.3% for detecting PIOL. [31, 49] In addition to cytology, immunohistochemistry, cytokine analysis, flow cytometry, and gene rearrangements by PCR are also performed on the specimens. [3]

#### *6.3.2. Tumor metastasis*

Tumor metastasis is the most common cause of intraocular malignancy in adults. [78] While their typical appearance and preexisting history of cancer typically lead to diagnosis, uveal metastases masquerading as intraocular inflammation have been reported. [21]

A few cases were reported on the use of aqueous sampling for cytology which led to the diagnosis of metastases masquerading as anterior uveitis. [79-84] Of patients undergoing diagnostic vitrectomy for uveitis of unknown cause, detection of metastasis from cytology results was rare in the literature, [11, 85, 86] with only one case reported in each of these series. In case reports of patients with the rare occurrence of tumors metastatic to the retina and vitreous, these conditions present as intermediate uveitis, vitreous hemorrhage, or retinal vasculitis with vitreous cytology and retinal biopsy assisting in diagnosis if no primary malignancy is identified. [21]

In a series of 159 cases by Shields et al, [87] transocular fine needle aspiration (FNA) biopsy led to an adequate sample collection in 88% of cases, with a sensitivity rate of 100% and specificity rate of 98%, leading to diagnosis of intraocular malignancies such as uveal mela‐ noma, uveal metastasis, retinoblastoma, lymphoma, and leukemia. In another series of 39 patients with uveal metastasis undergoing ocular biopsy of the tumor, 25 G vitrectomy had a yield of 100% for cytologic diagnosis. It indicated the site of origin in 24 out of 27 patients without a known primary tumor. [88]

### **7. Summary**

Diagnostic procedures in ophthalmology have gone a long way from the early days of pars plana vitrectomy, when instrumentation and diagnostic methods were limited and the amount of entities that could be diagnosed by invasive methods was restricted.

As this chapter has shown, the approach to a patient with a cryptic diagnosis, a rapidly deteriorating disease, or treatment failure has changed in the last decades and ophthalmolo‐ gists now have in their arsenal a battery of tools to help in the diagnosis of cases that were once considered unsolvable or untreatable.
