**5. Diagnostic testing of vitreous specimens**

With the advent of new laboratory techniques, a myriad of options are available for the clinician in the quest for obtaining a correct diagnosis of an unknown inflammatory, infectious, or neoplastic entity.

Of course not all tests should be performed in all cases, and tests should be chosen according to the suspected diagnosis.

#### **5.1. Histopathologic evaluation**

A sample is sent to a pathologist following the diagnostic procedure and is immediately processed. The specimen is generally divided into three portions: one third is fixed for routine histopathological evaluation, including light and electron microscopic examination. Another one third is frozen in optimal cutting temperature (OCT) embedding compound for immu‐ nopathology (phenotyping of cells by their surface markers) and molecular characterization. The last third portion is sent for culture of microorganisms. If the specimen is not adequate for all three procedures, frozen sections are recommended, as they can undergo routine histopa‐ thology, immunohistochemistry, and molecular analysis. [20]

#### **5.2. Cytology**

Cytological evaluation reveals the phenotypes of infiltrating cells in the vitreous. The vitreous specimen is centrifuged and cells are smeared onto glass slides, and then immersed in 95% ethanol for Papanicolaou (Pap) staining or left to dry for Giemsa staining. [20]

The reported sensitivity of cytology in the detection of intraocular malignancy ranges from 31% to 66.7%. This relatively low yield may be due to the presence of immune cells, necrotic cells, fibrin, and debris in the specimen, which may confound the examination. [31] Other reasons include small sample volumes with a low number of malignant cells, inadequate preparation of the sample, and previous administration of corticosteroids. [20]

Cytologic evaluation may also be used to distinguish between a malignant process and an inflammatory disease. An example of an inflammatory etiology that may be diagnosed with the aid of cytology is sarcoidosis. Kinoshita et al. demonstrated multinucleated giant cells in the vitreous in 85.7% of cases and lymphocytes and epithelioid cells in all cases of intraocular sarcoidosis. [32]

An advanced technique for cytology is the use of cell blocks. They are superior to cell smears since cells are accumulated by centrifugation and stored as paraffin blocks. The large number of cells in a compacted area of one section on a slide glass as opposed to sparse cells on a smear leads to a more accurate diagnosis. Paraffin sections also have the advantage of being used for immunocytochemical diagnosis and clonal analysis, such as amplification by PCR of the immunoglobulin heavy chain gene. [33]

#### **5.3. Microbiological analysis**

Microbiological cultures are considered the "gold standard" for diagnosis of infectious uveitis. There are different types of media for isolation of the causative agent, including blood agar (for gram-positive or fastidious gram-negative bacteria [34]), MacConkey agar (for most gramnegative rods [34]), and Brucella agar for bacterial infections; Sabouraud dextrose agar for pathogenic fungi and yeast; and shell vial culture for viral infections. Along with the culture, the sample is sent for Gram staining and antibiotic sensitivity tests. [20]

Some fastidious organisms, such as *Proprionibacterium acnes* and fungi require holding the culture for at least 1 month to avoid missing their diagnosis. [21]

The sensitivity of culture after diagnostic vitrectomy for diagnosis of chronic infectious uveitis has been reported between 16.7% and 96%. [21] In cases of acute endophthalmitis, the sensitivity of microbiological cultures and stains was shown to be 40-70%. [35] Higher yields are reported with vitreous rather than aqueous samples. [36] Processing both diluted and undiluted vitreous samples increases the sensitivity of vitreous cultures to 57.4%. [37]

The yield of positive cultures from vitreous samples is usually low in cases of fungal endoph‐ thalmitis. In a retrospective study by Tanaka et al., positive cultures were only found in 38% of vitreous specimens in patients with endogenous fungal endophthalmitis. [38]

While the utility of Gram stains is limited in comparison with culture (data from the Endoph‐ thalmitis Vitrectomy Study showed a yield of 66% for culture and 41% for Gram stain for patients undergoing vitrectomy [35]), they are useful for rapid initial diagnosis of intraocular infection and can help the clinician choose the appropriate antibiotic for the organism prior to culture results.

#### **5.4. Molecular analysis**

all three procedures, frozen sections are recommended, as they can undergo routine histopa‐

Cytological evaluation reveals the phenotypes of infiltrating cells in the vitreous. The vitreous specimen is centrifuged and cells are smeared onto glass slides, and then immersed in 95%

The reported sensitivity of cytology in the detection of intraocular malignancy ranges from 31% to 66.7%. This relatively low yield may be due to the presence of immune cells, necrotic cells, fibrin, and debris in the specimen, which may confound the examination. [31] Other reasons include small sample volumes with a low number of malignant cells, inadequate

Cytologic evaluation may also be used to distinguish between a malignant process and an inflammatory disease. An example of an inflammatory etiology that may be diagnosed with the aid of cytology is sarcoidosis. Kinoshita et al. demonstrated multinucleated giant cells in the vitreous in 85.7% of cases and lymphocytes and epithelioid cells in all cases of intraocular

An advanced technique for cytology is the use of cell blocks. They are superior to cell smears since cells are accumulated by centrifugation and stored as paraffin blocks. The large number of cells in a compacted area of one section on a slide glass as opposed to sparse cells on a smear leads to a more accurate diagnosis. Paraffin sections also have the advantage of being used for immunocytochemical diagnosis and clonal analysis, such as amplification by PCR of the

Microbiological cultures are considered the "gold standard" for diagnosis of infectious uveitis. There are different types of media for isolation of the causative agent, including blood agar (for gram-positive or fastidious gram-negative bacteria [34]), MacConkey agar (for most gramnegative rods [34]), and Brucella agar for bacterial infections; Sabouraud dextrose agar for pathogenic fungi and yeast; and shell vial culture for viral infections. Along with the culture,

Some fastidious organisms, such as *Proprionibacterium acnes* and fungi require holding the

The sensitivity of culture after diagnostic vitrectomy for diagnosis of chronic infectious uveitis has been reported between 16.7% and 96%. [21] In cases of acute endophthalmitis, the sensitivity of microbiological cultures and stains was shown to be 40-70%. [35] Higher yields are reported with vitreous rather than aqueous samples. [36] Processing both diluted and

undiluted vitreous samples increases the sensitivity of vitreous cultures to 57.4%. [37]

the sample is sent for Gram staining and antibiotic sensitivity tests. [20]

culture for at least 1 month to avoid missing their diagnosis. [21]

ethanol for Papanicolaou (Pap) staining or left to dry for Giemsa staining. [20]

preparation of the sample, and previous administration of corticosteroids. [20]

thology, immunohistochemistry, and molecular analysis. [20]

**5.2. Cytology**

168 Advances in Eye Surgery

sarcoidosis. [32]

immunoglobulin heavy chain gene. [33]

**5.3. Microbiological analysis**

Molecular analysis of a vitreous specimen is used for two main indications: 1) to diagnose PIOL 2) to detect the DNA of microorganisms in cases of infectious uveitis.

The techniques currently in use for molecular analysis include PCR, an in vitro technique used to amplify small quantities of nucleic acid into analytic amounts [39] and microdissection, which allows the selection and molecular analysis of malignant or atypical lymphoid cells from vitreous samples with a small amount of preserved cells. [31]

In cases of infectious uveitis, several PCR techniques may be used. Over the years new modifications to the basic method, such as real-time PCR and multiplex PCR have been developed. Real-time PCR allows for the characterization of an active infection versus lowgrade pathogenicity by quantifying the number of pathogen genomes in a sample. Multiplex PCR allows for the amplification and detection of a number of different sequences at the same time (such as two infectious agents from a single sample). [40]

The addition of PCR to microbiological analysis has been shown to increase the diagnostic sensitivity from 48% to more than 80%. [41] Prior short-term use of intravitreal antibiotics does not affect its ability to amplify DNA. In one series of patients with postoperative endophthal‐ mits treated with intravitreal antibiotics, PCR of vitreal specimens identified the causative organism in 10 of 16 patients (62%) versus only 3 (18%) with culture only. [42]

As the causative organism is not always known or suspected, a PCR technique that targets a specific microorganism is not always feasible. In such cases eubacterial PCR may be used. It targets the 16S ribosomal DNA (rRNA) common to all bacteria, thereby identifying a wider range of pathogens. [42-44] A similar approach detects the fungal genome in ocular fluids using probes that target the 18S rRNA present in the *Candida* and *Aspergillus* species, and probes that target the 28S rRNA also found in other species, including *Cryptococcus, Trichophyton, Mucor, Penicillium,* and *Pichia*. [45]

For PIOL diagnosis, PCR is used to detect monoclonality within the variable region of the third complementary determining region (CDR3) in the immunoglobulin heavy chain gene of malignant B cells. Single-band detection of immunoglobulin heavy chain rearrangement can be useful in PIOL. [20] In a study by Baehring et al., PCR was 64% sensitive for PIOL and identified immunoglobulin heavy chain gene rearrangements in four samples that were classified as negative for lymphoma based on cytopathology and flow cytometry. Cytology 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, suggesting a more aggressive treatment approach. [47]

#### **5.5. Flow cytometry**

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 surface markers that identify leukocytes. [20]

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

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 CD8<sup>+</sup> T lymphocytes were specific, but not sensitive. A CD4:CD8 ratio of ≥4 was highly sensitive and specific for inflammatory uveitis.

#### **5.6. Cytokine measurement**

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 treatment. [31]

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‐ icant.

Since the measurement of cytokine levels is fairly easy, measurement of IL-10 and IL-6 levels is recommended for patients with suspected PIOL. [20]

#### **5.7. Antibody measurement**

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

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 ratio of greater than 1.0 is abnormal and ratios of 2-3 are considered significant. [55] Its accuracy has been shown in the case of toxoplasmosis. [56] Errera et al have shown that GWC testing had better sensitivity than PCR in ocular toxoplasmosis, especially when the test was carried out in younger patients with quiet eyes, with smaller sized chorioretinal lesions. In contrast, they have shown that this test was not helpful in viral retinitis in comparison to PCR, as the sensitivity and positive predictive value (PPV) were lower for GWC. [57]
