RFPT011401

PCR and Flow CytometRy as diagnostiC aids FoR lymPhoid neoPlasmsLaura a. Snyder, dVM, dipLoMate aCVp

Establishing a diagnosis of lymphoma or lymphoid leukemia is frequently straightforward. When aspiration of enlarged lymph nodes and organs or evaluation of peripheral blood identifies high numbers of large lymphocytes or lymphoblasts, your diagnostic work is complete, unless you decide to pursue tissue biopsy and immunohistochemistry for further characterization, grading and immunophenotyping.

But every so often there are cases that aren’t quite so simple. The cytologic or hematologic diagnosis is equivocal. There are words in the pathology report like ‘suspicious’, ‘concern for’ and ‘cannot rule out’. Sometimes, even the biopsy intended to give you a definitive diagnosis comes back with uncertain findings. What options do you have?

Two different testing options can be particularly helpful in these circumstances. One is called PCR for antigen receptor rearrangements (abbreviated PARR); the other is flow cytometry. This article is intended to give a brief overview of these testing modalities and to help the reader understand the clinical context in which each test is best utilized.

PARR: How It Works

PCR for antigen receptor rearrangements (PARR) relies on the presence of unique VDJ (variable diversity joining region) rearrangements within the genes encoding the B and T cell receptors of lymphocytes (figure 1). These VDJ rearrangements are a critical component of the adaptive immune response. In

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PCR and Flow CytometRy as diagnostiC aids FoR lymPhoid neoPlasms ...............................1

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Primer

Conserved Region

V-D-J Rearrangement (encodes B and T cell receptors)

Figure 1. VDJ rearrangement shown with PCR primer binding to conserved regions.

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non-neoplastic conditions, lymphoid populations are polyclonal and contain cells with a wide variety of VDJ rearrangements. In contrast, neoplastic conditions contain a monoclonal population of lymphoid cells with an identical or nearly identical VDJ rearrangement.

PARR can be performed on cytology samples (stained or unstained slides, effusions) peripheral blood and paraffin embedded tissue biopsies. DNA is extracted from the submitted sample and primers for conserved regions of the B and T cell receptor gene are applied. The sample is then subjected to amplification in a thermocycler and the product applied to an agarose gel for separation of gene products. Upon separation, polyclonal/mixed VDJ segments from reactive lymphoid populations will form a ladder or smear of variably sized gene products. In contrast, monoclonal VDJ segments from a B or T-cell lymphoid neoplasm will create a solitary, thick, band from a single gene product (figure 2).

PARR: Diagnostic Utility

PARR is particularly helpful in cases of early/emerging acute leukemia and lymphoma where lymphoblast numbers may not yet comprise a sufficient percentage to establish a definitive diagnosis with cytology or blood smear evaluation. Similarly, PARR can be used to characterize a persistent or progressive increase in peripheral blood small lymphocytes and distinguish chronic lymphocytic leukemia from a lymphocytosis associated with antigenic stimulation or certain inflammatory conditions. In both instances the samples you have already submitted (cytology slides or whole blood) can be utilized for PARR analysis.

Though we tend to think of histopathology and immunohistochemistry as absolute and unequivocal, there are times when a definitive diagnosis of lymphoma can be challenging. In particular, the histologic appearance of gastrointestinal and epitheliotrophic lymphoma can overlap with inflammatory bowel disease and certain histiocytic/inflammatory skin conditions, respectively. In addition, cases of small cell and indolent lymphoma in the lymph nodes or spleen may present a diagnostic challenge. In these cases, employing PARR on the paraffin embedded tissue sample may help clarify the diagnosis.

PARR: Pros and Cons

As with any test, there are limitations that must be considered. The first is the sensitivity of the test. At present, the sensitivity of PARR is approximately 75-80% for dogs. This means

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Figure 2. Depiction of amplified gene products from PCR for antigen receptor rearrangement (PARR). Polyclonal: numerous, variably sized gene products from a reactive lymphoid population. Monclonal B and T-cell: a solitary gene product from a neoplastic B and T-cell population, respectively. Oligoclonal: a restricted population of gene products amplified from a dog with Ehrlichia canis infection.

Polyclonal MonoclonalB-Cell

MonoclonalT-Cell

Oligoclonal

that 1 out of 4 to 5 cases of lymphoma may be missed by this testing modality. There are several reasons why this may occur: 1) you have an NK cell lymphoid neoplasm that doesn’t have B or T cell receptor rearrangement, 2) the tumor is derived from very early lymphocyte precursors that do not have B or T cell receptor rearrangement, or 3) there are DNA mutations or deletions that lead to an inability to bind primers or allow amplification. The sensitivity of PARR for feline samples is significantly lower than their canine counterparts at only 65%. The reason for this comparatively poor sensitivity is somewhat of an enigma but is thought to be related to somatic hypermutation, particularly in the conserved region of the B-cell receptor gene, thus leading to impaired primer binding. For this reason, PARR may not be the best choice for ruling out leukemia/lymphoma in feline patients.

In contrast, the specificity of PARR is quite high at approximately 94%. The cause of the 6% false positive rate is consistently attributable to monoclonal or oligoclonal T-cell expansions associated with tick borne disease, particularly Ehrlichia canis and less commonly B-cell expansions with Lyme disease, Rocky Mountain Spotted Fever and bartonellosis (figure 2). In cases where tick borne disease is a consideration, particularly with lymphocytosis in the peripheral blood, serology or monitoring the CBC following treatment may be helpful for distinguishing neoplastic from non-neoplastic causes.

Flow Cytometry: How It Works

Flow cytometry relies on the surface or cytosolic expression of lymphoid antigens (CD4, CD8, CD21, CD79a, etc.). As previously mentioned, in non-neoplastic conditions, lymphoid populations are polyclonal and have mixed antigen expression patterns. Neoplastic lymphoid populations are monoclonal and will have a homogeneous pattern of antigen expression or possibly an aberrant pattern of expression not expected for normal/non-neoplastic cells.

In contrast to PARR, which requires only intact DNA, flow cytometry requires submission of viable cells. Samples may come from fine needle aspirates of solid tissue, effusions or peripheral blood; however cells must be collected into a specialized medium or combination of saline and serum (as specified by the lab performing the test) and shipped overnight for analysis. In brief, panels of fluorescent protein labeled monoclonal antibodies are incubated with cell suspensions. Labeled cells are passed through a flow channel where lasers enumerate, size and sub-classify cells by their fluorescent signal. Outputs are then charted and analyzed.

Flow Cytometry: Diagnostic Utility, Pros and Cons

Though flow cytometry may be performed on cell suspensions from any tissue, it is most frequently employed to characterize circulating lymphocyte populations in peripheral blood. As with PARR, flow cytometry may be used to characterize a persistent or progressive increase in peripheral blood small lymphocytes and distinguish chronic lymphocytic leukemia from a lymphocytosis associated with antigenic stimulation or certain infectious and inflammatory conditions.

At present, a diagnosis of neoplasia with a peripheral lymphocytosis requires meeting one of the following criteria: 1) the presence of ≥80% phenotypically homogeneous lymphocytes (e.g., all CD 8+ T-cells), 2) an expanded population of phenotypically abnormal lymphocytes (e.g., loss of T-cell markers), or 3) ≥60% phenotypically homogeneous lymphocytes with a concurrent positive clonality test (PARR).

Because flow cytometry is an interpretive test, there are no exact sensitivity and specificity values. However, as with PARR, Ehrlichia canis infection in dogs does present a diagnostic stumbling block. The monoclonal/oligoclonal expansion observed with PARR manifests as a homogeneous CD8+ lymphocytosis on flow cytometry of the peripheral blood. As previously mentioned, serology or serial monitoring of the peripheral blood following treatment may be considered to distinguish this unique inflammatory response from neoplasia. In cats, it is also

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worth noting that there are relatively fewer lymphoid antibodies available, which can make characterization of a peripheral lymphocytosis or lymphoid proliferation more challenging in this species. Consequently, there are also fewer studies on the efficacy of this test in the diagnosis of lymphoid neoplasms in the feline patient.

In contrast to PARR, which only provides confirmation of clonality and a determination of B or T cell lineage, flow cytometry provides a more complete characterization of immunophenotype, which may carry relevant or prognostically significant information in dogs. For example, a CD34+ lymphocytosis (precursor marker) carries a significantly poorer prognosis than CD34- cells. Additionally, phenotype can be combined with cell size and cell numbers to determine prognosis. For example, a CD21+ (B-cell) lymphocytosis comprised of small cells carries a significantly more favorable prognosis (median survival time not reached) than one comprised of large cells (median survival 129 days). Similarly, a CD 8+ (T-cell) small cell lymphocytosis with ≤30,000 cells/uL carries a more favorable prognosis (median survival 1098 days) than one with ≥30,000 cells/uL (median survival 131 days). At present, aside from identifying the LGL phenotype (which is often discernable via light microscopy alone), information regarding phenotypic impact on prognosis is lacking in cats with a persistent lymphocytosis.

In addition to providing prognostic information for some cases, the panels of antibodies utilized in flow cytometry may allow for identification of hematopoietic malignancies not detected by PARR, including non-B/T cell lymphoid neoplasms, leukemias of myelomonocytic origin and undifferentiated leukemias.

The Bottom Line:

As with most laboratory tests, the true utility lies in selection of the appropriate test for the appropriate case. At Marshfield Labs we are committed to helping you select the right tests for your patients. If you are wondering if PCR or flow cytometry are appropriate tests for a particular case, or if you simply have general questions about these tests, please feel free to contact one of our pathologists through customer service.

References:

• Molecular methods to distinguish reactive and neoplastic lymphocyte expansions and their importance in transitional neoplastic states. Vet Clin Pathol, 2004(4); 33:196-207.

• Canine lymphoproliferative disease characterized by lymphocytosis: Immunophenotypic markers of prognosis. J Vet Intern Med, 2008; 22:596-601.

• Determining the significance of persistent lymphocytosis. Vet Clin Small Animal 37(2007) 267-282.

• Diagnostic algorithm to differentiate lymphoma from inflammation in feline small intestinal biopsy samples. Vet Pathol; 28:212-222.

• Utility of polymerase chain reaction for analysis of antigen receptor rearrangement in staging and predicting prognosis in dogs with lymphoma. J Vet Intern Med 2006;20:329-334.

teChniCian’s CoRneRLaura a. Snyder, dVM, dipLoMate aCVpQuestions:

1. Name the erythrocyte morphologic abnormality identified by the red arrow (picture 1).

2. Describe how this morphologic abnormality forms.

3. Which animal is most susceptible to their formation?

4. What staining technique can be used to highlight this morphologic change?

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5. Name the erythrocyte morphologic abnormality identified by the blue arrows (picture 1).

6. Describe how this morphologic abnormality forms.

7. What do these two morphologic changes suggest?

8. What other changes might be seen on the blood smear if these morphologic abnormalities are observed in high number?

Answers:

1. The small protrusion identified by the red arrow is a Heinz body.

2. This morphologic change is caused by oxidative damage resulting in attachment of precipitated, oxidized hemoglobin to the internal erythrocyte membrane.

3. Cats are more susceptible to Heinz body formation than other species because their hemoglobin is more easily denatured by oxidants. In addition, the cat spleen is less efficient in removing these damaged cells from circulation. Note that up to 5% of cat erythrocytes may have Heinz bodies in health.

4. New methylene blue stained blood smears can be used to highlight Heinz bodies (picture 2, red circle).

5. These cells are called eccentrocytes.

6. Eccentrocytes form from oxidative damage to the internal erythrocyte membranes causing them to fuse and eccentrically displace the hemoglobin, leaving a clear area in the remaining part of the cell.

7. Seeing one or both of these morphologic changes raises strong concern for exposure to oxidants. Potential oxidants include onions, garlic, acetaminophen, anti-inflammatory drugs, vitamin K, naphthalene and prolonged propofol anesthesia. Certain disease states (particularly diabetic ketoacidosis, hyperthyroidism, lymphoma) may also precipitate Heinz body or eccentrocyte formation by increasing levels of endogenous oxidants.

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Picture 2

Picture 1

8. Both Heinz bodies and eccentrocytes increase the fragility of the affected cells, resulting in hemolysis. High numbers of cells can result in hemolytic anemia of varying degrees. If enough time has passed, evidence of regeneration may be evident on the blood smear. Note the polychromatophilic red blood cells (picture 1, asterisk) and the reticulocyte (picture 2, black circle), which are both indicators of regeneration.

what is youR diagnosis?dr. Jennifer L. BrazzeLL, dVM, MVetSC, dipLoMate. aCVp (CLiniCaL and anatoMiC), MrCVS

Table 1: Complete blood count from a 1 year 1 month old mixed breed dog.

Flag Test Results Units Reference Interval

RBC Count 5.09 x106/uL 4.48-8.53

Hemoglobin 13.0 g/dL 10.5-20.1

Hematocrit 38.4 % 33.0 - 58.7

Mean Corpuscular Volume 75.5 fL 63.0 - 78.3

Mean Corpuscular Hemoglobin 25.5 pg 21.0 - 27.0

Mean Corpuscular Hemoglobin Concentration

33.8 g/L 30.8-35.9

Red Cell Distribution Width 14.5 % 11.9-18.1

White Blood Cell Count 16.6 x103/uL 4.0-18.2

Segmented Neutrophil Absolute Number

15.44 x103/uL 2.50-15.70

Lymphocyte Absolute Number 0.50 x103/uL 0.30-3.90

Monocyte Absolute Number 0.33 x103/uL 0.00-1.40

Eosinophil Absolute Number 0.00 x103/uL 0.00-1.30

Basophil Absolute Number 0.00 x103/uL 0.00-0.10

Platelet Count 429 x103/uL 140-540

Answer:

In the table above, there are no numerical abnormalities on the CBC from this young dog but did you notice that there was no mention of red blood cell and white blood cell morphology in the results presented? This was a purposeful omission not meant to deceive but rather to illustrate the necessity of peripheral blood smear review for every CBC performed in a referral laboratory or in your clinic, regardless of the numerical data produced by the automated analyzer.

In the case presented, significant abnormalities were noted during the peripheral blood smear review (Table 2).

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Table 2: Manual Differential - modified absolute leukocyte counts based on the manual differential; red and white blood cell morphology notes.

Flag Test Results Units Reference Interval

Segmented Neutrophils 93 %

Band Neutrophils 2 %

Lymphocytes 2 %

Activated Lymphocytes 1 %

Monocytes 2 %

Eosinophils 0 %

Basophils 0 %

Segmented Neutrophil Absolute Number

15.44 x103/uL 2.50-15.70

H Band Neutrophil Absolute Number 0.33 x103/uL 0.00-0.20

Lymphocyte Absolute Number 0.33 x103/uL 0.30-3.90

Activated Lymphocyte Absolute Number

0.17 x103/uL 0.00-0.90

Monocyte Absolute Number 0.33 x103/uL 0.00-1.40

Eosinophil Absolute Number 0.00 x103/uL 0.00-1.30

Basophil Absolute Number 0.00 x103/uL 0.00-0.10

Platelet Count 429 x103/uL 140-540

RBC morphology—2+ poikilocytosis, 1+ polychromasia, 2+ spherocytes

WBC morphology—Dohle bodies noted

The most significant abnormality present on the peripheral blood smear examination is the presence of spherocytes. The presence of spherocytes in the peripheral blood should always be considered clinically significant and indicative of immune-mediated targeting and destruction of red blood cells. In this case, the presence of spherocytes would have gone unnoticed had a peripheral blood smear not been examined and the ultimate diagnosis of immune-mediated hemolytic anemia would have been missed. Consultation with the submitting veterinarian revealed that this dog was being treated with prednisone for a previous diagnosis of IMHA. In this case, despite the normal hemogram, the presence of spherocytes indicates that the immune-mediated destruction of the patient’s red blood cells was ongoing and that treatment should continue. Had the clinician relied solely on numerical results and the presence of a normal hematocrit, consideration may have been given to decreasing the dose of prednisone which is contraindicated in light of the continued spherocytosis.

In addition, a slight inflammatory leukogram evidenced by a neutrophilic left shift with toxic changes is present. This is commonly encountered as a secondary change in dogs with IMHA but monitoring of the CBC for worsening of the inflammatory leukogram was

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recommended due to the immunosuppressive doses of prednisone being administered to this dog and the potential for opportunistic infections.

Other significant findings that may be found on peripheral blood smear review in addition to red blood cell and white blood cell morphology changes include the presence or absence of platelet clumping, microfilaria, and hemoparasites.

At Marshfield Labs, accuracy and results you can trust are our highest priority. Thus, a manual differential count; review of red blood cell, white blood cell, and platelet morphology; and a hemoparasite screen is performed on every CBC regardless of the numerical data generated by the automated analyzer.

If you have any questions or comments regarding this case, please feel free to email Dr. Jennifer Brazzell at brazzell.jennifer@marshfieldclinic.org or call her via Customer Support at 1-800-222-5835.

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mailto: brazzell.jennifer@marshfieldclinic.org